Progress in Nucleic Acid Research and Molecular Biology, Volume 44

PROGRESS IN

Nucleic Acid Research and Molecular Biology Volume

44

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PROGRESS IN

Nucleic Acid Research and Molecular Biology edited by

WALDO E. COHN

KlVlE MOLDAVE

Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

Department of Molecular Biology and Biochemistry University of California, lrvine Irvine, Cal$ornia

Volume 44

ACADEMIC PRESS, INC. Harcourt Brace Jooanooich, Publishers San Diego New York Boston london Sydney Tokyo Toronto

This bock is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system. without permission in writing from the publisher.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-4311 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX

Library of Congress Catalog Number: 63- 15847 International Standard Book Number: 0-12-540044-6 PRINTED IN THE UNITED STATES OF AMERICA 9 3 9 4 9 5 9 6 9 7 9 8

BE

9 8 1 6 5 4 3 2

1

Contents

ABBREVIATIONSAND SYMBOLS ........................................

ix

.......................

xi

SOME

ARTICLES PLANNED FOR FUTUREVOLUMES

Structure and Action of Mammalian Ribonuclease (Angiogenin) Inhibitor Frank S . Lee and Bert L . Vallee I. I1. 111. IV. V.

Purification. Physicochemical Properties. and Occurrence . . . . . . . . . . Primary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitory Properties .......................................... Biologic Role ................................................ Concluding Remarks .......................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 6 10 20 24 26

Bacterial Adenylyl Cyclases Alan Peterkofsky. Aiala Reizer. Jonathan Reizer. Natan Gollop. Peng-Peng Zhu and Niranjana Amin I . The Action of cAMP as a Transcription Regulator in Escherichiu coli ............................................ I1. Regulation of cAMP Levels in E . coli .... I11. Structure and Expression of the E . coli Ade (cyu) Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Phosphoenolpyruvate:sugarPhosphotransferase System . . . . . . . . V. Regulation of E . coli Adenylyl Cyclase Activity by the Phosphoenolpyruvate:sugar Phosphotransferase System . . . . . . . . . . . . VI. Regulation of E . coli Adenylyl Cyclase Activity by Other Factors . . . . VII . AdenylyI Cyclases in Bacteria Other Than E . coli ................. VIII. Sequence Comparisons . . . . . . . . . . .................. IX. ATP-Binding Sites ...................... ................... X . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ..................................................

32 34 35 36

38 41 44 53 56

62 62

Initiation of Transcription by RNA Polymerase 11: A Multi-step Process Leigh Zawel and Danny Reinberg I . The Structure of Class I1 Promoters ............................. I1. RNA Polymerase I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

68 69

vi

CONTENTS

Transcription Factors and Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preinitiation and Initiation Complexes and Motifs . . . . . . . . . . . . . . . . . Activation and the General Transcription Factors . . . . . . . . . . . . . . . . . Repression of Class I1 Gene Transcription . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . , . . . . . . . . . , . . . . . . . . . . , . . . . . . . . . .

111.

IV. V. Vl.

75 94 100 102 105

Regulation of Repair of Alkylation Damage in Mammal ion Genomes Sankar Mitra and Bernd Kaina Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . Unusual Repair of Ofi-Alkylguanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multistep Repair of N-Alkylpurines . . . . . . . . . . . . . . . . . . . . . . IV. Properties of Mammalian Ofi-Methylguanine-DNA Methyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Cloning of Mammalian Alkylation Repair Genes by Phenotypic Rescue of E . coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... .. VI. Regulation of Mammalian M G M T and MPG VII. Role of DNA Methylation in Methyltransferas VIII. Inclucihility of Alkylation Repair Genes . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Alkylating Drug Resistance and Regulation of DlVA Repair . . x. Amplification of th e and Drug Resist ......... ................ XI. Outlook . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

11. 111.

109 112 114 116 118 120 128 129 132 135 136 137

Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides Jean Paul Leonetti, Genevihve Degols, Jean Pierre Clarenc, Nadir Mechti and Bernard Lebleu I. 11. 111.

IV. V. \'I.

VII.

Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From the Antisense Approach to the SNAIGE Concept Limitations of thc SNAIGE Approach . . . . . . . . . . . . . . . . . , . . . . . . . . . Internalization and Targeting of Oligonucleotides In tracelliilar Distribution of OIigonucIeotides . . . . . . . . . . . . . . . . . . . . . Mechanisms of Action of Antisense Oligonucleotides in the VSV Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Perspectives ...... Hefcrences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

143 145 148 153 160 161 163 164

Enzyme Organization in DNA Precursor Biosynthesis Christopher K . Mathews 1. 11.

Enzyme Organization and Contemporary Enzymology . . . . . . . . . . . . . E d y Evidence for dNTP Coinpartmentation . . . . . . . . . . . . . . . . . . . . ,

167 171

CONTENTS

I11. T4 dNTP Synthetase: A Multienzyme Complex for Deoxyribonucleotide Synthesis ................................. IV. Is the T4 dNTP Synthetase Complex Linked to DNA Replication Machinery? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Enzyme Organization in Bacterial Cells ......................... VI . Organization of dNTP Synthesis in Eukaryotic Cells . . . . . . . . . . . . . . . VII . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 177 181 186 187 200 201

Identification and Characterization of Novel Substrates for Protein Tyrosine Kinases Michael D . Schaller. Amy H . Bouton. Daniel C . Flynn and J. Thomas Parsons I . Detection of Phosphotyrosine-containing Proteins . . . . . . . . . . . . . . . . . I1. Receptor Protein Tyrosine Kinases .............................. I11. Oncogenic Protein Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Novel Strategies for the Identification of Substrates . . . . . . . . . . . . . . . V. Tyrosine Phosphorylation: Molecular Consequences . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 208 211 215 222 224 229

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Abbreviations and Symbols All contributors to this Series are asked to use the terminology (abbreviations and symbols) recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) and approved by IUPAC and IUB. and the Editors endeavor to assure conformity. These Recommendations have been published in many journals (I.2) and compendia (3) and are available in reprint form from the Office of Biochemical Nomenclature (OBN); t h 9 are therefore considered to be generally known. Those used in nucleic acid work, originally set out in section 5 of the first Recommendations ( I )and subsequently revised and expanded (2.3). are given in condensed form in the frontmatter of Volumes 9-33 of this series. A recent expansion of the one-letter system ( 5 ) follows. (5) SINGLE-LETTER CODERECOMMENDATIONS. Symbol

Meaning

Origin of symbol Guanosine Adenosine (ribo)Thymidine (Uridine) Cytidine

R Y M

K S Wb

G or A T(U) or C A or C G or T(U) G or C A or T(U)

puRine pyrimidine aMino Keto Strong interaction (3 H-bonds) Weak interaction (2 H-bonds)

C or T(U) T(U) or C C or A A or T(U)

D’

A or G or G or G or

N

G or A or T(U) or C

aNy nucleoside (i.e., unspecified)

Q

Q

Queuosine (nucleoside of queuine)

H B V

not not not not

G; H follows G in the alphabet A; B follows A T (not U); V follows U C; D follows C

‘Modified from h c . Natl. Acad. Sci. LISA. 83, 4 (1986). bW has been used for wyosine, the nucleoside of “base Y” (wye). ‘D has been used for dihydrouridine (hU or H, Urd).

Enzymes

In naming enzymes, the 1984 recommendations of the IUB Commission on Biochemical Nomenclature ( 4 ) are followed as far as possible. At first mention, each enzyme is described eirher by its systematic name or by the equation for the reaction catalyzed or by the recommended trivial name, followed by its EC number in parentheses. Thereafter, a trivial name may be used. Enzyme names are not to be abbreviated except when the substrate has an approved abbreviation (e.g., ATPase, but not LDH. is acceptable).

ix

ABBREVIATIONS AND SYMBOLS

X

REFERENCES 1. JBCtQ1.527 (1%); &hem 5, 1445 (1966); BIlO1. 1 (1966); ABB 115, I (1966), 129, I (1%9); and e1sewhere.t General. 2. EJB 15, 203 (1970); JBC 245, 5171 (1970); JMB 55. 299 (1971); and e1smhere.t 1. “Handbook of Biochemistry” (G. Fasman. ed.), 3rd ed. Chemical Rubber Co., Cleveland, Ohio, 1970. 1975, Nucleic Acids. Vols. I and 11, pp. 3-59. Nucleic acids. 4. “Enzyme Nomenclature“ [Recommendations (1984) of the Nomenclature Committee of the IUB]. Academic Pms. New York, 1984. 5. EIB 150, I (1985). Nucleic Acids (One-letter system).t Abbreviotions of Journal Titles

Journah

Abbreviotions used

Annu. Rev. Biochem. Annu. Rev. Genet. Arch. Biochem. Biophys. Biochem. Biophys. Res. Commun. Biochemistry Biochem. J. Biochim. Biophys. Acta Cold Spring Harbor Cold Spring Harbor Lab Cold Spring Harbor Symp. Quant. Biol. Eur. J. Biochem. Fed. Proc. Hoppe-Seyler’s Z. Physiol. Chem. J. h e r . Chem. Soc. J. Bacteriol. J. Biol. Chem. J. Chem. Soc J. Mol. Biol. J. Nat. Cancer Inst. Mol. Cell. Biol. Mol. Cell. Biochem. Mol. Gen. Genet. Naturs New Biology Nuclek Acid Research Proc Natl. h d . Sci. U.S.A. Proc Soc Exp. Riot. Med. progr. NucI. Acid. Res. Mol. Biol.

ARB ARGen ABB BBRC Bchem BJ BBA CSH CSHLab CSHSQB EJB FP ZpChem JACS J. Bact. JBC JCS

JMR JNCI MCBiol MCBchem MGG Nature NB NARCS PNAS PSEBM This Series

?Reprints available from the Office of Biochemical Nomenclature (W.E. Cohn, Director).

Some Articles Planned for Future Volumes The DNA Binding Domain of the Zn(ll)-containing Transcription Factors JOSEPH E. COLMAN AND T. PAN mRNA Binding Proteins in Eukaryotic Cells TOMDONAHUE AND K. GULYAS Specific Hormonal and Neoplastic Transcriptional Control of the Alpha 2u Globulin Gene Family

PHILIPFEIGELSON Molecular Biology in the Eicosanoid Field

COLIND. FUNK Cellular Transcriptional Factors Involved in the Regulation of HIV Gene Expression

RICHARDGAYNOR AND C. MUCHARDT tRNA Structure and Aminoacylation Efficiency

R~CHARD G I E G ~JOSEPH ~, D. PUGLISIAND CATHERINE FLORENTZ Control of Mitochondria1 Biogenesis in Yeast

LES GRIVELLAND H.

DE

WINDE

snRNA Genes: Transcription by RNA Polymerase II and RNA Polymerase 111 NOURIA HERNANDEZ AND LOBO

s.

Enzymology of Homologous Recombination in the Yeast Saccharornyces cerevisiae w. -D. HEYERAND RICHARDD. KOLODNER Regulation of mRNA Stability in Yeast

ALLANJACOBSON AND

s. PELTZ

Signal-transducing G Proteins: Basic and Clinical Implications

MICHAEL A. LEVINE Synthesis of Ribosomes LASSE LINDAHL AND

J. M. ZENGEL

Nitrogen Regulation in Bacteria and Yeast

BORIS MAGASANIK Immunoglobulin Gene Diversification by Gene Conversion WAYNE T. MCCORMACK, LARRY TJOELKER AND

w.

CRAIGB. THOMPSON xi

xii

SOME ARTICLES PLANNED FOR FUTURE VOLUMES

ADP-ribosylation Factors

JOEL MOSS AND MARTHA VAUGHAN Regulation of Eukaryotic mRNA Entry into Polysomes by Initiation Factor Phosphorylation

ROBERT E. RHOADES Mammalian 6-Phosphofructo-2-kinase/fructose-2,6-biphasphatase:A Bifunctional Enzyme

GUY G . ROUSSEAU AND LOUISHUE Analysis of Rice Genes in Natural and Transgenic Plants

R A Y WU, XIAOLAN

DUANAND

DEPING X U

Structure and Action of Mammalian Ribonuclease (Angiogenin) Inhibitor 1 FRANKs. LEE AND , BERT L. VAL LEE^

1 1

Center for Biochemical and Biophysical Sciences and Medin'ne Harvard Medical School Boston, Massachusetts 02115

I. Purification, Physicochemical Properties, and Occurrence . . . . . . . . . . . .

A. Inhibition Constants

2

....

.......................

20

References . . . . .

A exceptionally potent protein RNase2 inhibitor occurs in the tissues of mammalian species (1, 2). While this 50-kDa protein inhibitor is commonly employed experimentally to inhibit RNA degradation by adventitious RNases (3-18), it undoubtedly possesses a physiologic significance of its own. Among the functionally diverse mammalian RNases that it inhibits are some able to induce biologic activities that include neovascularization, ataxia, and paralysis, and others that possess potent helminthotoxic or antispermatogenic properties (reviewed in 19-22). Our original interest in this inhibitor stemmed from our efforts to identify factors involved in the regulation of organogenesis in general, and blood vessel development in particular. The culmination of this work was the isolation of angiogenin, a 14-kDa protein initially purified from the conditioned medium of the human adenocarcinoma cell line HT-29 and subsequently from normal human plasma (23,24).Angiogenin is a potent inducer 'To whom correspondence may be addressed. ZAbbreviations: RNase, ribonuclease; CAM, chorioallantoic membrane; RNase A, bovine pancreatic ribonuclease A; PRI, human placental RNase inhibitor; HRAS, Harvey ras oncogene homologue; IGF2, insulin-like growth factor 2; HBB, p-globin; INS, insulin.

1 Progress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright 0 1993 by Academic Press, Inc. All nghts of reproduction in any form reserved.

2

FRANK S. LEE AND BERT L. V A L I X E

of blood vessel growth in the chick chorioallantoic membrane (CAM) and the avascular rabbit cornea (23, 25, 26), with neovascularization in the CAM assay occurring at angiogenin doses as low as 0.5 ng. Angiogenin exhibits specific, saturable binding to calf pulmonary endothelial cells (27). It stimulates phospholipases C and A, in endothelial cells at concentrations as low as 0.1 ng/ml, but is not an endothelial cell mitogen (28, 29). The 35% identity of the angiogenin primary structure to that of bovine pancreatic ribonuclease A (RNase A) is a most unexpected feature (30, 31). Three residues catalytically essential in RNase A (Lys-41, His-12, and His-119) are fully conserved in angiogenin. Importantly, the catalytic activity of angiogen is distinct from that of RNase A or other RNases, and this, in turn, distinguishes it from all other angiogenic factors (32). These findings provided a unique opportunity to examine naturally occurring ribonuclease inhibitors for their antiangiogenic properties, and this resulted in the finding that the human protein RNase inhibitor abolishes both the angiogenic and ribonucleolytic and phospholipase C-stimulating activities of angiogenin (28, 33). Beyond pointing toward angiogentn regulation as a possible physiologic function for this inhibitor, these experiments indicated the importance of obtaining further information about this inhibitor as a basis for novel approaches toward antiangiogenesis. Antiangiogenic agents could potentially play important roles in the clinical treatment of a wide variety of diseases, including cancer, diabetic retinopathy, and rheumatoid arthritis (34). This review focuses on recent studies of the mammalian ribonuclease inhibitor and, in particular, human placental ribonuclease inhibitor (PRI). Far less is known about this protein inhibitor of ribonucleases than about protein inhibitors of proteinases, for which a vast literature exists (reviewed in 35-37). Nevertheless, recent studies have revealed distinctive properties of this family of proteins, properties of interest from the point of view of understanding their relation to the inhibition of the activities of the mammalian RNase superfamily of enzymes, including angiogenin in particular.

1. Purification, Physicochemical Properties, and Occurrence Early work on the protein RNase inhibitor from various mammalian tissues has been reviewed (I, 2). Hence the present discussion is confined to certain important aspects of that period and focuses on more recent studies. Historically, the existence of an RNase inhibitor was first inferred from the presence of latent RNase activity in the high-speed supernatant fraction from guinea pig liver homogenates (38).Acidification of the extract resulted in an increase in measurable RNase activity, presumably due to the dissociation of an RNase-inhibitor complex. Many critical studies (39-44) on the corre-

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

3

sponding inhibitor from rat liver demonstrated that it specifically inhibits bovine pancreatic ribonuclease A. These studies suggested that it was a protein, based on the susceptibility of the activity to proteases, acid conditions (0.05 M HCl), heat (65OC, 5 min), and sulfhydryl reagents [p-(hydroxymercuri)benzoate, N-ethylmaleimide, iodoacetate] (39, 41, 43). Initial attempts to isolate the inhibitor (40-42) were hampered by its sensitivity to air-oxidation, dilution to low protein concentrations, freezethaw cycles, and exposure to metals that often contaminated the RNA preparations employed to assay inhibitor activity (40, 42, 45-48). The use of reducing and metal-chelating agents in buffers employed during isolation facilitated maintenance of activity (42, 45, 48, 49). There have been many reports of partial purification of activity (11, 50-58). A critical advance in the purification of this inhibitor was the use of RNase A-Sepharose &nity chromatography (59).This allowed for the isolation of PRI and subsequently other inhibitors from mammalian sources in quantities sufficient for physicochemical analysis (59-62). The salient features of these inhibitors can be summarized as follows. They are acidic (pZ -4.7), heat- and acid-labile, sulfhydryl-dependent proteins of M, -50,000. They form 1:1 complexes with the highly basic (PI >9), acid-stable, mammalian pancreatic RNase (M, -14,000). They inhibit the enzymatic activity of this mammalian RNase but not that of Escherichia coli RNase I or the fungal RNases N,, T,, or U, (52, 63-65). Their amino-acid compositions are marked by high contents of Leu (-20% on a molar basis) and Cys (-7%) (Table I) (60-62, 66-68). Differences between these features and those of certain other inhibitors have been reported. For example, the inhibitor isolated from bovine lens is reported to have an M , of -30,000 (69, 70) while that from porcine brain is reported to have relatively low Leu and Cys contents (65).However, it is difficult to evaluate these observations because the stated specific activities of the protein preparations examined suggest that they were not pure. The inhibitor is a cytoplasmic protein, and the high-speed (60,000105,000 g) supernatant fraction of tissue homogenates is the source of virtually all inhibitory activity. Consistent with this, there is no evidence for signal peptides in the cDNA sequences encoding the human placental, HeLa cell, and porcine kidney inhibitors (66-67, 71).Free inhibitory activity can be detected directly in tissue homogenates. At the same time, latent RNase activity can be detected by treatment of the homogenates with sulfhydry1 reagents, presumably by inactivation of the sulfhydryl-dependent, bound inhibitor. Therefore, the inhibitor is apparently in stoichiometric excess over the enzyme(s) inhibited; the degree of molar excess has been estimated at three- to eight-fold (2, 72).The inhibitor constitutes -0.01% of the totaI protein in cytoplasmic extracts (2). Nuclear, mitochondrial, and

TABLE 1 AMINO-ACID(hMPOSITIONS

OF

MAMMALIAN HIHONLICLEASE

~NHIBI'IOHS"

~

Amino acid Asx

GlX Ser Cly His

Thr Ala Pro Tvr Val Met Ile Leu Phe

LYS CYS TrP Total Ref.

H 11 man placenta"

Huinan

HeLa"

Porcine liver","

Bovine brain"

44 59 45 32 5 24 13 32 14 3 25 2 12 92 4 16 32 6

44 59 46 32 5 23 13 32 14 3 25 2 12 92 4 16 32 6

40 60 38 37 7 22 23 32 16 4 19 2 9 98 0 14 30 5

43 65 40 53 5 20 20 38 18 5 22 2 9 88 3 15 30 5

460 66

460 67

456 68

481 60

aArnino-acid compositions reported in residues/mole protein. bFrom amino-acid sequence. CIdentical to porcine kidney inhibitor. dFrom amino-acid analysis of protein. "Not determined.

-.

nat

Ovinr liver"

Mti riiir liver"

Rat lived

47 6 19 21 37 1.5 5 24 2 9 91 4 14 30 5

48 62 42 51 5 20 19 35 17 4 23 1-2 10 92 4 15 27 6

55 63 13 33 3 19 22 30 I6 5 20 3 13 94 3 21 26-27 5

59 60 43 33 6 20 23 29 17 5 22 2 10 89 5 20 31 5

8 20 2 13 66 7 18 31 NUE

483

482

475

479

-

61

61

61

61

62

Bovine liver"

47 62 45

testis"

55 61 42 40 7 23 25 30 15

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

5

microsomal cell fractions, in contrast, contain little or none of the inhibitor (73, 74). Small amounts of inhibitor appear to be associated with mRNAribosomal particles (64,75). The RNase inhibitor has been detected in all mammals and in almost all tissues examined, which include lung, heart, parotid, esophagus, stomach, intestine, liver, pancreas, kidney, immature uterus, placenta, ovary, testis, thyroid, adrenal, thymus, spleen, reticulocyte, brain, muscle, and fat tissue (39, 58-63, 76-82). While most studies have assayed for the presence of inhibitor by inhibition of RNase activity, others have also documented its presence using anti-inhibitor antibodies (83) or the inhibitor cDNA (84). Among the tissues or cells examined, only mature uterus (82, 85, 86) and lymphocytes (87, 88) lack detectable levels of the inhibitor. It is not clear whether there is in fact a complete absence of it in these instances or whether it is present in quantities below the limit of experimental detection. In any case, it can be said that these inhibitors constitute a family of related, cytoplasmic proteins ubiquitous or nearly so in mammalian tissues. Extracellular fluids, which contain a variety of RNases, have also been examined for the presence of the inhibitor. Monoclonal antibodies directed against PRI detect immunoreactive material in human serum that would correspond to a concentration of 2 to 3 pg/ml if this material were PRI (89). However, when mammalian serum is either examined for latent RNase activity (90) or assayed directly for RNase inhibition (39, 58), no evidence for inhibitory activity is found. The RNase inhibitor may be present in serum in an inactive form; alternatively, it may be active but at undetectable levels. There is no evidence for the presence of inhibitor in either urine or saliva (55, 77). RNase inhibitory activity has been detected in nonmammalian organisms, including starfish nucleoli (91)and cytoplasmic extracts of insects (92), frogs (93,94), and chickens (78, 95, 96). As with the mammalian protein, the presence of the nonmammalian inhibitor has been demonstrated either by latent RNase activity or by direct inhibitory activity toward the endogenous RNase of the organism examined. These nonmammalian inhibitors do not appear to inhibit mammalian pancreatic RNase (49, 78). However, in the converse situation, the mammalian RNase inhibitor does inhibit at least one nonmammalian RNase, that from the pancreas of the snapping turtle (R. Shapiro, personal communication). This RNase is -30% identical in primary structure to mammalian pancreatic RNase; it is conceivable that some nonmammalian species may possess as yet undetermined inhibitors of mammalian pancreatic RNase. None of the nonmammalian inhibitors described have been purified to homogeneity. Gel filtration of crude cytoplasmic extracts suggests that the mass of the complex between frog inhibitor and its endogenous ribonuclease

6

FRANK S. LEE AND BERT L. VALLEE

is -130 kDa (93, 94). It is not known whether the difference in M, observed between this complex and the mammalian RNaseaRNase inhibitor complex reflects an intrinsic difference in the inhibitor, such as in molecular weight or inhibition stoichiometry.

II. Primary Structure The primary structures of RNase inhibitors from human placenta (66, 71), HeLa cells (69, and porcine kidney (84)have been derived from the cDNAs. In addition, that for the porcine liver inhibitor, identical to that from porcine kidney, comes from the amino-acid sequence (68).The primary structures of inhibitors from human placenta and HeLa cells are 99% identical and differ only at two residues, 422 and 423 (Arg and Gln, respectively, in the placental inhibitor; Ser and Glu, respectively, in the HeLa cell inhibitor). Those from human and porcine sources are 77% identical, and it may be noted that the human and porcine inhibitors bind to RNase A with comparable af€inity (see Section 111, Inhibitory Properties). The porcine inhibitor, 456 residues in length compared to 460 for the human placental inhibitor, is modified by Nacetylation at its N-terminus (68). The N-terminus of the human placental inhibitor is also refractory to Edman degradation and probably is modified by N-acetylation as well (66). The internal repeat structure is a salient feature of the inhibitor aminoacid sequence. It has been described variously as consisting of either (1) seven to eight 57-residue repeats, with each 57-residue repeat consisting of an internal duplication of a half-repeat (66, 67) or (2) 15 alternating repeats, with the alternating repeats consisting of either 28 or 29 residues each (68). The descriptions are essentially identical and for the sake of convenience the inhibitor is here described as consisting of seven direct, uninterrupted, internal repeat units, each exactly 57 amino acids in length (Fig. 1).These repeat units comprise nearly 90% of the molecule and are flanked by shorter N- and C-terminal segments that display a weak homology to the strong internal repeat motif. The average identity between any two repeat units is -40%. In the primary structure of PRI, 76-78% of either the leucines or the cysteines is conserved (present in at least four of the seven repeats) within the repeat units, compared to only 29% for the acidic residues (Asp Glu) (Fig. 1).Repetitive structural features reflected by the high degree of conservation of leucine and cysteine residues may constitute a common scaffold on which other residues, e.g., polar ones, determine specificity of interaction (67, 68). In this regard, it should be noted that the high conservation of cysteine residues does not imply the presence of conserved disulfide bonds, since chemical modification of these residues suggests that all are present in

+

LDIQCEELSDARWAE

1

-~

30 87

144 201

258 315 372 429

VESVR

SWRV I S

FIG. 1. Primary structure of PRI, highlighting the seven direct internal repeats (adapted from 66). The first residue in each line is numbered at the left. Shaded background is present for identical residues that occur in at least four of the seven repeats.

FRANK S. LEE AND BERT L. VALLEE

8

the reduced form (66, 84). The absence of disulfide bonds in PRI is consistent with its cytoplasmic localization, since the cysteines of cytoplasmic proteins are generally in the reduced form (97). Leucine-rich repeat units are not unique to RNase inhibitor. Related repeats, which are close to or exactly 21 residues long, have been identified in an increasing number of both mammalian and nonmammalian proteins (Fig. 2) (66-68). These proteins include the a and @ subunits of platelet glycoprotein Ib (the platelet receptor for von Willebrand factor) (98, 107, 108),the lutropin-choriogonadotropin receptor (103), the leucine-rich cxzglywprotein (99), the proteoglycan core protein (100,101,109, 110),the 83kDa subunit of human carboxypeptidase N (102), yeast adenylate cyclase ( I @ ) , and the Drosophila chaoptin and Toll-encoded proteins-the last two being factors involved in photoreceptor cell and embryonic development, respectively (105, 106). These proteins represent a widely divergent group of molecules, some that are membrane receptors and others that have catalytic or structural roles; collectively, they comprise extracellular, integral membrane, and intracellular proteins. In many of these proteins-such as the RNase inhibitor, the @-subunit of platelet glycoprotein Ib, the extracellular domain of the lutropin-choriogonadotropin receptor, the leucine-rich a,-glycoprotein, and chaoptin-the leucine-rich repeat units constitute most of the protein. Hence, these units may be intimately involved in the function of the protein. In others, such as the 83-kDa subunit of human carboxypeptidase N and yeast adenylate cyclase, the leucine-rich repeat units are distinct from

PRI (human) GPIba (human) LRG (human)

L-s -G

--v

-B

-a -&

- I

-@

PG40 (human) CPN (human) LHCGR (rat) AdCyc (yeast) Chaoptin (Drosophila) Toll (Drosophila)

FIG.2. Comparison between a portion of the repeat wnsensus sequence of PRI and those of the a subunit of platelet glycoprotein Ib (GPlba) (98),leucine-rich a2-glywprotein (LRG) (99),fibroblast proteoglycan core protein ( P O ) (100,101). the 83-kDa subunit of carboxypeptidase N (CPN) (102). the lutropin-choriogonadotropin receptor (LHCGR) (103), adenylate cyclase (AdCyc) (104). chaoptin (109, and the ToUencoded protein (106). Shaded background indicates residues identical among consensus sequences. Aliphatic residues (V, L, and I) are indicated by a.

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

9

the catalytic portions of the molecule. Thus, they may not be involved in the primary activity of the protein, and may have other functions. Though the three-dimensional structures of any of these proteins, including RNase inhibitor, have yet to be determined, the high degree of conservation of certain residues, particularly leucine, in the repeats of all of these proteins suggests the possibility of common structural motifs. It has been hypothesized that the repeats may be important for protein-membrane or proteineprotein interactions (99, 105,106). The implication, then, is that this leucine-rich repeat unit may provide a versatile motif for the attainment of diverse, specific molecular interactions. It remains to be seen whether any of these proteins share common functional properties attributable to the leucine-rich repeat. In this regard, it should be noted that this leucine-rich repeat is distinct from the leucine-zipper motif associated with the dimerization of various transcription factors, in which there is a regular periodicity of leucines every seventh residue (111). Northern blot analysis of human placental, monocyte, or various porcine tissue mRNAs employing the inhibitor cDNA as a probe reveals in all cases a single message of -1.8 kb in length (66, 67,71,84).While a single message size is detected in these studies, there is molecular heterogeneity in the inhibitor mRNA, since the inhibitor cDNA sequences from human placenta and HeLa cells differ in both sequence and length at their 5’ ends, and there are at least three additional human 5’ variants (71).The difference in size between the longest and shortest of these variants in 383 nucleotides, and the sequences suggest that up to four exons are potentially spliced into a site in the 5’ untranslated region, raising the possibility of translational regulation (71).Curiously, one of the 5‘ variants encodes for a protein lacking the first five residues, which comprise a pentapeptide sequence duplicated at the N-terminus of PRI. Whether this alternative cDNA produces a functional protein in uiuo is not known, but it should be noted that the porcine inhibitor, which lacks this N-terminal pentapeptide repeat, binds to RNase with an affinity comparable to that of PRI (see Section 111, Inhibitory Properties). Southern blot analysis of human DNA shows that the inhibitor is encoded by a single locus (71).Further localization studies employing in situ hybridization techniques and human-rodent hybrid cell lines indicate that the inhibitor gene is on chromosome subband llp15.5 (112,113). Other genes mapped to this subband include those for the Harvey ras oncogene homologue (HRAS), insulin-like growth factor 2 (IGF2), P-globin (HBB), and insulin (INS) (114). Chromosomal abnormalities involving band l l p 1 5 have been identified in a variety of neoplasms, including Wilm’s tumor, rhabdomyosarcoma, breast tumors, acute lymphoblastic leukemia, and acute myeloid leukemia (113).It will be of interest to examine whether RNase inhib-

10

FRANK S. LEE AND BERT L. VALLEE

itor expression, which could conceivably affect intracellular RNA turnover (see Section IV, Biologic Role), is altered in any of these disorders. The inhibitors from both human placenta and porcine kidney have been expressed as recombinant proteins. That from human placenta has been expressed from E. coli in a yield of 50 Fg/g of wet cells (1 mg/4 liters of culture) (115), while that from porcine kidney has been expressed from Saccharomyces cerecisiae in a yield of 200 Fg/g of wet cells (84).As with the native inhibitor protein, affinity chromatography employing RNase ASepharose is a critical step in the isolation of the recombinant protein. The protein expressed from E. coli lacks a modified N-terminus, in contrast to that expressed from S. cerecisiae and the native protein, both of which are modified at the N-terminus and refractory to Edman degradation. Otherwise, the physicochemical, immunological, and inhibitory characteristics of the recombinant inhibitors are virtually identical to those of the native proteins. This equivalence in the case of the inhibitor expressed in E. coli confirms that eukaryotic posttranslational modifications are not essential for inhibitory activity. In this regard, there is no evidence of glycosylation of the native protein (59).

111. Inhibitory Properties A. Inhibition Constants A striking feature of the mammalian ribonuclease inhibitor is its exceptionally potent inhibition of RNase, with extremely low K,values that reflect both rapid association and very slow dissociation rates. Kinetic studies concerning the angiogenin. PRI interaction illuminate these features. Association rate constants, obtained by stopped-flow kinetic measurements of the 50% fluorescence enhancement that accompanies the binding of PRI to angiogenin, indicate a two-step binding mechanism (116). The first step involves rapid formation of an enzymeeinhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: E

+I

Ki +

EX

ki? S

El*

k-2

The values of K , and k, are 0.53 F M and 97 s - l , respectively, while the apparent second-order rate constant of association at protein concentrations
11

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

The consequence is that the K, value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is extremely tight, 7.1 x 10- l6 M (0.71 fM). This value is among the lowest reported for the binding of two proteins and is comparable to the d n i t y of avidin for biotin, -10- l5 M (118).Other RNase-RNaseinhibitor interactions have been examined in comparable detail and display similar kinetic features. The association rate constants are all high and close to the diffusioncontrolled limit (Table 11) (84,116, 119).The dissociation rate constants are extremely low, with half-lives on the order of hours to months. The K , values in all cases are below 10- l3 M. PRI binds to angiogenin about as tightly as it does to placental RNase, in spite of only a 24% identity between the two enzymes. These 4 values, in turn, are & t o -&j of that for RNase A. of those originally reported These 4 values are between 10-1 and by others for various RNase AaRNase inhibitor interactions (59,60,65,120, 121).In some of the studies (59, 60,65),the use of Lineweaver-Burk and Dixon plots could be one possible cause of the discrepancy, since these plots do not take inhibitor depletion into account (122).In several of the cases, the fact that the inhibitor was isolated from a different source could be another cause (60, 65,120, 121).It is apparent that the tight-binding nature of the inhibitor interaction suggests that conventional kinetic analysis of the inhibitor may significantly underestimate the strength of binding. The K, value for the RNase AaPRI interaction increases from 44 fM in 0.1 M NaCl to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl (117). The 20,000-fold increase in & value from 0.1 to 1 M NaCl suggests that ionic interactions play a significant role in the binding of PRI to RNase. This provides a rationale for the efficacy of 3 M NaCl in the elution of PRI from its affinity absorbant, RNase A-Sepharose (59). At least part of the change in K, value is probably due to a decrease in association rate constants, since inTABLE I1 KINETIC PARAMETERS OF INHIBITION BY RIBONUCLEASE INHIBITORS" ~~~

~

k, x Inhibitor source

Enzyme

Human placenta Human placenta Human placenta Porcine liver

Angiogenin Placental RNaseb RNase A RNase A

10-8

(M-1s-1)

1.8 1.9 3.4 1.7

kd x

107

(s-1)

1.3 1.8 150 98

Ki

(fM)

Ref.

0.71 0.94 44 59

116, 117 119 116, 117 84

"Conditions are 100 mM Mes, pH 6, 100 mM NaCl, 1 mM EDTA, %"C, except for studies on porcine liver inhibitor and RNase A, where conditions are 50 mM Mes, pH 6, 125 mM NaCl, 1 mM EDTA, 25°C. k, and kd denote apparent association and dissociation rate constants, respectively. *Placental RNase is identical to hepatic alkaline RNase (119).

12

FRANK S. LEE AND BERT L. VALLEE

creasing the NaCl concentration from 75 mM to 1 M decreases the apparent second-order rate constant of association of PRI with angiogenin 140-fold (116). The two-step binding mechanism by which PRI binds to angiogenin has been observed in the interaction of numerous other enzymes with tightbinding inhibitors, and may represent a relatively common kinetic mechanism by which these inhibitors bind (123). Examples include the inhibition of trypsin by soybean trypsin inhibitor (124), a-chymotrypsin by pancreatic trypsin inhibitor (125), dihydrofolate reductase by methotrexate (126), and angiotensin-converting enzyme by captopril and enalapril (127, 128). With the serine proteases, small conformational changes in the inhibitor have been observed in the X-ray crystal structures of several enzymeeinhibitor complexes; thus, the second step may involve a small conformational change in these inhibitors (35, 129).

B. Mode of Inhibition It is now clear that the mode of inhibition of RNase A by the mammalian RNase inhibitor is competitive, as determined from the effects of either an RNase A competitive inhibitor or substrate on the apparent association rate (117) or on the inhibition constant (84), respectively, for the interaction. In the former study, the data are consistent with competition occurring between substrate and inhibitor at the first step of the two-step binding mechanism (119,while the latter suggests competition at the second step (84). Whether this difference reflects intrinsic differences in the binding of RNase to the two inhibitors employed (human and porcine, respectively) or a difference in experimental procedures is not clear.

C. RNase Binding Site for RNase Inhibitor Studies on the binding site in RNase A for PRI have made use of the wealth of knowledge available on RNase A, historically the subject of many classic protein structure-function studies (2). Among the three critical active-site residues of RNase A-Lys-41, His-12, and His-119-only the first appears to be an important contact residue with PRI; carboxymethylation of Lys-41 is reported to weaken the interaction 10-fold, while carboxymethylation of His-12 and His-119 reportedly strengthens it 1.3- and 3.6-fold, respectively (130, 131). In addition, three proteolytically modified RNase A derivatives-RNase des-(1-20), des-(121-124), and des-(119--124)-all appear to bind as tightly to PRI as RNase A (130).Therefore, it was concluded that the residues removed are not part of the contact region. However, it should be noted that these studies may have underestimated the strength of binding of native RNase A to PRI and therefore may have significantly underestimated the effect of these RNase A modifications. Denatured, reduced,

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

13

and alkylated RNase A does not interact at all with the inhibitor, demonstrating that the native tertiary structure of the enzyme is necessary for binding (130, 132). Chemical modification of the RNase A-PRI complex with methyl acetimidate followed by disruption of the complex, isolation of the modified RNase, and tryptic peptide mapping reveals that in the complex, lysines 7, 31, 37, 41, 61, and 91 of RNase A are protected (133)but Iysines 1, 66, 98, and 104 are not. Based on these studies, it has been suggested that the binding site of RNase A for PRI is extensive (2).In the three-dimensional structure of RNase A, the contact points have been grouped as follows: (1) Lys-7, Lys-41, Pro-42, Val-43, Lys-91, Tyr-92, Pro-93; (2) Lys-31, Lys-37; and (3)Lys-61 and adjacent residues. Many of the aforementioned residues in angiogenin are identical or similar (33):angiogenin possesses identical residues at positions corresponding to Lys-41, Pro-93, Lys-61, and Asn-62, and similar residues at positions corresponding to Lys-7 (His), Val-43 (Ile), Tyr-92 (Trp), Lys-31 (Arg), and Gln-60 (Asn). In addition, energy minimization calculations suggest that the backbones of angiogenin and RNase A are similar (134).These observations make it of interest to compare the binding site of angiogenin for PRI with that of RNase A. Angiogenin derivatives prepared by either mutagenesis or chemical modification bind to PRI with &nities that vary over four orders of magnitude (Table 111). The computed three-dimensional structure for angiogenin (134) predicts that the residues modified include some at the active site (Lys-40, His-13, and His-114), some near the active site (Lys-60, Trp-89, and those flanking His-131, and one more distant &om the active site (Lys-50). We review the findings with these derivatives. The most notable result in these studies is that the single amino acid substitution of Gln for the active-site Lys-40 weakens the interaction fully 1300-fold (Table 111) (135).The effect corresponds to a free energy change of 4.3 kcal/mol, in the range of experimental values, 2 to 6 kcal/mol, for the removal of either a salt-bridge partner or a hydrogen-bond partner to a charged group (139-143). This suggests the existence of a similar situation in this instance and, in turn, implies the presence of an anionic partner to Lys-40 in PRI. It may be noted that even preservation of the positive charge by the seemingly conservative Lys-40 +-Arg substitution still weakens the interaction 120-fold, suggesting that the interaction of Lys-40 with PRI is highly specific and can be accommodated only partially by an arginine (136). Carboxymethylation of either His-13 or His-114 of angiogenin, the two other residues that together with Lys-40 constitute the catalytic triad of residues, results in a substantial 13fold weakening of the interaction with PRI (135). However, when either His-13 or His-114 is replaced by an al-

14

FRANK S. LEE AND BERT L. VALLEE

TABLE 111

BINDINGOF ANCIOCENIN DERIVATIVESTO PLACENTALRIBONUCLEASE INHIBITOR^ Native residue(s) Native Lys-40 Lys-40 His-13;114 His-I3 His- 114 Trp-89 Lys-50 Lys-60 His-A to Asp-22

Modified residues(s)

Gln Arg

C M -H isr Ala Ah Oia“ DNP-Lyse DNP-Lysr Lys-7 to Ser-2l-f

K,

Ki, mod

(fM)

Ki.unniod

0.71 930

38 11 0.20 1.0 1.7 0.63 0.65 (0.07

1300 120 1s

0.6 3.0 2.4 0.9 0.9 (0.1

Ref.

117 135 136 135 137 137 135 135 135 138

Conditions are 100 mM Mes, pH6, 100 mM NaCI, 1 mM EDTA, 25°C. The subscripts “mod” and “unrnod” refer to modified and unmodified angiogenin, respectively. “The Lys-40 -+ Arg, His-13 + Ala, and His-I14 + Ala angiogenin derivatives also differ from native angiogenin with respect to its N-terminus: Met-(-1)Glu-1 versus (Glu-1, respectively. Thus, the appropriate comparison for obtaining Ki.,,M,/Ki,unm
anine, there is no more than a 3-fold change in the K, value (137). This therefore indicates that the changes in binding observed following carboxymethylation of these histidine residues do not necessarily imply critical contacts with them. These changes may reflect, for example, the introduction of a negative charge or perturbation of the PRI interaction with Lys-40 (135). The Trp-89 of angiogenin has been predicted to be on the same face of the molecule as the active site (134) and the following observations suggest that it may be part of the contact region with PRI. First, tryptophan fluorescence is enhanced 50% upon angiogenin-PRI complex formation, and this change is abolished when Trp-89 is first oxidized by dimethyl sulfoxide and HCI, suggesting a marked alteration of the Trp-89 environment in angiogenin in its complex with PRI (116).Second, in the PRI complex, Trp-89 becomes less accessible to acrylamide (116). While oxidation of Trp-89 to oxindolalanine results in only a modest 2.4-fold weakening of the interaction {Table HI), it should be noted that this modification introduces only a modest change in chemical structure (116).

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

15

Residues flanking His-13 in the peptide sequence from residues 8 to 22 of angiogenin have also been predicted to be in the vicinity of the active site, and replacement of this peptide sequence by residues 7 to 21 of RNase A unexpectedly strengthens the binding at least 10-fold (138).The measured K, value is less than M. While the multiple simultaneous substitutions make it dimcult to attribute the change to any particular residue, it seems that Lys-8 and Arg-10 in this modified angiogenin are possible candidates for forming hydrogen bonds or salt bridges with the acidic PRI molecule, thereby strengthening the interaction, since their side chains would be expected to extend directly out from the active site, based on the RNase A structure. Perturbations in the region around Lys-60 of angiogenin, corresponding to that around Lys-61 of RNase A, may be expected to affect binding, based on the studies on the RNase A-PRI interaction. However, dinitrophenylation of Lys-60 with the bulky reagent l-fluoro-2, 4-dinitrobenzene does not significantly alter the K, value (135). In addition, an angiogenin molecule in which residues 59 to 73 of RNase A substitute for residues 58 to 70 of angiogenin binds as tightly to PRI as does native angiogenin (144). It is possible that Lys-60 of angiogenin occupies a position in three-dimensional space that is different from that of Lys-61 in RNase A. Alternatively, the region around Lys-60 of angiogenin may not be a critical contact site for PRI. In summary, these results (135-138) indicate that PRI contacts a discrete region of the angiogenin molecule, which may be smaller than that suggested for RNase A (2, 133). Prominent among the critical contacts is one with Lys-40 of angiogenin (Lys-41 of RNase A). Contact with Trp-89, a residue predicted to be near the active site, appears to occur as well. However, Lys-60, another residue predicted to be near the active site, does not appear to be a critical contact. It may also be noted that in placental RNase, which binds 50-fold more tightly to PRI than does RNase A, the proposed RNase A contact residues Lys-7, Lys-31, Lys-37, Lys-91, and Pro-93 are substituted nonconservatively by Trp-10, Gln-28, Gln-34, Pro-90, and Ser-94, respectively (2O),thus perhaps raising questions regarding the importance of some of the former residues in the interaction with PRI.

D. RNase Inhibitor Binding Site for RNase The examination of the complementary binding site involved in the interaction, i.e., that on the RNase inhibitor, is particularly intriguing given the internal repeat structure of the inhibitor; while the inhibitor contains seven repeat units, it binds only one molecule of RNase or angiogenin. This contrasts with proteinaseeproteinase inhibitor interactions, where the stoichiometry of binding and the number of inhibitor domains deduced from the primary structure usually coincide (35). Therefore, it is of interest to know if the entire internal repeat structure of the inhibitor is necessary for

16

FRANK S. LEE AND BERT L. VALLEE

activity, or whether activity can be assigned to a smaller portion of the molecule. With proteinase inhibitors, limited proteolysis of the native protein is one means to define functional domains. In the RNase inhibitor, the heterologous expression of porcine RNase inhibitor in the yeast S. cerevisiae affords, in part, a somewhat comparable approach, for two active protein species isolated during purification of the full-length protein both appear to be products of limited proteolysis of the latter during fermentation (145). One protein lacks the first 90 residues of the porcine inhibitor while the other lacks the first 93. A mixture of the two proteins (2:1, respectively) binds to and inhibits RNase A with a K,value of 154 fM,only 2.3-fold weaker that that of the full-length protein (Table IV). Therefore, the first 93 residues of the porcine inhibitor (correspondingto the first 97 residues of PRI) are not essential for activity. In contrast to the expression of porcine inhibitor in S. cerevisiae, expression of PRI in E. coli yields no proteolytically cleaved fragments with activity (F. S. Lee and B. L. Vallee, unpublished data). In addition, expression of a series of N- and C-terminal deletion mutants of PRI, including one in which only the 62 N-terminal residues were removed, reveals no evidence either for inhibition of RNase A or for binding to RNase ASepharose with any of these mutants (Fig. 3A) (147). One possible explanation for the lack of activity in any of these PRI mutants is that surface properties of the mutant proteins may have been altered such that activity is

TABLE IV

INHIBITION CONSTANTSFOR RIBOUCLEASEINHIBITOR DERIVATIVES" K , (PW Inhibitor source

Derivativeb

Porcine liver' Porcine liver Human placentan Human placenta Human placenta

AN A3-4 A6

RNase A 0 067

0.154 0.068 170 13

Angiogenin

Ref.

NDe NW

84 145

O.OOO29

117

0.72

146 146

22

QConditionsare 100 mM Mes, pH 6, 100 mM NaCI, 1 mM EDTA, 2 S T , except for studies on porcine liver inhibitor or its derivative and RNase A, where conditions are SO mM Mes, pH 6. 125 m M NaCI, 1 mM EDTA, 0.02%(w/v) Tween 20, 25°C. &Theresidues deleted are as follows: AN, 1-90 or 1-93 (2:1 mixture); 83-4, 144-257;

A6, 315-371. CExpresssed in S. cerevisiae. dExpressed in E . coli. eNot determined.

17

MAMMALIAN FUBONUCLEASE (ANGIOGENIN) INHIBITOR

A PRI

I - I --

I -

PRI ANa PRI AN@

I -

PRI ANY PRI A N & PRI ACa PRI ACg PRI ACY PRI ACb

B PRI PRI A 1 PRIA1-3

--

PRI A 2

I

PRI A 2 - 4

+

PRI A 3-5

I I

l + 1

PRlA5

t

r +

-

I

I +

+

PRI A 3 4

PRI A 5-6 PRI A6 PRI A4-7 PRI A7

+ +

-

P-

PRI A 1 -7

FIG.3. RNase A binding and inhibitory activities of PRI mutants (146).(A) N- and Cterminal deletion mutants. In the diagram of native PRI, numbers refer to repeats defined as follows: 1, residues 30-86;2, 87-143; 3, 144-200; 4, 201-257; 5, 258-314; 6, 315-371; 7, 372428. Residues deleted are 1-62 (ANa), 1-225 (ANB), 1-298 (ANy), 1-394 (ANG), 435-460 (ACa), 396-460 (ACp), 227-460 (Ahcy), and 64-460 (ACG). (B)Internal-deletion mutants. In the nomenclature of internal-deletion mutants, numbers refer to deleted repeats as defined above. Aside from the portions of PRI deleted, the native PRI amino-acid sequence has been preserved. Exceptions are as follows, with the residue numbers denoting their positions in the native protein: PRIA4-7, Gln-430 + Ala and Val-432 4 Lys; PRIA1-7, Glu-429 + Gln, Gln-430 + Ala, and Val-432 + Lys; PRIAl-3, Glu-201 + Gln; PRIANa, Arg-63 + Cly; PRIACa, Tyr-434 -+ Ser; PRIACG, Arg-63 + Pro.

18

FRANK S. LEE AND BERT L. VALLEE

only fully expressed in the presence of a surface-active agent, such as a detergent (145). In the studies of the N-terminally truncated porcine inhibitor, full inhibitor activity was only obtained in the presence of 0.02% Tween 20. Another possibility is that the genetically engineered truncations of the inhibitor may have yielded polypeptides incapable of folding into a nativelike structure, a situation probably averted in the expression and subsequent limited proteolysis in uiuo of inhibitor in the yeast. In this regard, though, it should be noted that limited proteolysis of native PRI employing trypsin, chymotrypsin, V8 protease, Arg-C protease, elastase, and subtilisin fails to generate active inhibitor fragments (F. S. Lee and B. L. Vallee, unpublished data). As an alternative approach toward mapping the RNase binding site in PRI, the internal-repeat structure of PRI was employed as a basis of systematic deletions of these repeats, either singly or in combination (147). The features of this “modular mutagenesis” approach were as follows (Fig. 3B). (1) Precise integral multiples of 57 residues were deleted from PRI, the exact length of a repeat unit. (2) Deletion boundaries were based on a set of repeat unit boundaries that begins at residue 30 and ends at residue 428, the beginning and end of the strong repeat motif of PRI. Most surprisingly, certain deletions of PRI are compatible with activity while others completely abolish it (Fig. 3B). Thus, mutants containing deletions of repeats 3 plus 4 (PRIA3-4) or repeat 6 (PRIAG) both inhibit RNase A, whereas all others do not. Extension of these deletions to include adjacent repeats abolishes inhibitory activity as does deletion of other protein regions of comparable size. PRIA3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively (Table IV) (146).The corresponding values for PRIAG are 22 and 43 pM, respectively. Since recombinant PRI binds to angiogenin and RNase A with K, values of 0.29 and 68 fM, respectively (117), deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. In spite of these weakened Ki values, these mutants are still potent inhibitors of the two enzymes. This suggests, in turn, that in both mutants the main contact area in PRI is intact and cannot be located in the deleted repeats-i.e., 3, 4, or 6. Taking into consideration the active inhibitor with the N-terminal truncation (145), the RNase binding site on the inhibitor must be located predominantly or entirely in repeats 2, 5, 7, the C-terminal segment, or a combination of these (Fig. 4). Further, the contact region cannot reside exclusively in repeat 5 because three mutants in which this repeat has been deleted (PRIA3-5, PRIA5, and PRIA5-6) all bind to RNase A. These three mutants, however, do not inhibit RNase A (Fig. 3B), and there are two plausible explanations. (1) Binding to RNase A is sufficiently lessened so that activity is observed

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

19

only in the binding assay, the more sensitive of the two assays (see 147). This would imply that these mutant proteins may still be inhibitory. (2) Binding to RNase A occurs at a site outside of its active site and does not result in inhibition. These results bear upon the structural significance of the PRI repeat units. PRIA3-4 is active, suggesting that repeat 2 can substitute functionally for repeat 4 because its position in the mutant protein is similar, with respect to repeat 5, to that of repeat 4 in the native protein. Along the same lines, repeat 5 can substitute functionally for repeat 3; applying this reasoning to the active mutant PRIA6, repeats 5 and 7 each can substitute functionally for repeat 6. This analysis can be extended to the three additional mutants that display binding activity, PRIA3-5, PRIA5, and PRIA5-6. Collectively, the results suggest a significant degree of flexibility with respect to repeat substitutions that can be tolerated in the region of repeats 3-6. This flexibility, which allows functional substitutions, in turn suggests that PRI has a modular structure, where one 57-residue repeat constitutes one structural module. The boundaries defining repeats 1-7 may define or nearly define these modules because selective deletions based on these boundaries result in functional proteins. It will be of interest to determine whether deletions of nonintegral multiples of 57 residues or regions based on different repeat boundaries are compatible with activity. In this regard, it may be noted that the active porcine inhibitor with N-terminal truncation exhibits a new N-terminus within eight residues of the proposed boundary between repeats one and two. It will also be of interest to determine whether intron-exon boundaries in the inhibitor gene fall near these proposed module boundaries. Chemical modification of the RNase A*porcine inhibitor complex with 4-N,N-dimethylaminoazobenzene-4'-iodoacetimido-2'-sulfuric acid, followed by disruption of the complex, isolation of the modified inhibitor, and peptide mapping and sequencing, reveals that Cys-371 (Trp-375 in PRI) and Cys-404 (Cys-408) are protected in the complex (148). These two residues may therefore form part of the inhibitor contact site with RNase A, and it may be noted that both are present in repeat 7, a repeat that, when deleted,

FIG.4. Mapping of the RNase binding site in PRI (adapted from 145 and 147). Numbers refer to repeat units, N and C designate the N- and C-termini, respectively, and a shaded background indicates nonessential repeats. The RNase binding site resides predominantly, or entirely, in repeats 2, 5, 7, the C-terminal segment, or a combination of these. Of those, the binding site cannot reside exclusively in repeat 5.

20

FRANK S. LEE AND BERT L. VALLEE

results in loss of activity (Fig. 3). Eleven additional cysteines are also protected in the complex; however, interpretation of these results is complicated by the low level of labeling of these inhibitor residues (c0.2mol/mol) when RNase A is absent (148). The internal-repeat structure of PRI probably arose by gene duplication. These results suggest that this duplication may have proceeded through functionally active intermediates that have less than a full complement of repeat units, similar perhaps to some of the active mutants described. Based on the lower Ki values observed for native PRI compared to the mutants PRIA3-4 and PRIAG, it is tempting to speculate that the evolution of the protein may have been marked by increasingly tighter binding properties. However, it is conceivable that the actual evolutionary intermediates may have been equally as potent as the native protein. In addition, the repeats not essential for inhibition may serve as yet unknown functions.

E. Experimental Applications The broad experimental efficacy of PRI and related inhibitors in the preservation of RNA integrity is consistent with its activity. These inhibitors stabilize nuclear RNA (3) and polyribosomes (4-12) during their isolation and preserve the translational capacity of the latter. They improve the yield of higher molecular-weight products of in uitro translation (13-16), transcription (17), and cDNA synthesis (18)systems and increase the incorporation of amino acids in the first of these systems (14-16).

IV. Biologic Role While the literature concerning the possible biologic roles of the mammalian RNase inhibitor is extensive, a definite function for this inhibitor remains unknown. Speculations concerning the biologic role of the RNase inhibitor have revolved around inhibition of either cytoplasmic or noncytoplasmic RNases. While inhibition of the latter is well documented (see below), that of the former remains to be demonstrated. Nevertheless, inhibition of cytoplasmic RNases has been the focus of much of the earlier work on the inhibitor, which we review before turning to more recent work drawing attention to inhibition of noncytoplasmic RNases. The earliest observations relating to a possible physiologic role for the RNase inhibitor were the increased inhibitor levels in regenerating rat liver following partial hepatectomy (53,149). This led several investigators to hypothesize a more general correlation of anabolic cellular activity with increased inhibitor levels, and catabolic cellular activity with decreased levels (78,87, 149, 150).The implication was that increased inhibitor levels may decrease cytoplasmic RNase activity, decrease RNA catabolism, and thereby

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21

increase RNA levels. Conversely, decreased inhibitor levels may decrease RNA levels. Hence, the inhibitor might play a role in the regulation of cytoplasmic RNA turnover, an area about which little is known of the proteins (enzymes, inhibitors, or otherwise) involved (151, 152). Subsequent studies support this correlation between cellular activity and inhibitor levels, the most compelling being those examining tissues that undergo anabolic or catabolic changes in response to defined stimuli. Studies of anabolic changes include observations of increased inhibitor levels in rat adrenal glands following adrenocorticotropin hormone (ACTH) administration (150) and cultured T cells following lectin transformation (87, 88), and decreased RNase activity in rat liver following growth hormone administration to hypophysectomized rats (153).Studies of catabolic changes include observations of increased RNase activity in rat liver following low-protein diets (154, 155). Similarly, inhibitor levels in rat mammary gland rise during pregnancy and lactation and fall during postlactational involution (73); they rise in rat thyroid during goiter induction by thiouracil and fall during subsequent T3 administration (80, 156, 157). A number of other observations further support this hypothesis. They include the measurement of increased inhibitor levels in leukemic mouse thymus (78), rat mammary tumors (73), rat liver following treatment with 2acetamidofluorene (158), and rat brain and liver during neonatal development (159, 160); they also include decreased inhibitor levels in rat lymph nodes, liver, and thymus with aging (78, 87) and rabbit skeletal muscle following immobilization (161). Teleologically, these observations raise the question of why the inhibitor levels, which are sufficient to inhibit the enzyme completely, should increase further in different physiological states; the extremely low K , values (see Section 111, Inhibitory Properties) suggest that only a slight molar excess of inhibitor over enzyme is necessary for complete inhibition. One possible explanation is that the inhibitor binds to the cytoplasmic enzymes substantially more weakly than to the noncytoplasmic ones. Thus, stoichiometric amounts of inhibitor may not inhibit completely and inhibitor levels could conceivably modulate cytoplasmic RNase activity. Another explanation could be that the inhibitor and its bindings &nity may be modified under certain circumstances; proteolysis (92, 161, 162), exposure to metals (Sa), and changes in the oxidation state of its sulfhydryl groups (62,92,150) have been suggested as possible control mechanisms for inhibitor activity. A number of investigators have drawn attention to a possible role for the inhibitor in the pathogenesis of a variety of disease states. Thus, decreased concentrations of poly(A) RNA, increased amounts of free RNase, and decreased concentrations of inhibitor-bound RNase have been reported in the cerebral cortex from patients with Alzheimer’s disease (163). This sug+

22

FRANK S. LEE AND BERT L. VALLEE

gests that an increased RNase:inhibitor ratio, leading to increased RNA turnover, might play a role in the development of this degenerative disease. However, other observers have failed to demonstrate altered levels of RNA ( I @ ) , RNase (165),or RNase inhibitor (166) in autopsy brains from Alzheimer’s disease patients. Decreased inhibitor concentrations have also been reported in other diseases, including muscular dystrophy (16T), hyperthyroidism (168), and cataracts (169),and have been found to result from, and may play a role in, the toxicity of a number of agents, including p- or X-irradiation (170-172), cycloheximide (173), 5-fluorouracil (174), actinomycin D (175), and electroconvulsive shock (176).The extent to which altered inhibitor levels contribute to the development of these different processes remains to be established. A general correlation between anabolic or catabolic states and increased and decreased inhibitor levels, respectively, is not universal. For example, changes in inhibitor levels are not observed in steroid-sensitive mouse lymphosarcomas upon Sol-fluoroprednisolone-inducedregression (174), in rat liver following triamcinoline-induced gluconeogenesis (177), or in erythroleukemia cells, SV4O-transformed hamster embryo fibroblast cells, and H L 6 0 cells upon phorbol ester stimulation (88).In addition, many hepatoma cell lines do not exhibit increased inhibitor concentrations (57, 178), and there is a decrease in free inhibitor levels in immature rat uterus following 17p-estradiol treatment despite an increase in cellular RNA (72, 82). The studies cited should be interpreted with caution because of considerations relating to the experimental methods. First, noncytoplasmic RNases may have been present as contaminants in the cytoplasmic extracts examined in most, if not all, of these studies. In fact, purification and sequence analysis of two RNases from porcine liver “cytoplasmic” extracts that interact with the inhibitor indicate that the RNases probably contain disulfide bonds characteristic of secreted RNases (179).Therefore, these two RNases were probably noncytoplasmic rather than cytoplasmic proteins. Hence, the measured levels of free and bound inhibitor or ribonuclease in other studies may not reflect those of the cytoplasm in t-ico. A second consideration is that free inhibitor levels in extracts are measured typically by direct inhibition of RNase A, while bound inhibitor is measured by the increase in endogenous RNase activity following inactivation of the inhibitor. RNase activity, in turn, is usually measured using a single substrate, often with the implicit assumption that the specific activities of endogenous free and latent RNase(s) toward the substrate are identical, in some cases, it is assumed further that these are identical to that of RNase A. At this time, however, the cytoplasmic RNases inhibited have

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23

not yet been characterized kinetically or even isolated, thus making problematic the assessment of the measured free and bound ribonuclease or inhibitor concentrations in these studies. Indeed, while the inhibitor is a cytoplasmic protein, the RNases that have been isolated thus far and shown to be inhibited by it all belong to the mammalian noncytoplasmic RNase superfamily (19, 20, 119). This superfamily includes (1) pancreatic RNase (2), (2) hepatic alkaline RNase (119, 179), (3) angiogenin (23), (4) tumor-derived RNase (180), and (5) eosinophil cationic protein (181), among others. The enzymatic activities of the first four of these RNases and probably that of the fifth are potently inhibited by the to RNase inhibitor, with K , values on the order of M (Table 11). One possibility is that these inhibitors may serve a role in inhibiting noncytoplasmic RNases should they enter the cytoplasmic compartment (19, 20, 40, 119, 150). Many RNases, such as RNase A or angiogenin, are potent inhibitors of cell-free protein synthesis by virtue of their cleavage of ribosomal RNA (182). Cytotoxicity from inadvertant entry of these RNases into the cytoplasm could therefore be averted by the presence of the inhibitor. Alternatively, the inhibitor may have a direct regulatory role, particularly given recent work demonstrating distinct physiologic activities for several of these enzymes. For example, eosinophil-derived neurotoxin, identical to hepatic alkaline RNase (183, 184), and eosinophil cationic protein are potent neurotoxins that induce the Gordon phenomenon, a neurological syndrome characterized by ataxia and paralysis, when injected intrathecally into rabbits (181). These proteins are also both helminthotoxic, with activity against parasites such as Schistosoma mansoni and Trypanosoma cruzi (185-187). Bovine seminal RNase, a member of the pancreatic RNase family, is a dimeric enzyme with antispermatogenic, antitumor, and immunosuppresive activities (22,188,189). The implication therefore is that the RNase inhibitor may be involved in the regulation of distinct physiologic activities associated with these enzymes. Possibilities for a distinct regulatory role for the RNase inhibitor are best exemplified by its potent inhibition of angiogenin (116, 117). For instance, the inhibitor may serve to terminate the action of angiogenin. In this respect, the extremely low & value for the binding of PRI to angiogenin is appropriate for inhibition of a protein capable of inducing neovascularization at femtomolar levels (23). Alternatively, angiogenin could also be present as an enzyme-inhibitor complex releasing free angiogenin when the inhibitor is inactivated. In addition, other RNases may indirectly affect the interaction by decreasing levels of inhibitor that would otherwise be capable of binding angiogenin. Given the extremely long half-lives of the various

24

FRANK S . LEE AND BERT L. VALLEE

RNase-inhibitor complexes examined (Table II), the association rate of inhibitor with angiogenin relative to that of inhibitor with other RNases may, in large part, determine the partitioning of PRI between angiogenin and competing HNases. In this regard, it should be noted that material reactive with anti-PRI antibodies is detected in serum (89), a fluid in which many mammalian HNases are present. However, while this is an intriguing observation, it remains to be seen whether RNase inhibitory activity is actually present in that, or for that matter any, extracellular fluid. Thus, the temporal and spatial expression of the inhibitor, the nature of its inhibition of the varied noncytoplasmic RNases that it binds, and the physiologic consequences of this inhibition remain to be defined.

V. Concluding Remarks The HNase.RNase inhibitor interaction is among the tightest reported for proteinaprotein interactions. With regard to the inhibition of angiogenin, this provides a unique approach toward antiangiogenesis, with potentially far-reaching applications, including the treatment of cancer, diabetic retinopathy, rheumatoid arthritis, and chronic inflammatory disorders. Toward this end, future studies incorporating mutagenesis and X-ray crystallographic approaches should further characterize the RNase and RNase inhibitor binding sites for each other and additionally provide the basis for designing novel RNase inhibitors. These studies may also provide insight into the acid and sulfhydryl lability of the native protein, features that conceivably could be diminished or altered by rational protein design. Such structural studies would also establish the three-dimensional structure of the leucine-rich repeat unit in the RNase inhibitor and thereby facilitate a comparison between it and the as yet undetermined structures of other leucine-rich repeats found in other proteins. Since it appears that only a finite number of protein structural motifs are utilized in nature, it will be of interest to determine why this leucine-rich motif is also present in other proteins with such diverse physiological functions as blood coagulation, hormone action, extracellular matrix stabilization, protein degradation, second messenger responses, and photoreceptor cell and embryonic development. A biologic role of this protein remains to be defined. Whether the protein inhibits cytoplasmic or noncytoplasmic enzymes remains a fundamental question. With regard to the latter, the exceptionally tight binding between the inhibitor and various noncytoplasmic RNases argues strongly for a physiologic role involving inhibition of noncytoplasmic RNases. The KNase inhibitor may have a role in the extracellular regulation of the diverse biologic activities of the noncytoplasmic RNases, which include the induction of

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

25

neovascularization or paralysis and the inhibition of spermatogenesis or parasite growth. With regard to a potential physiologic role for the inhibitor, many questions remain. If the inhibitor displays regulatory activity in an extracellular environment, by what means is it transported out of the cytoplasm? Is this activity compromised by the acid- or sulfhydryl-lability of the protein, or conversely, is this lability a means of regulating the inhibitor? Does the inhibitor exert its activity locally, e.g., within a given tissue or organ, or are there means for systemic, e.g., vascular, distribution of the protein? Is there regulation of inhibitor gene expression by the enzymes it inhibits? The inhibitor may have a distinctly different function in the inhibition of cytoplasmic RNases with resultant effects on RNA turnover. While this particular function has been the primary focus of RNase inhibitor research for much of the past 30 years, it has yet to be demonstrated that a cytoplasmic RNase does in fact bind to the inhibitor. Certainly, the isolation and characterization of cytoplasmic RNases inhibited by the inhibitor are critical both for elucidating this possible in uivo role of the inhibitor as well as for providing the basis for quantitating changes in cytoplasmic RNases under different physiologic states. The use of the RNase inhibitor cDNA as a probe should provide a complementary means of examining inhibitor gene expression as well. This review reveals that there is but one RNase inhibitor protein &at has been identified so far. This protein binds to many mammalian RNases whose catalytic specificities differ, thereby potentially inhibiting many diverse biological processes. This contrasts markedly with the protein inhibitors of proteinases. In that case, the multiplicity of inhibitors matches that of the proteinases (35-37). Moreover, these proteinase inhibitors vary markedly in size, from the 4-kDa potato carboxypeptidase inhibitor to the 725-kDa cxzmacroglobulin. In contrast, the mammalian RNase inhibitor is a single 50kDa protein that seems to function for many enzymes. One reason for these differences may simply be that many classes of proteinases have been examined for inhibitors while only one superfamily of mammalian ribonucleases has been found, perhaps because there has been a less intensive search for others. Thus, other protein RNase inhibitors may yet remain to be discovered. Alternatively, evolutionary pressures for maintenance of tightbinding properties have resulted in the selection of but one unique arrangement with a leucine-rich motif that has exceptional potential and variability of structure for multiple interactions. ACKNOWLEDGMENTS We thank Drs. James F. Riordan and Robert Shapiro for helpful discussions. The original work here reported was supported by funds from Hoechst H. G . under an agreement with Harvard University.

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P. Blackburn and B. L. Jailkhani, JBC 254, 12488 (1979). P. Blackburn and J. G. Gavilanes, JBC 255, 10959 (1980). J. S. Roth and D. Hurley, BJ 101, 112 (1966). P. Blackburn and J. G. Gavilanes, JBC 257, 316 (1982). K. A. Palmer, H. A. Scheraga, J. F. Riordan and B. L. Vallee, PNAS 83, 1965 (1986). F. S. Lee and B. L. Vallee, B c h m 28, 3556 (1989). R. Shapiro, E. A. Fox and J. F. Birordan, Bchem 28, 1726 (1989). R. Shapiro and B. L. Vallee, Bchem 28, 7401 (1989). M. D. Bond and B. L. Vallee, Bchem 29, 3341 (1990). A. R. Fersht, JMB 64, 497 (1972). A. R. Fersht, Bchem 26, 8031 (1987). A. R. Fersht, J.-P. Shi, J. Knill-Jones, D. M. Lowe, A. J. Wilkinson, D. M. Blow, P. Brick, P. Carter, M.M.Y. Waye and G. Winter, Nature 314, 235 (1985). 142. D. M. Lowe, G. Winter and A. R. Fersht, Bchem 26, 6038 (1987). 143. J. A. Wells, D. B. Powers, R. R. Bott, T. P. Graycar and D. A. Estell, PNAS 84, 1219 (1987). 144. J. W. Harper and B. L. Vallee, Bchem 28, 1875 (1989). 145. J. Hofsteenge, A. Vicentini and S. R. Stone, BJ 275, 541 (1991). 146. F. S. Lee and B. L. Vallee, Bchem 29, 6633 (1990). 147. F. S. Lee and B. L. Vallee, PNAS 87, 1879 (1990). 148. J. Hofsteenge, C . Servis and S. R. Stone, JBC 266, 24198 (1991). 149. K. Shortman, BBA 61, 50 (1962). 150. R. C. Imrie and W. C. Hutchison, BBA 108, 106 (1965). 151. R. Raghow, Trends Bioch. Sci. 12, 358 (1987). 152. G . Brawerman, Cell 57, 9 (1989). 153. E. N. Brewer, L. B. Foster and B. H. Sells, JBC 244, 1389 (1969). 154. C. Quirin-Stricker, M. Gross and P. Mandel, BBA 159, 75 (1968). 155. P. Ross0 and M. Winick, J. Nutri. 105, 1104 (1975). 156. R. L. Grief and E. F. Eich, FP 30, 360 (1971). 157. P.V.N. Murthy and J. M. McKenzie, Endocrinol. 94, 74 (1974). 158. R. J. Wojnar and J. S. Roth, Cancer Res. 25, 1913 (1965). 159. Y. Suzuki and Y. Takahashi, J. Neurochem. 17, 1521 (1970). 160. D. K. Liu, E. E. McKee and P. J. Fritz, Growth 39, 167 (1975). 161. 2. Kiss and F. Guba, FEBS Lett. 108, 185 (1979). 162. P. Fuhge and K. Otto, ZpChem 358, 1203 (1977). 163. E. M. Sajdel-Sulkowska and C. A. Marotta, Science 225, 947 (1984). 164. M. R. Morrison, S. Pardue, K. Maschoff, W.S.T. Griffin, C. L. White, J. Gilbert and A. Roses, Biochem. SOC. Trans. 15, 133 (1987). 165. K. Maschoff, C. L. White, L. W. Jennings and M. R. Morrison-Bogorad, J. Neurochem. 52, 1071 (1989). 166. L. M. Jones and J. T. Knowler, J. Neurochem. 53, 1341 (1989). 167. B. W. Little and W. L. Meyer, Science 170, 747 (1970). 168. R. L. Grief and E. F. Eich, Metabolism 26, 851 (1977). 169. B. J. Ortwerth and R. J. Byrnes, Exp. Eye Res. 12, 120 (1971). 170. J. S. Roth, ABB 60, 7 (1956). 171. J. Tabachnick, Radiat. Res. 15, 785 (1961). 172. N. Kraft, K. Shortman and D. Jamieson, Radiot. Res. 39, 655 (1969). 173. H. Hilz, M.-M. Oldekop and B. Bertram, ZpChem 349, 1475 (1968). 174. E. Ambellan and V. P. Hollander, PSEBM 127, 482 (1968). 175. R. G. von Tigerstrom, Can J. Biochem. 50, 244 (1972). 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141.

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176. S. C. Park, S. S. Choi and K. Y. Lee, SeouEJ. Med. 18, 31 (1977). 177. N . K. Sarkar, FEBS Leff. 4, 37 (1969). 178. J. S. Roth, S . Hilton and H. P. Morns, Cancer Res. 24, 294 (1964). 179. J. Hofsteenge, R. hlatthies and S. R. Stone, Bchem 28, 9806 (1989). 180. R. Shapiro, J. W. Fett, D. J. Strydom and B. L. Vallee, Bchem 25, 7255 (1986). 181. G . J. Gleich, D. A. Loegering, M. P. Bell, J. L. Checkel, S. J. Ackerman and D. J. McKean, PNAS 83,3146 (1986). 182. D. K. St. Clair, S. M . Rybak. J. F. Riordan and B. L. Vallee, PNAS 84, 8330 (1987). 183. K. J. Hamann, R . L. Barker, D. A. Loegering, L. R. Pease and G. J. Gleich, Gene 83,161 (1989). 184. H. F. Rosenberg, D. G . Tenen and S. J. Ackerman, PNAS 86, 4460 (1989). 185. D. J. McLaren, C.G.B. Peterson and P. Venge, Parasitology 88, 491 (1984). 186. G. J. Gleich and C. R. Adolphson, Adu. Itnmunol. 39, 177 (1986). 187. H. .4.Molina, F. Kierszenbaum, K. J. Hamann and 6 . J. Gleich, Am. 1.Trop. Med. Hyg. 38, 327 (1988). 188. E. Leone, L. Greco, R. K. Rastogi and L. Iela, J. Reprod. Fed. 34, 197 (1973). 189. J. Matousek, Experientia 29, 858 (1973).

Bacterial Adenylyl Cyclases ALAN PETERKOFSKY*,~ AIALA REIZER,~JONATHAN REIZER,1NATANGOLLOP,* PENG-PENG ZHU* AND NIRANJANAAMIN* *Laboratory of Biochemical Genetics National Heart, Lung and Blood Institute Bethesda, Maryland 20892 ?Department of Biology University of Cal!&niu, San Diego La Jolla, Calqornia 92093 I. The Action of cAMP as a Transcription Regulator in Escherichia coli 11. Regulation of CAMP Levels in E. coli . . . . .

IV. The Phosphoeno1pyruvate:sugarPhosphotransferase System . . V. Regulation of E. coli Adenylyl Cyclase Activity by the Phosphoeno1pyruvate:sugarPhosphotransferase System . . . . . . . . VI. Regulation of E. coli Adenylyl Cyclase Activity by Other Factors A. Activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...

35 36 38

......

.............................................

.............

..........................

X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...........

32

34

..........................

41 41 43 44 44 44 49 53 56 61 62 62

Adenosine 3',5'-cyclic monophosphate M CAMP)^ is a signaling molecule found in many, but not all, bacterial species. It has also been identified as an 'To whom correspondence may be addressed. 2Abbreviations: CAMP, adenosine 3',5'-cyclic monophosphate; CRP, cAMP receptor protein; R, regulatory subunit; C, catalytic subunit; CRE, cAMP response element; CREB, cAMP response element binding protein; P2, major promoter of cyu gene; ITS, phosphoenolpyruvate:sugar phosphotransferase system; Enzymes 11, membrane-bound sugar-specific transporters (permeases) of the PTS; protein 111, soluble sugar-specific phosphocarrier protein of the PTS; IIIdc, glucose-specific soluble phosphocarrier of the PTS; Enzyme I, PTS phosphocarrier protein, phosphorylated by PEP; PEP, phosphoenolpyruvate; HPr, PTS phosphocarrier protein phosphorylated by the phosphorylated form of Enzyme I (Enzyme I-P); crr, the gene for IIIdc (mutation of this gene confers on E. coli the catabolite repression-resistance phenotype); PDE, CAMP phosphodiesterase.

31 Progress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright 0 1993 by Academic Press, Inc. All rights of reproductlon in any form reserved.

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important regtilator in eukaryotic cells. The mode of action of the nucleotide in bacteria is to convert the transcription factor known as cAMP receptor protein (CKP) from an inactive to an active form. The active form of CRP promotes the expression of unique sets of genes, generally referred to as inducible genes. The focus of this review is on the enzyme (adenylyl cyclase) that effects the synthesis of CAMP in various bacterial species. It is not intended to be an exhaustive review of the literature in this area, consonant with the style for this series. Kather, the content will reflect our current major interests. The adenylyl cyclase from Escherichiu coli has been a major subject of research interest ever since it was identified as the probable point for physiological regulation of CAMP levels in that organism and therefore a prime candidate for a protein mediator of the catabolite repression response mechanisin. Much of the emphasis in our laboratory in Bethesda has been on the elucidation of the factors controlling the activity of adenylyl cyclase in E . coli. The emphasis on that area is reflected in the review. The elucidation of the sequences of numerous adenylyl cyclases has made it possible to pose questions concerning the relatedness of the enzymes from various sources. The review contains a compendium, not previously assembled, of sequence homology comparisons for all the adenylyl cyclases whose sequences are currently known. On the basis of these analyses, we have organized some of these enzymes into four groups that suggest relatedness in either an evolutionary or functional sense. We have utilized a combination of “eyeball” and computer-assisted analyses to search for an ATP-binding site characteristic of these cyclases. The liberty is taken of suggesting an adenylyl cyclase “consensus,” which we are currently evaluating. Our aim is to provide an overview of bacterial adenylyl cyclases to the generalist as well as to provide food for thought for the adenylyl cyclase aficionados.

I. The Action of cAMP as a Transcription Regulator in Escherichiu coli CAMPfunctions as a cytoplasmic element mediating some reactions crucial for efficient cellular function. An activity that has been found only in eukaryotic cells is the CAMP-dependent protein kinase ( 1 ) . This enzyme is a well-known target of the action of cAMP as a second messenger, in which action this ligand transmits a signal generated by an extracellular hormone. The manner in which cAMP acts on the CAMP-dependent protein kinase invohees a release of the catalytic moiety of the enzyme from a complex in which its activity is inhibited as a result of binding to a regulatory subunit. The structure of the inactive holoenzyme is generally described as R,C,

BACTERIAL ADENYLYL CYCLASES

33

(where R indicates a regulatory subunit and C indicates a catalytic subunit); the interaction with cAMP leads to the formation of a complex of CAMP with R, and free active C. An important downstream function for cAMP in many eukaryotic cells involves the regulation of a family of CAMP-inducible genes (2).The cAMP response element (CRE) on such genes is believed to function as an enhancer, that is to say, it is located at a substantial distance from the regulated gene and can exhibit activity in either orientation. The mechanism by which these elements respond to cAMP involves the action of protein kinase A (3). The current model for the activation of these genes via cAMP requires a phosphorylation by protein kinase A of a transcription factor known as CREB (CAMPresponse element binding protein). The phosphorylation changes the conformation of CREB, allowing it to interact with a transcription complex and to thereby stimulate gene expression. The biochemistry of the action of cAMP in E . coli has been thoroughly defined and shown to be different than that in eukaryotic cells. In E . coli, cAMP serves as a coactivator of gene transcription for a substantial number of inducible genes (see Fig. 1).The key feature of this mechanism requires the interaction of CAMP with the pleiotropic transcription activator CRP (4). Genes coding for the enzymes required for the catabolism of a variety of sugars, such as lactose, are important but not exclusive targets for CRP. One essential regulatory feature of the structure of CRP is that the protein exists as a homodimer of the 210-aminoacid polypeptide. This dimeric structure is characterized by the presence of two domains: one for DNA binding (in the carboxyl-terminal region), and one for interaction with

ATP

-

-D

cAMP @LAMP

FIG. 1. The action of CAMP as a transcription regulator. cAMP interacts with cAMP receptor protein (CRP) to convert it to a form that binds specifically to certain promoters (crosshatched region). This binding stimulates RNA-polymerase-dependent transcription of unique genes (solid bar). The result is the accumulation of specific protein products.

34

ALAN PETERKOFSKY ET AL.

cAMP (in the amino-terminal region). In the absence of CAMP, CRP takes on a conformation that eliminates its capability to interact effectively with specific promoters; this conformation (the inactive conformation) also makes CRP relatively resistant to degradation by proteolytic enzymes. Upon binding ofcAMP, CRP changes its conformation to the active one, which shows an enhanced affinity for double-stranded DNA. Thus, the function of cAMP in transcription in E . coli is to change the structure of CRP from the inactive state to the active one. The demonstration that it is possible to isolate mutated forms of CRP that can function as transcriptional regulators in the absence of cAMP (5a) proves that it is the activated structure of CRP, rather than CAMP, that is essential for transcription activation. In general, the promoters that are activated by CRP are characterized by the sequence 5'-TGTGAN4KACA-3' (5b),suggesting that this sequence is essential for CRP binding and thereby activation of transcription of CAMPinducible genes. CRP contains a helix-turn-helix DNA-binding motif. Recognition helices (one from each monomer) insert into the major groove of DNA at the TGTGA sequence. This interaction (as well as other contacts) promotes a bending of the DNA. The CRP-DNA complex facilitates the binding of RNA polymerase, which further increases the bending of the promoter DNA. It is currently assumed that this highly bent form of DNA (the open complex) is essential for the initiation of transcription of inducible genes.

II. Regulation of cAMP levels in E. coli As pointed out previously (6), CAMP levels can be regulated in three obvious ways. These patterns are depicted in Fig. 2 . The rate of the conversion of ATP to cAMP by the enzyme adenylyl cyclase might be regulated up or down by a variety of effectors. This type of regulation has been a major focus of interest for us, and was reviewed in 1989 (7). It is discussed in detail in Sections V and VI. The synthesized cAMP can, under the appropriate conditions, be eliminated from the cells. A variety of studies (8) have determined that there is an energy-dependent discharge of the nucleotide, and that the rate of this discharge is not proportional to its rate of synthesis (9). The relationship derived is that the intracellular cAMP pool size is proportional to the combined rate of cAMP degradation and excretion. Studies (10)using E . coli membrane vesicles suggest that the excretion of cAMP is carrier mediated and that the rate of nucleotide efflux is subject to regulation by both the magnitude of membrane energization as well as dose of carrier. The third factor involved in the regulation of cAMP levels is the degradation of cellular CAMP by cAMP phosphodiesterase (EC 3.1.4.19), the product of the cpd gene. Mutants

35

BACTERIAL ADENYLYL CYCLASES

energy

q

ATP --@-CAMP

FIG.2. Regulation of cAMP levels in E . coli. cAMP is produced from ATP by the enzyme adenylyl cyclase (AC). As described in Sections V and VI, the activity of this enzyme is controlled by numerous factors (also, see Fig. 4). cAMP is a substrate for the enzyme cAMP phophodiesterase (PDE), the product being 5' AMP. The effect of this enzyme on cAMP levels is discussed in Section 11. Another factor that plays a role in regulating intracellular cAMP levels is the energy-dependent excretion of the nucleotide (wavy arrow). This aspect of CAMP metabolism is alluded to in Section 11.

deficient in this enzyme have higher steady-state levels of CAMP, indicating the importance of this activity as a regulator of nucleotide levels. An additional modulatory factor in maintaining cellular levels of CAMP is the cAMP binding protein, CRP. Strains carrying mutations in the gene (crp)for this protein accumulate larger than normal levels of cAMP both intracellularly and extracellularly (11). A typical crp mutant synthesizes cAMP approximately 25 times faster than does a wild-type strain. A strain deficient in both CRP and cAMP phosphodiesterase had a 100-fold higher intracellular cAMP level and an excretion rate 150-fold higher than the wildtype straing (12). The double-mutant strain was suggested to be a possible source for the industrial production of CAMP,although even higher levels of CAMP in culture media could probably be obtained using strains in which the gene for adenylyl cyclase is incorporated into a plasmid wherein the expression of the gene is under the control of a powerful promoter (13).

111. Structure and Expression of the E. coli Adenylyl Cyclase (cya) Gene Both the structural gene and promoter elements of the E. coli cya gene have been cloned (14, 15). The strategy used for the cloning involved the recognition that a host strain of E. coli deficient in the cya gene forms white colonies on a lactose MacConkey plate, while a strain expressing the cya gene forms red colonies. In this way, hybrid plasmids expressing the cya gene were easily selected. Isolated cya clones programmed the synthesis of a protein of M, -85,000. The analysis of the promoter region of such clones indicated the presence

36

ALAN PETERKOFSKY ET AL.

of two sequences capable of binding CRP. This observation was of interest in the context of providing a possible explanation for the finding mentioned in Secticin I1 that E . coli strains deficient in CRP overproduce CAMP.The idea was then pursued that transcription of the cya gene might be negatively regulated by the complex of CRP with CAMP, a logical type of feedback inhibition. The transcription from the major promoter (called P2) of the cya gene was inhibited in Gitro by the CAMP-CRP complex (16). Further evidence for an interaction of CAMP-CRP with the cya promoter was derived from DNAse-I footprinting studies that showed an overlapping interaction of CAMP-CRP and RNA polymerase with the promoter region. The magnitude of the repression of cya transcription by CAMP-CRP in Salmonella typhimurium, a close relative of E . coli, has recently been quantitated (1 7). The fraction of cya expression through the P2 promoter (80%of total transcription) was repressed approximately eightfold by CAMP-CRP. A clone of the cya gene from E . coli was used to determine the complete nucleotide sequence of the structural gene (18).The data showed that the enzyme contains 848 amino acids, and indicated that the translation initiation codon is TTG, rather than the typical ATG. This observation, together with the finding that the ribosome binding site is weak, provided a partial explanation for the low level of adenylyl cyclase protein found in cells (19). The notion that the TTG initiation codon imposes a negative translational regulation was validated (20) by showing that mutating the TTG codon to ATG increased the expression of the cya gene three- to sixfold. The first extensive purification of E . coli adenylyl cyclase used as starting material a strain that overproduces the enzyme fivefold due to an episome carrying the cya gene in combination with a crp deletion background. The preparation of essentially homogeneous protein required a seven-step procedure that achieved an approximately 17,000-fold purification. More recently, a construct was made in which the cya gene was placed under the control of powerful promoter and ribosome binding sites derived from the A bacteriophage (13). After promoter activation in a strain containing such a construct, the amount of adenylyl cyelase corresponded to approximately 16% of the total protein. A three-step purification procedure was developed for the preparation of substantial amounts of essentially homogeneous enzyme.

IV. The Phosphoenolpyruvate: Sugar Phosphotransferase System Cellular levels of CAMP in E . coli are relatively high in cells depleted of a carbon source and are decreased substantially when such cells are exposed to a variety of transportable sugars (21). This regulation derives from a sugar-

37

BACTERIAL ADENYLYL CYCLASES

SUGAR

FIG. 3. The phosphoenoZpyruvate:sugarphosphotransferase system. This transport system utilizes phophoenolpyruvate (PEP) to concomitantly translocate and phosphorylate numerous sugars into E. coli as well as some other organisms (see Section IV). The proteins Enzyme I (EI) and HPr are cytoplasmic proteins (not sugar-specific)used for the transport of all PTS sugars, while enzymes designated 11 and/or I11 are sugar-specific and, in general, membrane-associated. As indicated in the figure and discussed in Section V, a complex of the PTS proteins, designated by the bracket, is believed to regulate the activity of E . coli adenylyl cyclase (AC).

dependent inhibition of adenylyl cyclase activity. Numerous studies have implicated the sugar transport system known as the phosphoenolpyruvatemgar phosphotransferase system (PTS) as an important regulatory element for the control of adenylyl cyclase activity in E. coli. A general description of the PTS is outlined in Fig. 3. The multiprotein system catalyzes the following overall reaction: phosphoenolpyruvate

+ sugar

(out) -+ pyruvate

+ sugar-P

(in)

It is important to note that the total reaction involves both a sugar phosphorylation and a transmembrane movement of the sugar. The membrane-

38

ALAN PETERKOFSKY ET AL.

associated recognition molecules that are specific for the family of sugars transported by this system are referred to as Enzymes 11; the genes for these enzymes are scattered on the E . coli genetic map (22). There appears to be at least one active cysteine residue in the typical Enzyme 11, and it has been proposed that this may be a site of phosphorylation. This suggests that the phosphoprotein is an intermediate in the sugar transport pathway. Consistent with the notion that there is an active phosphoenzyme intermediate, there is some evidence that Enzymes I1 can catalyze an exchange transphosphorylation reaction from a sugar-]? to free sugar. Transport of some sugar substrates of the PTS (for example, glucose) requires a soluble sugar-specific protein referred to as protein 111 (in the case of glucose, the designation is IIIaic). We point out in Section V that IIIgIc functions both as a phophocarrier in the transport reaction as well as a regulator of some physiological processes. The activity of E . coli adenylyl cyclase is one of the processes regulated by IIIglC. There are two phosphocarrier PTS proteins that are soluble and function with all the Enzymes 11; that is, they are not sugar-specific. The protein directly phosphorylated by PEP is designated Enzyme I and the phosphorylated form of Enzyme I can phosphorylate the protein known as HPr. In E . coli, the genes for Enzyme I, HPr, and protein IIIglCare linked in an operon in the order listed above. These genes have been cloned (23).Transcription from the promoter region driving the expression of the genes for Enzyme I and HPr is influenced positively by CRP (24). Extracellular glucose increases transcription of the pts operon; the mechanism of this effect appears to involve the conversion of the Enzyme I1 for glucose to the dephosphorylated form, which serves as a transcription activator. The operon for the general PTS proteins has been sequenced (25, 26). Recently, expression vectors have been constructed for overexpression of the genes for Enzyme I, HPr, and IIIgIc (13),and methods for the purification of the proteins on a large scale from extracts have been reported (27). The availability of large quantities of these proteins is expected to assist in the clarification of their roles in the regulation of adenylyl cyclase activity (7).

V. Re ulation of E. coli Aden lyl C clase Activity by the

9,

J r :

Phosp oeno1pyruvate:sugar hosp otransferase System As mentioned in Section IV, addition of glucose to a suspension of washed E . coli cells provokes a rapid decrease in the level of CAMP in the cells, an effect that is due to an inhibition of adenylyl cyclase activity. A variety of other sugars can substitute for glucose to cause this inhibition (21). The important property determining the specificity for inhibition is that the sugar can be transported. Therefore, induction of the transport system for a

BACTERIAL ADENYLYL CYCLASES

39

number of sugars, such as mannitol or fructose, confers on cells the ability of those sugars to inhibit adenylyl cyclase activity. These studies provided a basis for the notion that the process of sugar transport across the cell membrane is responsible for the inhibition of adenylyl cyclase activity, consistent with the original idea (28) that passage of catabolites into cells is an essential aspect of the process of catabolite repression. A direct demonstration that glucose inhibits adenylyl cyelase activity was made by using E. coli cells made permeable by treatment with toluene (29). In this system, the adenylyl cyclase catalytic unit is presumed to be in a complex with appropriate regulatory factors that permit the signal transduction from glucose to the requisite components of the system. Consistent with the notion of a regulatory system being responsible for the glucose-dependent inhibition of adenylyl cyclase activity is the observation that glucose does not inhibit the enzymatic activity in broken cell extracts. The requirement for unique Enzyme-I1 components of the PTS for sugar-dependent inhibition of adenylyl cyclase activity was established by using mutants in one or the other of the Enzymes I1 specific for glucose. The product of the ptsG gene is a carrier that accommodates both glucose and amethylglucoside; a strain carrying mutated ptsG loses both the ability to phosphorylate a-methylglucoside via phosphoenolpyruvate (PEP) and to show a-methylglucoside-dependent inhibition of adenylyl cyclase activity. Similarly, the product of the ptsM gene is a carrier that promotes the transport of both glucose and 2-deoxyglucose; a strain of E. coli mutated in ptsM is deficient in both phosphorylation of 2-deoxyglucose and 2-deoxyglucosedependent inhibition of adenylyl cyclase activity (30).An important ramification of the observation that either one of these mutants allowed complete inhibition of adenylyl cyclase activity by glucose or the appropriate glucose analog was that there is only one population of adenylyl cyclase molecules and that the signal for sugar inhibition of the enzyme through different sugar-specific carriers was probably due to a common downstream component in the transport system. The realization that a common factor of the PTS might be responsible for either an activation or an inhibition of adenylyl cyclase activity, depending on the presence or absence of a sugar substrate for the PTS, led to the hypothesis that the condition of phosphorylation of one or more of the PTS phosphocarrier proteins could modulate adenylyl cyclase activity (31).The idea proposed was that the phosphocarrier(s) had to be in the phosphorylated condition to support a high level of enzyme activity; therefore, the explanation for sugar-dependent inhibition of adenylyl cyclase activity was that the process of PTS-dependent sugar phosphorylation resulted in a concomitant dephosphorylation of the PTS carrier. In the framework of the phosphorylation-dephosphorylation regulation

40

ALAN PETERKOFSKY ET AL.

model for adenylyl cyclase (32),there was consideration of the role in physiologic regulation of enzyme activity of the other small components of the PTS. It was pointed out that PEP is the phosphate donor in the PTS and, conversely, pyruvate and PTS sugars are phosphate acceptors in the PTS. There are data in support of the idea of a push-pull or opposing mechanism for regulation of adenylyl cyclase activity in which phosphate donors (PEP) activate and ph6sphate acceptors (glucose or pyruvate) deactivate the enzyme. Further, a system for trapping PEP (ADP plus pyruvate kinase) can inhibit adenylyl cyclase activity in permeabilized cells; this suggests that there is normally a dynamic mechanism for maintaining a flux of phosphate groups into the PTS proteins that results in a modulated level of adenylyl cyclase activity. The precise nature of the interaction of adenylyl cyclase with the PTS has not heen cemented. Based on the repression behavior of E . coli mutants deficient in protein IIIglC( 3 4 , it was proposed that IIIRlc forms a regulatory complex with adenylyl cyclase, promoting an increase in enzyme activity when it is in the phosphorylated condition (34, 35). The position that other investigators, including our group, have taken is that a complex of the general PTS proteins (Enzyme I, HPr, and IIIglc)with adenylyl cyclase is the physiologically relevant species of the enzyme (36). This idea is represented in Fig. 4. Experimental support for that model has come from studies (36)in which homogeneous preparations of the PTS proteins Enzyme I, HPr, and IIIg'. were added to crude extracts of E . coli, resulting in a reconstitution of regulatory properties of the enzyme. It is

Inhibitors

Adenylyl Cyclase Activators FIG. 4. Inhibitors and activators of E . coli adenylyl cyclase activity. As described in Section V, the proteins of the PTS designated Enzyme I, HPr, and Enzyme IIIglc are proposed to form a complex at the cell membrane. Adenylyl cyclase is believed to interact with the complex, resulting in a diminution of the enzyme activity. The evidence for an inhibitory effect of the CAMP receptor protein (CRP) is discussed in Section VI. The roles of a variety of activators of adenylyl cyclase activity are dso enumerated in Section \'I. These activators are EF-Tu, nucleotides, PEP, and Pi.

41

BACTERIAL ADENYLYL CYCLASES

noteworthy that the addition of Enzyme I to the extracts led to a dosedependent inhibition of adenylyl cyclase activity and that this inhibition became more severe when all three PTS proteins were added to the extracts; the interpretation of this finding was that adenylyl cyclase has interaction sites for not only IIIg'" but also for Enzyme I and HPr. Since the effects observed were with proteins in the unphosphorylated condition, the possibility must be considered that the mechanism of regulation of adenylyl cyclase activity involves an inhibition by unphosphorylated PTS proteins that is relieved by phosphorylation of the proteins. The properties of a family of precisely constructed mutants in which specific portions of the pts operon were deleted provided further evidence for the importance of PTS proteins other than II1glc for the regulation of adenylyl cyclase activity (37). A strain in which the crr gene (for IIIgl') was deleted was characterized by a level of cAMP 14-fold lower than a wild-type strain. Importantly, a further deletion of the gene for Enzyme I led to a further (2- to %fold) decrease in the level of cAMP production. Another aspect of cellular organization relevant to the regulation of adenylyl cyclase activity involves the possible assembly of a complex of PTS proteins at the cytoplasmic membrane. A follow-up to previous studies showing that membrane vesicles, which are essentially completely depleted of cytoplasmic proteins, actually contain some Enzyme I and HPr (38, 39) was carried out (40). Immunoelectron microscopy of frozen thin sections of E . coli was performed using antibody directed against Enzyme I. This type of study showed that a substantial fraction of the cellular Enzyme I was localized at or close to the cytoplasmic membrane. It seems reasonable to assume, therefore, that under physiological conditions the general proteins of the PTS may gather together at the membrane in a functional complex and that this complex may serve as a matrix for interaction with adenylyl cyclase (see Fig. 4).

VI. Regulation of E. coli Aden lyl Cyclase Activity

'f:

by Other actors

A. Activators As mentioned in Section V, there is a role for PEP as an activator of adenylyl cyclase activity. The mechanism of this activation relates to the role of this metabolite as a phosphate donor to PTS phosphocarriers. The notion is that PTS proteins in the phosphorylated condition promote a higher activity state of adenylyl cyclase than do PTS proteins in the unphosphorylated condition (32). The importance of inorganic orthophosphate as an activator of adenylyl

42

ALAN PETERKOFSKY ET AL.

cyclase was realized as an offshoot of the development of the permeabilized cell system for the study of regulatory aspects of adenylyl cyclase activity (29). Treatment of cells with toluene allows the cellular pool of small factors to become depleted by dilution into the surrounding medium while maintaining physiologically significant protein-protein interactions. In this system, the assay for adenylyl cyclase in the absence of added phosphate reveals an activity that is characteristically low and insensitive to inhibition by PTS sugars. Since the addition of phosphate to French-press extracts of E. coli does not stimulate adenylyl cyclase activity, the locus of action of Pi appears not to be directly on the catalytic unit of the enzyme. The observation that potassium phosphate stimulates both adenylyl cyclase and PTS activities in permeabilized cell preparations (41) led to the proposal that the effects of these ions on adenylyl cyclase activity are mediated via some effect on the PTS. In this regard, it is noteworthy that phosphate stimulation of the cyclase is not observed in permeable cell preparations from a mutant strain of E . coli deficient in PTS proteins. The characteristic features of adenylyl cyclase in toluene-treated cells in the presence of Pi are that both the V,, and the K , for ATP are increased. Since the stimulatory effect of phosphate on adenylyl cyclase activity is abolished by transportable PTS sugars, it was proposed (29) that the PTS proteins exercise a dual regulation of adenylyl cyclase activity. First, the phosphorylation state of the PTS proteins, determined by the availability of a transportable sugar, dictates the activity level of the complex. Second, when sugars are transported via the PTS, the pool size of Pi is decreased due to the accumulation of sugar phosphates. It should be emphasized that the maximal stimulation of adenylyl cyclase activity requires Pi concentrations of 20-40 mM, concentrations that are within the normal physiological range (42). Exposure of intact cells to glucose or other PTS sugars results in a rapid decrease of the internal phosphate pool to approximately 20%of the original level (43).These effects have been convincingly demonstrated by the use of nlP nuclear magnetic resonance (NMR) spectroscopy (44). The kinetic properties of adenylyl cyclase vary substantially depending on whether the activity is measured in permeabilized cells, where the enzyme is assumed to be interacting with appropriate physiological regulators, or in broken cell extracts, where the enzyme is assumed to be dissociated from such regulators (41). In the permeable cell system, adenylyl cyclase produces sigmoidal substrate-vs. -velocity plots, suggesting an allosteric interaction. These studies provide a basis for thinking that, under physiological conditions, the adenylyl cyclase complex has two ATP-binding sites, one catalytic and the other regulatory. Since the allosteric kinetics requires the presence of PTS proteins, it has not yet been clarified whether the allosteric regulatory site is actually located on the adenylyl cyclase protein or on one of the PTS proteins.

BACTERIAL ADENYLYL CYCLASES

43

Since eukaryotic adenylyl cyclases are generally regulated by proteins that specifically bind GTP, an investigation was made of the possibility that E. coli adenylyl cyclase exhibits a G-protein interaction as well (45). The discovery was made that EF-Tu, which is the most abundant protein in E. coli and serves as an elongation factor in the process of protein synthesis, specifically activates adenylyl cyclase. At a weight ratio of approximately 250:l (EF-Tu:cyclase), which is close to the normal cellular ratio, the stimulation of adenylyl cyclase was approximately 70%. The likely association of EFTu with adenylyl cyclase may provide a partial explanation for the previously observed association of a portion of the cellular EF-Tu pool with the cell membrane (46).The suggestion has been made, but not yet proved, that EFTu forms an essential part of the adenylyl cyclase regulatory complex, and that the allosteric kinetics reported with ATP may normally be due to the interaction of GTP with EF-Tu. Further studies in reconstituted systems may resolve this issue.

B. inhibiton Some factors that promote inhibitory effects on adenylyl cyclase have been described. It was mentioned in Section V that exposure of intact or permeable cells to a transportable PTS sugar results in an inhibition of adenylyl cyclase. The mechanism of this effect (a combination of dephosphorylation of PTS proteins and decrease of the cellular Pi pool) has been discussed in Section V. Pyruvate is also an inhibitor of adenylyl cyclase when the enzyme is in its coupled form. The mechanism of this regulation (36) is believed to be analogous to that involving the transportable PTS sugars; it drains phosphate from PTS proteins through a reversal of the PTS, and it concomitantly decreases the pool size of Pi. CRP is the mediator of the physiological effects of CAMP (see Section I); the complex cAMPCRP is a transcription activator. Interestingly, mutants deficient in CRP produce abnormally large amounts of CAMP (11).This observation has led to the proposal that CRP functions ordinarily as a downregulator of the adenylyl cyclase complex, although this protein has no effect on the activity of partially purified preparations of the enzyme (45). In keeping with the notion that CRP is a component of a multiprotein adenylyl cyclase complex, the elevation of CAMP levels characteristic of the absence of CRP requires the presence of Enzyme I, HPr, and II1glc of the PTS (47). The mechanism of the apparent interaction of CRP with the adenylyl cyclase complex has not yet been clarified, although a likely scenario is that CRP inhibits adenylyl cyclase by interacting with the complex when the PTS proteins are phosphorylated, resulting in only a suboptimal degree of enzyme activation. In the absence of CRP, the phosphorylated PTS proteins would be expected to lead to a higher level of enzyme activity. It is tempting to speculate that the physiological significance of the interaction of CRP with

44

ALAN PETERKOFSKY ET AL.

the adenylyl cvclase complex is to mediate a down-regulation of the enzyme by CAMP;this would effectively be a type of product inhibition. The implication of this model would be that only CAMP-CRP, but not free CRP, would serve as an inhibitor of the adenylyl cyclase complex. This is clearly an area that deserves further analysis.

VII. Adenylyl Cyclases in Bacteria Other Than E. coli A. Mycoplasma Members of the genus Mycophina belong to the class Mollicutes (organisms with no cell walls) (48). These bacteria, with a genome size of 1155 kb, corresponding to one-quarter to one-fifth of the genome size of E . coli or Bacillus subtilis (@), are regarded as the smallest and simplest self-replicating organisms (50).It has been suggested (51) that they arose by evolution with loss of some of the genome from a branch of the eubacterial tree that contains gram-positive eubacteria containing DNA with a low G + C content. The assumption has been made (52) that the minimum number of genes in Mycoplasma (approximately 350) represents the conservation of only essential functions. Mycoplas~nucapricolum contains CAMP (53).In wild-type strains of this organism, the intracellular level of cAMP is reduced after exposure of the cells to sugars transported by the PTS. As is the case in E . coli, the level of cAMP in the cells is inversely proportional to the amount of PTS sugar substrate in the growth medium; depletion of the sugar in the medium concomitant with cessation of growth leads to an increase in the cellular cAMP level, and addition of sugars back to the cells results in a drop in the concentration of CAMP. The conclusion from these studies (53) is that the activity of the Mycoplasma adenylyl cyclase is regulated by the PTS as it is in E . coli and that this type of metabolic regulation must be very important since it is evolutionarily preserved in an organism with only “essential” genes. Since the level of cAMP was found to be consistently higher in glucose-grown compared to fructose-grown cells, it is likely that the expression of adenylyl cyclase is metabolite-controlled. The analysis of PTS components in M . capricolum indicated that this system may be even more complex than that in E . coli, since the Enzyme-I protein appears to be a large product (220 kDa) containing three different subunits. In contrast, the E . coli Enzyme-I protein in its physiologically active form is a homodimer consisting of subunits of M, approximately 70,000.

B. Sordetella pertussis and Bacillus anthracis Adenylyl cyclases are secreted by the pathogenic microorganisms Bacillus anthracis, Bordetella pertussis, and Brevibacteriurn liquefaciens.

45

BACTERIAL ADENYLYL CYCLASES

The enzymes from B . pertussis and B . anthracis are considerably stimulated by calmodulin, a protein supplied by the target cell but absent from the bacteria. The uncontrolled levels of CAMP generated as a result of the bacterial invasion of the target eukaryotic cell reduce the capability of leukocytes and macrophages to kill the bacteria (54). The mechanism by which the B . pertussis toxin, containing adenylyl cyclase, penetrates cells is directly through the plasma membrane rather than by the expected receptor-mediated endocytosis (55)(see Fig. 5). Uptake of the toxin is inhibited by gangliosides, indicating a lipid-dependent mecha-

I-]

secretion

Bacteria

*

200 kDa

ofAC

protein C

n

binding (Ca-dependent) and penetration I

Activation by Calmodulin

Host cell

FIG.5 . The mechanism of action of Bordetellu pertussis adenylyl cyclase. As described in Section VII,B, adenylyl cyclase (AC) is secreted from bacteria as a protein of approximately 200 kDa. This enzyme can, in the presence of calcium (Ca), bind to and penetrate eukaryotic cells. As a result of the penetration, domains for the binding of calmodulin (CaM) and for catalytic activity become localized in the intracellular space. The result is that toxic levels of CAMPcan accumulate in the cells. Eukaryotic cells are equipped with an ATP-dependent mechanism for degrading the intracellular portion of the AC; this degradation mechanism allows eukaryotic cells to alleviate the toxicity produced by the elevation of CAMP levels. N and C represent the N-terminus and C-terminus of the protein, respectively.

46

ALAN PETERKOFSKY ET AL.

nism of target cell invasion (56). In contrast to that of the B . pertussis enzyme, the mechanism of penetration of the B . unthrucis adenylyl cyclase is by way of receptor-mediated endocytosis (56);this endocytic uptake depends on the presence of an additional protein. The entry into cells of the B . pertussis adenylyl cyclase requires millimolar concentrations of calcium. The suggestion has been made (54)that the metal requirement is for the interaction of the enzyme with the membrane of the target cells. The current thinking is that the adenylyl cyclase penetrates the plasma membrane of the target cells but remains associated with the membrane in such a way that the catalytic and calmodulin binding domains become exposed to the cellular cytoplasm. It should be noted that exposure of Chinese hamster ovary cells to B . pertussis led to the expected elevation of CAMP in the cells (58).Unexpectedly, electron microscope analysis indicated that intact bacteria invade the cells. These studies suggested an alternate mechanism for cell intoxication involving a sequential adherence of the bacteria to the cells followed by entry of the bacteria, rather than a transmembrane transport of the bacterial toxin. I n B . pertussis, adenylyl cyclase is initially synthesized as a 200-kDa protein (61).The molecular mass of the invasive form of the enzyme determined by equilibrium sedimentation is 175-178 kDa (57).The 200-kDa form of the cyclase is necessary for invasive activity (57). Digestion by trypsin converts this large form to a smaller protein (45-50 kDa), a size similar to that generally found in culture supernatants as the secreted form of the enzyme. The fraction of cell-associated adenylyl cyclase ( M , 215,000) was 28%, with the remaining 72% of the catalytic activity found in culture supernatants ( M , 45,OOO). When the cell-associated activity was incubated with an extract of the bacteria, the 215-kDa species was degraded to the 45-kDa form (62). Interestingly, the bulk of the adenylyl cyclase found in cell culture media appears to be the 47-kDa species; however, this form of the protein is not toxic and is therefore probably not important in the etiology of whooping cough. The weight of evidence is that the smaller form is derived from the larger one by proteolytic digestion. Column chromatography on wheat germ lectin-agarose was used to separate, from the B . pertussis culture medium, an additional protein factor that conferred on the purified adenylyl cyclase the ability to invade neuroblastoina cells (59)with a concomitant increase in cellular CAMP concentration. Once inside the target cells, the adenylyl cyclase is rather unstable. The maintenance of continued high intracellular levels of CAMP depends on the continual presence of the cyclase in the extracellular space, in order to effect the constant replacement of enzyme inactivated by proteolysis. This proteolysis by a host-cell enzyme appears to occur by an ATP-dependent mecha-

BACTERIAL ADENYLYL CYCLASES

47

nism (60). Nonhydrolyzable analogs of ATP can substitute for ATP, suggesting that the binding of the substrate induces some conformational change that makes the enzyme susceptible to degradation. Genes related to adenylyl cyclase synthesis and secretion in B . pertussis are located on an operon composed of four genes (63-65). cyaA codes for cyclolysin (200 kDa), which contains both adenylyl cyclase and hemolytic activities (66).The hemolytic determinant has been localized to the 3' region of the molecule by the examination of deletions. The other three genes (cyaB, cyaD, and cyaE) are involved with the transport of the cyclolysin protein. The gene for the adenylyl cyclase has been cloned in E . coli (67). The translation product is a precursor of the active enzyme, which is 1706 amino acids long. The amino-terminal end of this precursor (450amino acids) contains the calmodulin-activated enzyme activity. In a similar vein, the adenylyl cyclase gene from B . anthracis was cloned in E . coli. In this case (68), the clever selection method used depended on the restoration of adenylyl cyclase activity of a cya- strain that expressed the gene for calmodulin, which substantially activates the B . anthracis enzyme. The 43-kDa form of adenylyl cyclase from B . pertussis has two domains. The N-terminal region (residues 1-235) harbors the catalytic activity; the Cterminal region (residues 236-399) contains the calmodulin binding domain. Both domains are essential for the enzyme to display a high activity (69). These two domains of the enzyme may be separated after cleavage of the protein by trypsin into two fragments (70). The domain that interacts with calmodulin shows a tryptophan 242 fluorescence shift as a result of binding the activator. The B . pertussis adenylyl cyclase is similar to the enzyme from rat brain in that both are activated by calmodulin. Antibodies produced by the two enzymes cross-react, indicating a structural similarity (71). It has therefore been suggested that the two enzymes may be related evolutionarily. (However, see Section VIII for a discussion of the protein-sequence comparisons.) The site-directed mutagenesis approach was taken to identify amino-acid residues essential for binding calmodulin by the B . pertussis cyclase (72). Tryptophan 242 was identified as an amino acid important for tight binding of the activator. A change of the Trp to Asp reduced the &nity for calmodulin by a factor of 1000. These data are consistent with the previous suggestions that the C-terminal tryptic fragment (residues 236-399) of the adenylyl cyclase harbors the calmodulin binding domain. The DNA sequence of the B . anthracis edema factor (which has adenylyl cyclase activity) has an open reading frame of 2400 bp encoding an 800aminoacid protein. This precursor protein is processed to form a mature protein of 767 amino acids by a secretory mechanism (73). A 24-aminoacid sequence in the central region is homologous to a comparable sequence in

48

ALAN

PETERKOFSKY ET AI,.

the N-terminal portion of the cyclase from B . pertussis (74)(see Section VIII for a discussion of sequence homologies of these two proteins). The calmodulin binding domain of the B . anthracis adenylyl cyclase was labeled with a photofinity crosslinker that was coupled to calmodulin. Analysis of CNBr and N-chlorosuccinimide cleavage products allowed the localization of the binding region to the 150 amino acids at the C-terminus of that protcin. The N-terminal region of the B . anthracis cyclase encodes the binding site for the protective antigen, one of the components of the exotoxin that is a receptor binding protein, mediating the entry of the cyclase into target cells. There is no sequence in the B . anthracis adenylyl cyclase showing a similarity to the calmodulin binding site of the B . pertussis adenylyl cyclase, suggesting different mechanisms for calmodulin activation in the two enzymes (75) (see Section VIII and Fig. 7 therein). The cyu genes from B . anthrucis and B . pertussis differ in the percentage of G + C ( B . anthrucis = 65% G + C, B . pertussis = 29% G + C). However, the two adeiiylyl cyclases contain three highly conserved amino-acid domains. Both contain consensus sequences similar to that found in many ATPbinding proteins (76)(see Section IX, ATP-Binding Sites). 3’-Anthraniloyl-2’deoxyATP, a fluorescent analog of ATP, is a competitive inhibitor of the adeiiylyl cyclases from B . pertussis or B . unthracis (77)with a K , of about 10 pM. The fluorescent nucleotide was displaced by either ATP or 3’-dATP. The fluorescence of the nucleotide was enhanced when the adenylyl cyclase was complexed to Ca2+ and calmodulin. Mutagenesis studies (78) of the B . pertussis adenylyl cyclase show that lysine 58 is essential for catalytic activity. Replacement of this residue by inethionine resulted in loss of activity. Further studies (73) showed that mutagenesis of lysine 58 or lysine 65 to glutamine led to a decrease of catalytic activity by a factor of approximately 1000. These data are in keeping with the model that the N-terminal tryptic fragment of the adenylyl cyclase contains the catalytic activity. The enzyme also contains a conserved sequence typical of some ATP-binding proteins (DxD, corresponding to aspartic acid residues 188 and 190). Amino-acid replacement studies (79) led to the proposal that the carboxyl side chains of these aspartic acid residues coordinate with the Mg2+ of the Mg-ATP complex. The adenylyl cyclase of B . anthracis (edema factor) contains 800 amino acids, compared to a chain length of 1706 amino acids for the B . pertussis enzyme. Residues 342-358 of the B . anthracis enzyme and 54-70 of the B . perkussis enzyme contain sequences that represent ATP-binding consensus sequences (76). These two regions are almost identical (15 out of 17), indicating a high degree of conservation. i t has been suggested that lysines 346 arid 353 of the B . anthrucis cyclase may be essential since the corresponding

BACTERIAL ADENYLYL CYCLASES

49

lysine residues of B . pertussis (58 and 65) have been shown by site-directed mutagenesis experiments to be required for activity. Mutation of Lys-346 to Gln led to a major decrease in the capability of a truncated form of the B . anthracis enzyme to bind a fluorescent analog of ATP (3’-anthraniloyl-2’-deoxyATP),resulting in loss of catalytic activity (80). These data suggest that a major function for Lys-346 is to enhance the binding of ATP to adenylyl cyclase. The protein has two probable ATPbinding A-type consensus sequences (see Section IX) with lysines at positions 346 and 353 as well as a B-type consensus sequence +X++[P] (where 4 is a hydrophobic residue) (100) containing a glutamate at position 436. The two proposed A-consensus sequences are overlapping and opposite (81). Mutagenesis of Lys-346 or -353 to Met resulted in loss of adenylyl cyclase activity. Mutagenesis of Glu-436 to Gln resulted in only a 25% loss of activity, suggesting that the proposed B-type sequence is not essential for activity. In summary, the adenylyl cyclases from B . pertussis and B . anthracis form a unique class of enzymes designed for expression of their activities in eukaryotic cells. Although they share with the adenylyl cyclase from bovine brain the property of being activated by calmodulin, there is no extensive sequence similarity (see Section VIII on sequence homology) of the bacterial and brain enzymes. These bacterial adenylyl cyclases constitute an interesting example of a parasitic relationship whereby the enzymes produced in the bacteria are activated by a protein (calmodulin) produced in the host cell.

C. Rhizobium Rhizobium species are of great practical interest due to their potential for a symbiotic relationship with specific plant tissues. The conversion of Rhizobium species from free-living cultures to the bacteroid form in plant tissues represents a type of differentiation. The involvement of CAMP in this process as well as in the regulation of gene expression in the different phases of this organism has been the subject of numerous investigations. It has been speculated that rhizobia, growing under symbiotic conditions, have the capability to simultaneously repress ammonia assimilatory enzymes (i.e., glutamine synthetase, glutamate synthase, and glutamate dehydrogenase) concomitant with the derepression of nitrogenase. Studies of the effect of CAMPon the growth of Rhizobiumjaponicum (82)showed that while 5 mM cAMP is toxic to the cells, 1 mM cAMP leads to a repression of the three NH4+ assimilatory enzymes. The effects were two- to threefold for glutamine synthetase and glutamate synthase and two- to ninefold for glutamate dehydrogenase. Since the inclusion of 1 mM AMP, ADP, or ATP in the growth medium did not show these effects, it was concluded that the repression is specific for CAMP. A model was suggested in which glutamate is transported from the plant to the Rhizobium bacteroid as a possible energy

50

ALAN PETERKOFSKY E T AL.

source that also represses ammonia assimilation, and that cAMP is an intracellular messenger that mediates the repression of the NH, assimilatory enzymes. A n important characteristic of Rhizobium meliloti is its capability to fix CO,. The fixation of CO, is repressed by a number of citrate cycle intermediates. There appears to be no connection of CAMP with this repression, since the cellular levels of the nucleotide are invariant under conditions of repression or derepression (83). On the other hand, hydrogen utilization by R . japonicum is influenced by the carbon source in the growth medium. Growth in the presence of malate has been associated with significantly lower hydrogenase levels than growth in the presence of glucose, suggesting some form of cataholite repression (84). Addition of CAMP (1 mM) to the growth medium overcame the malate-dependent inhibition of hydrogenase. Since the effect of CAMP was eliminated in the presence of protein or RNA synthesis inhibitors, it was concluded that new protein synthesis was required for the antagonism of the malate effect on hydrogenase activity. Transformation of the cells with a plasmid expressing the cya gene (the gene for adenylyl cyclase) of R . meliloti relieved malate-dependent repression of hydrogen uptake. The alleviation of the repression was accompanied by an approximately fourfold increase in the cellular CAMP level. While these observations suggested a role for CAMP in the regulation of H, metabolism in Rhizobium, the mechanism for modulation of CAMP levels in the organism remains undefined. It was speculated (84) that malate serves as a catabolite repressor in R. juponicum by leading to a reduction in cAMP levels, which then results in a decrease in hydrogenase activity by a mechanism that remains to be clarified. Using a gene bank from R. meliloti (comprising 1200 clones) in the broad host-range vector pRK290, the cyu gene was expressed in E . coZi, where it weakly complemented a cyu deletion (85, 86). Hybridization studies indicated that the cya gene is conserved in a variety of R . meliloti strains but not in other Rhizobiutn strains or in E . coli. While the cya gene from E . coli is repressed by growth in media supplemented with glucose, no such effect was observed for the Rhizobium cyu gene. The CAMP levels in the complemented cyu deletion strains were approximately 14% of that found in wildtype strains. The Rhizobium adenylyl cyclase expressed in E . coli was subject to catabolite repression. P-Galactosidase activity decreased after the addition of glucose in both wild-type strains as well as in the cyu deletion strain complcniented with the Rhizobium enzyme, although the repression was not as severe in the complemented strain. It is not clear from these studies what the mechanism is of catabolite repression mediated by the Rhizobium adenylyl cyclase. First, it has been suggested that rhizobia are devoid of the PTS +

BACTERIAL ADENYLYL CYCLASES

51

(for glucose and fructose), a necessary component for catabolite repression in

E. coli. Second, the adenylyl cyclase of Rhizobium appears to be smaller than the E . coli enzyme ( M , -20,000), a size that may not include a domain for regulation by the PTS. It should be pointed out that no one has reported the presence of a cAMP receptor protein in any species of Rhizobium. A function for cAMP in transcription regulation should require the presence of such a cAMP binding protein. The sequence of the cya gene from R. meliloti revealed that the gene product has an M, of about 20,000 (87). The low M, of the protein suggests that it is composed of a catalytic domain with no regulatory domain, although the possible existence of a larger form carrying a regulatory domain is not excluded. The enzyme was purified and characterized as a lac fusion protein. It is unusual in its catalytic behavior, in that the K , is 4 mM compared to the K , of 0.6 mM for the E. coli cyclase. Another unusual property of the enzyme is its sensitivity to inhibition by GTP (a concentration of GTP equal to the substrate concentration gave approximately 35% inhibition). While the enzyme could interact with GTP, it did not synthesize cCMP. A comparison of the amino-acid sequence of the protein with that of other adenylyl or guanylyl cyclases reveals no obvious similarity with other bacterial adenylyl cyclases. However, there is a significant level of similarity with the adenylyl cyclases from yeast and bovine brain as well as the guanylyl cyclases of rat and bovine origin. These comparisons suggest a common origin for the adenylyl cyclase from Rhizobium and an enzyme from eukaryotes that might originally have had the capacity to synthesize both cAMP or cCMp (see Section VIII for a discussion of sequence similarities). Some experiments suggest that R. meliloti may have two cya genes (88). A cloned cya gene from this organism was used to construct cya-lac fusions in order to study the expression of the gene. The finding that there was little expression of the fusions prompted the construction of mutations in the cya gene. The authors discovered that a gene disruption of the cya gene decreased the levels of cAMP in the cells from 30 to 70%, but did not totally abolish the accumulation of the nucleotide. Therefore, they proposed that there may be an additional cya gene in these cells. It was noteworthy that the cya gene disruptions had no significant effects on growth, nodulation, or nitrogen fixation. A restriction fragment of DNA from Bradyrhizobium japonicum (a slowgrowing species) containing the cya gene was cloned into pBR322 (89).The clone was detected by complementation of Acya strains of E. coli. The transformants produced cAMP and reversed the Acya-encoded phenotype by allowing growth on sugars that require cAMP (arabinose, mannitol, ribose, and xylose) and regaining motility, which also requires the presence of CAMP. The cloned cya gene showed no sequence similarity to the E. coli

52

ALAN PETERKOFSKY ET AL.

cya gene, as determined by DNA hybridization. The expression of the B . juponicuin cyu gene in E . coli led to lower cAMP levels (approximately 30%) than in wild-type E . coli. Slow-growing strains of Rhizohium have been examined for their caCell cultures took up the sugar by a mechapability to transport glucose (90). nism that depended on membrane energization, while bacteroids showed essentially no glucose uptake. The mechanism of glucose uptake was further explored in cell-free preparations. The cytoplasmic fraction of cultured cells phosphorylated glucose by an ATP-dependent reaction; there was no PEPdependent sugar phosphorylation. The bacteroids showed an ATP-dependent phosphorylation reaction in both the membrane and soluble fractions. A major conclusion from these studies was that there is no demonstrable PTS activity for glucose in these slow-growing rhizobiu. Rhizobium legzmtinosarurntakes up fructose by a process that is inhibited by azide, 2,4dinitrophenol, or carbonyl cyanide m-chlorophenylhydrazine, suggesting that the mechanism of the transport involves membrane energization (92).In a mutant that does not metabolize the transported fructose, the sugar accumulates in the nonphosphorylated form. These studies indicate that this organism does not transport fructose by the PTS. The properties of adenylyl cyclase and cAMP phosphodiesterase (PDE) in bacteroids of B . japonicum have been investigated (91). Adenylyl cyclase activity was found in the membrane fraction of the bacteroids, but not in any plant fraction. CAMP phosphodiesterase was found both in the soluble and membrane fractions. The adenylyl cyclase activity was stimulated four- to fivefold by sodium dodecyl sulfate (0.01%). Adenylyl cyclase activity increased approximately threefold during aging of the nodules (low at day 17 and higher at day 21), while the membrane-bound phosphodiesterase decreased about 40%. The authors suggest that CAMP may play a role in symbiosis. Adenylyl cyclase activity increased at least 10-fold after the bacteroids were broken in a French-pressure cell, indicating that the enzyme is located within the bacteroids. None of the following compounds affected the bacteroid adenylyl cyclase activity: Gpp(NH)p, forskolin, fluoride, glutamate, glutamine, hydroxybutyrate, pyruvate, pyrophosphate, PEP, Ca2+, NH4+, NL41)+,and NADH. Pi, which is a potent stimulator of E . coli adenyiyi cyclase activity, was not tested. The patterns of adenylyl cyclase and CAMP phosphodiesterase activities suggest that the level of CAMP increases during the development of symbiotic N, fixation. In summary, it is likely that cAMP functions in Rhizobium physiology as a mediator of catabolite repression as well as in the bacteroid-plant interaction in nodules. Since it appears that rhizobia do not transport sugars by the PTS, and the unusually small size of the adenylyl cyclase in this organism may not allow for a PTS-dependent type of regulation of adenylyl cyclase activity, it is

BACTERIAL ADENYLYL CYCLASES

53

reasonable to speculate that a new mechanism for regulation of the activity of adenylyl cyclase in Rhizobium may be uncovered by further studies of this organism.

VIII. Sequence Comparisons The complete coding sequences of 12 adenylyl cyclases from a variety of sources have been deduced (in the case of Salmonella typhimurium, 50% of the sequence, the amino-terminal half, has been reported). In the published reports of these sequences, the authors have frequently shown sequence comparisons with other proteins. We have constructed a complete matrix of all the binary sequence similarity analyses, using the FASTA program (93). The results are shown in Fig. 6, together with pertinent information about the sizes of the proteins. It is noteworthy that there is considerable variation in the sizes of the different adenylyl cyclases; the chain lengths vary from 193 amino acids (A.meliloti) to 2026 amino acids (Saccharomyces cereuisiae). Presumably, this is a reflection of the differences in regulation mechanisms associated with the various enzymes. The results of the binary comparisons indicate that there is no common extensive sequence motif that is characteristic of all adenylyl cyclases, even though they catalyze the same enzymatic reaction. The data in Fig. 6 do indicate, however, that there are certain groups (four of them) that demonstrate significant relatedness. The members of these groups are denoted by shaded cells in Fig. 6 and the sequence similarities within each group are schematically depicted in Fig. 7. The first group (A, Fig. 7) consists of the adenylyl cyclases from S . typhimurium, E . coli, Erwinia chysanthemi, and Pasteurella multocida. All of these are gram-negative bacteria and may be closely related in an evolutionary sense. The regions of identity of these adenylyl cyclases encompass essentially the complete length of the coding sequences. The second group (B, Fig. 7) includes adenylyl cyclases from B . anthracis and B . pertussis. These two organisms produce toxins, of which adenylyl cyclase is one component. Further, these two enzymes are activated by calmodulin. The enzymes from B. anthracis and B . pertussis are dissimilar in that the former contains 800 amino acids, while the latter contains 1706 amino acids. Only the N-terminal 450-aminoacid region (isolated as a 45-kDa protein) of the B . pertussis adenylyl cyclase is necessary for calmodulin-activated catalytic activity. The alignment shown in Fig. 7 indicates that the region of the B . anthracis sequence from residue 303 to residue 688 is homologous to the region of the B . pertussis sequence from residue 15 to residue 407. It was pointed out in Section VII,B that the N-terminal portion of the B. pertussis adenylyl cyclase (residues 1-235) contains the catalytic domain while the

FIG.6. Sequence comparisons of prokaryotic and euhryotic adenylyl cyclases. The figures in each cell represent sequence comparisons of the adenylyl cyclases from the two organisms intersecting the matrix (defined as a binary comparison), The numbers in the table correspond to the percent identity in the segments compared, and to the number of amino acids in the aligned segment (in parentheses). Values in brackets denote the number of standard deviations higher than that obtained with 100 comparisons of randomized sequences of these protein segments. The shaded cells represent the 11 examples of homologies that are statistically significant. The FASTA (word size = 1) and the RDF2 programs (93)were used to assess similarity and to determine the comparison scores, respectively. The numbers in parentheses below the sources of the enzymes indicate the number of amino-acid residues in the enzyme, except in the case of S. typhimurium, where only the amino-terminal 419 residues out of a total of approximately 850 amino acids of the sequence have been published. Full genus names and references for the published sequences of the various enzymes are as follows: Rhizobium meliloti (89, Salmonella typhimuriuin (109, Escherichia coli (18), Erwinia chrysanthemi (108), Pasteurella multocida ( l o g ) , Bacillus anthracis (73), Bordetella pertussis (67), Breoibacterium liquefaciens (110), Saccharomyces cerevisiae (ill), Schizosaccharomyces ponibe (103), Bos taurus (104), and Rattus norcjegicus (112).

55

BACTERIAL ADENYLYL CYCLASES

0

2

4

6

8

10

12

14

16

18

20

Chainlength

I

I

(xl 00)

Regions of sequence homology

A1

&16

E. coli E. chrysanthemi P.rnuitocida

833

B

C1

1706

997~~26 1$ ,7 , 1692

861

I

1

447

679

B anthracis 6.pemssis

1144

1134

S. cerevisiae

s

pomae

R. norvegicus B. taurus R. meblon

FIG.7. Schematic depiction of homologous regions in four distinct groups of eukaryotic and prokaryotic adenylyl cyclases. The four depicted classes are (A) gram-negative bacteria, (B) gram-positive pathogens, (C) yeast, and (D) mammalian Rhizobiurn. [A sequence alignment for the adenylyl cyclase from Salmonella typhirnuriurn is omitted from this figure because the complete sequence has not been published (see legend to Fig. 6 ) , ]The cross-hatched areas represent the regions of sequence homology. The numbers at the left and right termini of the sequences correspond to the amino-terminal and carboxy-terminal residues, respectively. The numbers at the left and right of the cross-hatched regions represent the amino-terminal and carboxy-terminal ends of the homologous sequences. The apparent discrepancies in the number of residues shown in the homologous regions in this figure and in the data of Fig. 6 are due to the placement of gaps in the sequence alignments by the FASTA program.

region from residues 236-399 harbors the calmodulin-binding domain. The 150 amino acids at the C-terminus of the B . anthracis enzyme contain the calmodulin-binding region. These data are consistent with the idea that there is a common ancestral origin for the two catalytic and calmodulinbinding domains, in contrast to the previous suggestion (82) that there is no homology in the calmodulin-binding sites of the two enzymes. The third group (C, Fig. 7) of adenylyl cyclases corresponds to the enzymes from s. cerevisiae and Schizosaccharomyces pombe. It has previously been reported that the catalytic domains of both of these adenylyl cyclases reside in the carboxyl-terminal regions. It is therefore not surprising that the region of sequence homology of these two proteins is in the C-terminal portion. The amino-terminal regions of these proteins are presumably involved with regulatory activities, which may differ in the two proteins. It has been pointed out that the adenylyl cyclase from S. cerevisiae is a target for

56

ALAN PETERKOFSKY ET AL.

the action of the RAS protein while the adenylyl cyclase from S. pombe is not. The lack of sequence homology in the N-terminal regions of these two cyclases supports the notion that the regulation of adenylyl cyclase activity in these two organisms proceeds by different mechanisms. The last group of adenylyl cyclases (D, Fig. 7) includes the enzymes produced by rat and bovine species and by the bacterium R. meliloti. The N-terminal halves of the rat and bovine enzymes contain the regions of sequence homologies, suggesting that the catalytic centers or calmodulinbinding domains are located in those segments of the enzymes. It is noteworthy that there is a stretch of approximately 130 amino acids beginning at residue 313 of the bovine enzyme that is highly homologous to the Nterminal 130 amino acids (two-thirds of the complete sequence) of the adenylyl cyclase from R. meliloti. These observations suggest that there is an essential conserved region in these three proteins. It is noted in Section IX that the presumptive regions for ATP binding in these three proteins occur in other regions of these sequences. It is a surprising observation that the sequence of the R. meliloti enzyme is more closely related to eukaryotic enzymes than to other bacterial enzymes. The significance of this relationship remains to be established.

IX. ATP-Binding Sites Since the substrate for the adenylyl cyclase reaction is ATP, it might be surmised that a common feature of all adenylyl cyclases should be a sequence or domain that recognizes this nucleotide. X-Ray crystallographic (94) and NMR studies (95) as well as affinity-labeling experiments (96) on rabbit muscle adenylate kinase have helped to define the ATP-binding site for this enzyme. Additional studies with a variety of other enzymes that interact with ATP have led to the proposal that a typical ATP-binding domain is glycine-rich, contains a basic residue, and can undergo a conformational change upon binding ATP (95, 97-99). The typical so-called A-type ATP-binding domain can be expressed by the following consensus sequence (where slashes indicate “either/or”): (G/A)(X,)(G/A)(H/K/R)(X,--J(S/T/K/R/H) (100).This sequence is referred to as pattern 2 in Table I. Mutagenesis studies of a variety of enzymes that interact with ATP suggest that the basic residue (usually lysine) adjacent to the conserved G or A in the flexible loop sequence is important for activity. Replacement of this amino acid by a variety of others (Met, Ile, Glu, Gln, or Arg) results in changes in activity ranging from 2.5- to %fold decrease (I?. coli F,-ATPase) (101)to essentially complete loss of activity (B. pertussis or B. anthrucis adenylyl cyclase) (53, 54). We have carried out an alignment analysis of the all published adenylyl cyclase sequences (of which there are 12) as indicated in Table I and Fig. 8.

TABLE I OCCURRENCE OF CONSENSUS SEQUENCES IN

Bovine brain (type 1)

Rat olfactory tissue (type 111)

S. pombe S . cereoisiae 8 . pertussis

B . unthrucis

P. multocidu E . chrysanthemi

E . coli

S. typhimurium"

R. meliloti B . liquefuciens

VARIOUS

ADENYLYL CYCLASES

1042 GvsvKGkgemLT 1048 GKGemLT

26 AagpgGRR 202 AtlvpAKR 433 AaglpGKvH 780 AgaisGRS 989 AgvigARR

1104 GpifvKGkgeLlT

447 AggipGRvH

1110 GKGeLlT

1051 AgvigARK

1423 GsknevlyRGLS 1521 GefklKGLdT 1871 GehklKGLeT 54 GvatKGLgvhakS 825 GgidiasrKGerpaLT 826 GidiasrKGerpaLT 1165 GrggddilRGgLgldT 1167 GgddilRGgLgldT 1168 GddilRGgLgldT 1490 GrgldagaKGvfLS 1492 GldagaKGvfLS 1496 GaKGvfLS 1499 GvflslgKGfasLmdepeT 1505 GKGfasLmdepeT 174 GKGisLdiiS 342 GvatKGLnehgkS 375 GqqlaveKGnLenkkS None 51 GylegkvpHGicLfS 55 GkvpHGicLfS 82 GelsapdrKGeLpiT 51 GyldgnvpKGicLyT 55 GnvpKGicLyT 82 GmsvqdppKGeLpiT 708 GaishnklHGLS 82 GmtpqdppKGeLpiT None None

None None 26 GikavAKeK 59 GlgvhAKS 252 AvgteARR 349 AygvaGKS

347 GlnehGKS 417 GiilkGKK None 189 AvrmaGKR

190 AvrlaGKR

190 AvrlaGKR None None

~

"Pattern 1 is the proposed consensus sequence characteristic of adenylyl cyclases (see text), while pattern 2 is the consensus sequence proposed previously (100)to be a typical ATP-binding site. The numbers correspond to the first residue of the segment shown. Amino-acid residues shown in bold capital letters correspond to conserved residues of the consensus sequences, while amino-acid residues shown in lowercase letters correspond to nonconserved residues (indicated as X in the patterns). The data were generated by use of the FINDPATTERNS program (102). Wnly the first 419 amino acids of the sequence have been published.

58

ALAN PETERKOFSKY ET AL.

ENIYXE SOURCE

SEOUENCE

RamB

------- )

bovine brain (type 1)

1048-1054

G(

rat olfactory (type 111)

1104-1116

G(---pifv) K G(-kge) L(----1) T

S. pombe

1521-1530

G(---efkl) X G(----) L(----d) T

S. cerevisiae B. pertussis

1871-1880

G(---ehkl) K a(----) L(----e) T

54-66

(3 (---- vat) EG(----)

B. anthracis

342-354

G (---- vat) g G(----) L(nvhgk) 8

P. multocida

249-265

G(aslwg1y) K a(----) I(dapyk) 8

E. chrvsanthemi

82-96

G(e1sapdr) K G(---e) L(---pi) T

E. coli

82-96

G(msvgdpp) K G(---e) L(---pi) T

S . tvDhimurium

82-96

G(mtpgdpp) X G(---e) L(---pi) T

R. meliloti B. lisufaciens

161-172

G(---taka) K G(rsta) L

126-134

G(-----mv) ReG(---a) L(-----) T

CONSENSUS

X G(--em) L(-----) T

L(gvhalc) S

G X o - 7 (H/X/R)GXo-,LX,-,(S/T)

Ftc. 8. Sequence of presumptive regions for ATP binding in various adenylyl cyclases. For a discnssion of this figure, see Section IX. The underlined lysine residues of the adenylyl cyclases from B. pertussis and B. anthrocis have been shown, by site-directed mutagenesis experiments, to be essential for activity.

{However, see Addendum at the end of this section.) The results of this type of analysis have allowed us to develop an alternative consensus sequence, which is found in essentially all these enzymes [G(qP7)(H/K/R)G (q-4) L(q)-5)(S/T)].This sequence is referred to as pattern 1 in Table I. A unique feature of this consensus sequence is that a basic residue precedes a glycine residue, whereas in the A-type sequence (pattern 2) a basic residue follows a glycine residue. It may be that this inversion of conserved amino acids is due to the nature of the adenylyl cyclase reaction (cleavage of ATP between the 01 and p phosphates) compared to that of the typical kinase or ATPase reaction that involves cleavage of ATP between the p and y phosphates. It should also be noted that this consensus contains a conserved leucine (or isoleucine) residue between the second conserved glycine and the conserved serine/threonine residue. On the basis of this proposal, it would be predicted that the indicated sequences for the various adenylyl cyclases would play an important role in the enzymes’ function. As noted above, mutagenesis stud-

BACTERLAL ADENYLYL CYCLASES

59

ies on the adenylyl cyclases from B . pertussis or B . anthracis have indicated the importance of the conserved lysine in this sequence [see the two underlined lysine (K) residues in Fig. 81. A search using the FINDPATTERNS program in the GCG software package (Version 7.0) (102)was made of the 12 adenylyl cyclase sequences to determine the presence or absence of the modified consensus described here (pattern 1)or the previously defined ATP-binding consensus (100)(pattern 2; see Table I). The type-I adenylyl cyclase of bovine brain contains one region (beginning at either residue 1042 or 1048) that matches pattern 1 (Table I and Fig. 8). There are five examples of adherence to the pattern 2 consensus. Two of these sequences (beginningat residues 202 and 780) occur in transmembrane regions and two of the sequences (beginning at residues 433 and 989) occur in cytoplasmic regions (104,113). It is possible that either or both of these sequences play a role in catalysis. The adenylyl cyclase from rat olfactory tissue (type 111) contains one region (beginning at either residue 1104 or 1110) that matches pattern 1. There are two sequences (beginning at residues 447 and 1051) that fit pattern 2. Since all of these sequences occur in regions proposed to be cytoplasmic (113),any one or more of them are possible candidates for essential ATPbinding sites. It is important to note that, in both S. pombe and S . cerevisiae, there are no sequences that match pattern 2. Since the sequence in S. pombe beginning at residue 1521 is homologous to the only match to pattern 1 of s. cerevisiae (103),that sequence has been selected as the best ATP-binding site candidate of S. pombe (Fig. 8). Since the active site of the S. cerevisiae adenylyl cyclase lies in the region of residues 1609-2026, the identification of the consensus sequence from residues 1871-1880 as an ATP-binding site is reasonable. Table I shows that there are 11 sequences in B. pertussis and three in B . anthracis that match pattern 1. Since the B . pertussis sequence beginning at residue 54 is homologous to the B . anthracis sequence beginning at residue 342, these two sequences have been selected as the best candidates for essential ATP-binding sites (Fig. 8). It is noteworthy, in this regard, that both lysine residues in each of these sequences have been shown by site-directed mutagenesis studies to be essential for catalytic activity (53,54). Another reason for choosing the indicated sequences is that they fall within the catalytic domains of the proteins. The P. rnultocidu adenylyl cyclase sequence has no perfect matches to either pattern 1 or 2. The sequence from residues 249-265 has been selected as a possible ATP-binding site since it only deviates from a pattern 1 consensus by a change of the conserved leucine to an isoleucine residue (Fig. 8). The sequences of the adenylyl cyclases from E . chrysanthemi, E . coli,

60

ALAN PETERKOFSKY ET AL.

and S. typhimurium are nearly identical. The sequences beginning at residue S5 of the E . chysanthemi and E . coli adenylyl cyclases match pattern 1 and are possible ATP-binding sites, but the sequences beginning at positions 82 adhere to the consensus for all three proteins and therefore are more likely to be ATP-binding regions. In the case of pattern 2 sequences, there is one match to the consensus for all three proteins (beginning at residue 189 or 190); these sequences cannot be eliminated from consideration as ATP-binding regions sirice they all fall in the N-terminal domains of the cyclases, which are believed to harbor the catalytic site (115). The sequence of the R . rneliloti adenylyl cyclase (87) has no perfect matches to either the Chin et al. (100)consensus or the consensus proposed here [fable I). However, the sequences that begin at residues 132 (GMNKDYGTSVL) and 161 (GTAKAKGRSTAL)are variants of the consensus proposed it1 this study. In this variation, the Lys-Gly sequence (shown in bold type) may be interrupted by as many as two amino acids, and the conserved serine or threonine @old italics) precedes rather than follows the conserved leucine (bold type). In Fig. 8, the sequence beginning at residue 161 has been selected as the best candidate for an ATP-binding site. It is noteworthy that the R . meliloti enzyme is the smallest reported adenylyl cyclase (193 amino acids) and also has an unusually high K , (4mM), compared to the value of approximately 0.6 mM for the comparable E . coli lac2 fusion enzytne (87) or 0.21 mM for the wild-type enzyme (41). It is therefore possible that the unusually high Km is a reflection of the nonadherence in Rhizobirim to the ATP-binding consensus observed in other adenylyl cyclases. The B . liquefaciens adenylyl cyclase has no perfect matches to either pattern 1 or 2 (Table I). The sequence beginning at residue 126 has been selected as a good candidate for the ATP-binding site since it deviates from the pattern 1 consensus only by placement of a single amino acid (glutamic acid) between the conserved R and G (Fig. 8). Obviously, much experimental work will be necessary to evaluate these predictions of the location of ATP-binding sites in adenylyl cyclases. The question arises of whether the suggested consensus for adenylyl cyclases is restricted only to that class of enzymes. A search was made through a protein-sequence database (SwissProt) for the occurrence of the consensus sequence using the FINDPATTERNS program in the GCG package from the University of Wisconsin. Approximately 60 bacterial sequences contained the consensus sequence. Of the 60 sequences, 36 were for proteins that bind nucleotides. The search revealed that many of these proteins also contain the motif in which the basic amino acid follows a Gly or Ala residue (100). Interestingly, some of the sequences identified were for aminoacyl-tKNA synthetases (for alanine, phenylalanine, and lysine), enzymes

BACTERIAL ADENYLYL CYCLASES

61

that are similar to adenylyl cyclases in that they produce PP, from ATP. The enzyme responsible for the synthesis of ppGpp, which involves a pyrophosphoric transfer, also contains the adenylyl cyclase consensus sequence. A number of E. coli enzymes that interact with NAD, which contains a pyrophosphoric unit (e.g., NADP-specific glutamate dehydrogenase and NADH dehydrogenase), have the consensus sequence. Another enzyme found to contain the consensus sequence is guanylyl cyclase (bovine and rat), an enzyme that is quite analagous in reaction mechanism to that of adenylyl cyclase. It therefore appears that the consensus sequence found in all adenylyl cyclases sequenced thus far is not a specific signature for those enzymes, but is found as a more general sequence in a variety of enzymes that bind nucleotides as well as in numerous enzymes that have not yet been shown to interact with nucleotides. Using this consensus paradigm, it will be useful to further explore the importance of Gly, Lys (or His or Arg), Leu, and Ser (or Thr) residues for the function of various adenylyl cyclases or guanylyl cyclases. We speculate that the function of glycines is to impart flexibility, that of the lysine is to interact with one of the phosphoric groups of ATP, that of the leucine is to provide some hydrophobic interaction with the adenosine moiety of ATP, and that of the serine (or threonine) residue is to facilitate the a-phosphoric transfer to the 3’-OH of the adenosine of ATP. A recent characterization of a calmodulin-activated adenylyl cyclase from bovine brain (105) demonstrated that two domains of the protein are required for full catalytic activity. The authors suggested that adenylyl cyclases and guanylyl cyclases may require an interaction of two domains for maximal catalysis. It is likely that E. coli adenylyl cyclase requires a domain in addition to the Gly-82-Thr-96 region for activity. In support of this idea is the demonstration that an in-frame deletion of a 25-aminoacid segment corresponding to residues 118-142 of E. coli adenylyl cyclase produces a protein that is catalytically inactive (106).The Trp-118-Asn-142 region has a helical structure (predicted by a Chou-Fasman analysis performed using the PEPTIDESTRUCTURE program in the University of Wisconsin GCG analysis package) with three repeated leucines spaced seven residues apart, reminiscent of a leucine-zipper motif, It might be speculated that the Gly-82-Thr-96 domain interacts with the Trp-118-Asn-142 domain to form a complete catalytic unit.

Addendum Since these data were assembled, the sequences of two additional eukaryotic adenylyl cyclases (the rat-brain, type-11, calmodulin-insensitive enzyme and the rat-testis, type-IV, calmodulin-insensitive enzyme) have been published (113, 114). Examination of the two new sequences indicates that

62

ALAN PETERKOFSKY ET

AL.

they both contain regions in their C-terminal cytoplasmic domains that adhere to the characteristic adenylyl cyclase consensus proposed in this section. The relevant sequences in the type-I1 enzyme (114)include residues 1060- 1072 (GiinvKGkgdLkT) (conserved residues in bold capital letters). The comparable sequences in the type-IV adenylyl cyclase (113) includes residues 1036- 1048 (GvikvKGkgqLcT). These data provide compelling support for the importance of the consensus sequence [ G(&-7)(H/K/R)G(X,--4)L(q-,)(S/T)] or a slight modification of it for the catalytic activity of adenylyl cyclases. As more adenylyl cyclase sequences become available, it may be useful to modify the proposed sequence motif. A more general consensus that accommodates all the available sequence data would be G(&-7)(H/KW(&- ~ ) G ( ~ - ~ ) ( L / I ) ( & - ~ ) ( S / T ) .

X. Conclusions Adenylyl cyclases are responsible for the synthesis of an important small regulatory factor. Since variations of the cAMP level at the targets have profound effects on cell metabolism, it is of utmost importance to strictly control the activities of the adenylyl cyclases. In the case of E . coZi, the mechanism for regulating the enzyme activity involves a phosphorylationdephosphorylation of PTS proteins. The state of phosphorylation of these proteins is determined by the relative concentrations of the substrates phosphoenolpyruvate and sugar. There are, in addition, several other factors, of which P, is noteworthy, that exert an important influence on the activity of E. coli adenylyl cyclase. The adenylyl cyclases produced by the invasive B. anthracis or B . pertussis are relatively inactive outside of the eukaryotic host cells. After entry, the host contributes the activators calcium and calmodulin, resulting in a profound increase in adenylyl cyclase activity. The net result is that toxic levels of cAMP accumulate. Numerous genes for adenylyl cyclases have now been cloned and sequenced. Studies have been initiated in a number of laboratories to delineate structure-function relations of some of these proteins. The popular approach being utilized takes advantage of the techniques of site-directed mutagenesis of the structural genes for the enzyme under study. It is anticipated that, over the next few years, an enormous insight into the manner in which adenylyl cyclases interact with substrates and modulators will be achieved.

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63

3. J. F. Habener, Mol. Endocrinol. 4, 1087 (1990). 4 . B. de Crombrugghe, S. Busby and H. Buc, Science 224, 831 (1984). 5a. J. G. Harman, K. McKenney and A. Peterkofsky, JBC 261, 16332 (1986). 5b. D. M. J. Lilley, Nature 354, 359 (1991). 6 . A. Peterkofsky, Ado. Cyclic Nucleotide Res. 7, 1(1976). 7 . A. Peterkofsky, I. Svenson and N. Amin, FEMS Microbiol. Reu. 63, 103 (1989). 8. M. H. Saier, Jr., B. U. Feucht and M. T. McCaman, JBC 250, 7593 (1975). 9 . A.D.E. Fraser and H. Yamazaki, Can. J. Biochem. 57, 1073 (1979). 10. P. E. Goldenbaum and G. A. Hal1,J. Bact. 140,459 (1979). 11. K. Potter, G . Chaloner-Larsson and H. Yamazaki, BBRC 57, 379 (1974). 12. A.D.E. Fraser and H. Yamazaki, Can. J . Microbiol. 24, 1423 (1978). 13. P. Reddy, A. Peterkofsky and K. McKenney, NARes 17, 10473 (1989). 14. H. Aiba, M. Kawamukai and A. Ishihama, NARes 11, 3451 (1983). 15. A. Roy and A. Danchin, MGG 188, 465 (1982). 16. H. Aiba, JBC 260, 3063 (1985). 17. J. P. Fandl, L. K. Thorner and S. W. Artz, Genetics 125, 719 (1990). 18. H. Aiba, K. Mori, M. Tanaka, T. Ooi, A. Roy and A. Danchin, NARes 12, 9427 (1984). 19. J. K. Yang and W. Epstein, JBC 258, 3750 (1983). 20. P. Reddy, A. Peterkofsky and K. McKenney, PNAS 82, 5656 (1985). 21. A. Peterkofsky, J. Hawood and C. Gazdar, J . Cyclic Nucleotkh Res. 1, 11(1975). 22. N. D. Meadow, D. K. Fox and S. Roseman, ARB 59, 497 (1990). 23. H. De Reuse, E. Huttner and A. Danchin, Gene 32, 31 (1984). 24. H. De Reuse and A. Danchin, J. B a d . 173, 727 (1991). 25. D. W. SafFen, K. A. Presper, T. L. Doering and S. Roseman, JBC 262, 16241 (1987). 26. H. De Reuse and A. Danchin, J. Bact. 170, 3827 (1988). 27. P. Reddy, N. Fredd-Kuldell, E. Liberman and A. Peterkofsky, Protein Express. Purijcat. 2, 179 (1991). 28. B. Tyler and B. Magasanik, J . Bact. 102, 411 (1970). 29. J. P. Harwood and A. Peterkofsky, JBC 250, 4656 (1975). 30. J. P. Harwood, C. Gazdar, C. Prasad, A. Peterkofsky, S. J. Curtis and W. Epstein, JBC 251, 2462 (1976). 31. A. Peterkofsky, Trends Biosci. 2, 12 (1977). 32. A Peterkofsky and C. Gazdar, J . Supramolec. Struct. 9, 219 (1978). 33. M. H. Saier, J . and S. Roseman, JBC 251, 6598 (1976). 34. S. 0. Nelson, B. J. Scholte and P. W. Postma, J . Bact. 150, 604 (1982). 35. B. U. Feucht and M. H. Saier, Jr., J . Bact. 141, 603 (1980). 36. P. Reddy, N. Meadow, S. Roseman and A. Peterkofsky, PNAS 82, 8300 (1985). 37. S. Lkvy, G . Zeng and A. Danchin, Gene 86, 27 (1990). 38. H. R. Kaback, JBC 243, 3711 (1968). 39. M. H. Saier, Jr., D. F. Cox, B. U. Feucht and M. J. Novotny, J. Cell. Biochem. 18, 231 (1982). 40. B. K. Ghosh, K. Owens, R. Pietri and A. Peterkofsky, PNAS 86, 849 (1989). 41. E. Liberman, P. Reddy, C. Gazdar and A. Peterkofsky, JBC 260, 4075 (1985). 42. H. Rosenberg, L. M. Russell, P. A. JacombandK. Chegwidden,J. Bact. 149, 123(1982). 43. K. Ugurbil, H. Rottenberg, P. Glynn and R. G. Shulman, PNAS 75, 2244 (1978). 44. P. Reddy, E. Liberman, C. Gazdar and A. Peterkofsky, Factors Regulating the Activity of Escherichia coli Adenylate Cyclase. In “Gene Manipulation and Expression” (R. E. Class and J. Spizek, eds.), pp. 318-338. Croom Helm, Kent, United Kingdom, 1985. 45. P. Reddy, D. Miller and A. Peterkofsky, JBC 261, 11448 (1986). 46. G . R. Jacobson and J. P. Rosenbusch, Nature 261, 23 (1976).

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47. M. Crasnier and A. Danchin, J . Gen. Microbiol. 136, 1825 (1990). 48. S. Razin, Microbiol. Rev. 49, 419 (1985). 49. A. Muto, H. Hori, M. Sawada, Y. Kawauchi, M. Iwami, F. Yamao and S. Osawa, Yale]. B i d . Med. 56, 373 (1983). 5Q. M. Miyata, L. Wang and T. Fukumura. FEMS Microbwl. Lett. 79, 329 (1991). 51. C. C . Dascher, S. K. Poddar and J. Maniloff, J . Bact. 172, 1823 (1990). 52. A . Muto, lsrael J . Med. Sci. 23, 334 (1987). 53. U. Mngharbil and V. P. Cirillo, J . Bact. 133, 203 (1978). 54. E. Hanski, Trends Biosci. 14, 459 (1989). 55. M. G. Donovan and D. R. Storm, J . Cell. Physiul. 145, 444 (1990). 56. V. M. Gordon, W. W. Young, Jr., S. M. Lechler, M. C. Gray, S. H. Leppla and E. L. Hewlett, JBC 264, 14792 (1989). 57. F. Gentile, L. G. Knipling, D. L. Sackett and J. WoM, JBC 265, 10686 (1990). 58. M . Mouallem, Z. Farfel and E. Hanski, Infect. Zmmun. 58, 3759 (1990). 59. M. G . Donovan, H. R. Masure and D. R. Storm, Bchem 28, 8124 (1989). 60. A. Cilboa-Ron, A. Rogel and E. Hanski, BJ 262, 25 (1989). 61. J. Bellalou, D. Ladant and H. Sakamoto, Infect. Immun. 58, 1195 (1990). 62. H. R. Masure and D. R. Storm, Bchem 28, 438 (1989). 63. R. M. Laoide and A. Ullmann, EMBOJ. 9, 999 (1990). 64. P. Glaser, A. Uanchin, D. Ladant, 0. BBrzu and A. Ullmann, Tokai J . Exp. Clin. Med. 13 (Suppl.), ,239 (1988). 65. P. Glaser, H. Sakamoto, J. Bellalou, A. Ullmann and A. Danchin, EMBO J . 7,3997 (1988). 66. J. Bellalou. H. Sakamoto. D. Ladant, C. Geoffroy and A. Ullmann, Infect. Immun. 58, 3242 (1990). 67. P. Glaser, D. Ladant, 0. Sezer, F. Pichot, A. Ullmann and A. Danchin, Mol. Microbiol. 2, 19 (1988). 68. M. Mock, E. Labruyere, P. Glaser, A. Danchin and A. Ullmann, Gene 64, 277 (1988). 69. D. Ladant, S. Miclielson, R. Sarfati, A. M. Gilles, R. Predeleanuando. BBrzu,JBC264, 4015 (1989). 70. A.-M. Gilles, H. Munier, T. Rose, P. Glaser, E. Krin, A. Danchin, C. Pellecuer and 0. BPrzu, Bchem 29, 8126 (1990). 71. A . Monneron, D. Ladant, J. dAlayer, J. Bellalou, 0. BBrzu and A. Ullmann, Bchem 27, 536 (1988). 72. P. Glaser, A . Elmaoglou-Lazaridou, E. Krin, D. Ladant, 0. Birzu and A. Danchin, EMBO J . 8, 967 (1989). 73. D. L. Robertson, M. T. Tippetts and S. H. Leppla, Gene 73, 363 (1988). 74. V. Escuyer, E. Ihflot, 0. Sezer, A. Danchin and M. Mock, Gene 71, 293 (1988). 75. E. Labruyere, M. Mock, D. Ladant, S . Michelson, A. M. Gilles, B. LaoideandO. BPrzu, Bchem 29, 4922 (1990). 76. D. L,. Robertson, BBRC 157, 1027 (1988). 77. R. S. Sarfati, V. K. Kansal, H. Munier, P. Glaser, A.-M. Gilles, E. Labmyere, M. Mock, A. Danchin and 0. BBrzu, JBC 265, 18902 (1990). 78. 13. C. Au, H. R. Masure and D. R. Storm, Bchem 28, 2772 (1989). 79. P. Glaser, H. Munier, A. M.Gilles, E. Krin, T. Porumb, 0. Bkzu, R. Sarfati, C. Pellecuer and A. Danchin, EMBO J . 10, 1683 (1991). HO. E. Labruyere, M. Mock, W. K. Surewicz, H. H. Mantsch, T. Rose, H. Munier, R. S. Sarfati and 0. Bdrzu, Bchem 30, 2619 (1991). 81. Z. Xia and D. R. Storm, JBC 265, 6517 (1990). 82. R. G . Upchurch and G. H. Elkan, BBA 538, 244 (1978). 8.3. A. M. McGetrick, C. F. Goulding, S. S. Manian and F. O'Gara,]. Bact. 163, 1282 (1985).

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84. S. T. Lim and K. T.Shanmugam, BBA 584, 479 (1979). 85. B. Kiely and F. O’Gara, MGG 192, 230 (1983). 86. R. Lathigra, M. O’Regan, B. Kiely, B. Boesten and F. O’Gara, Gene 44, 89 (1986). 87. A. Beuve, B. Boesten, M. Crasnier, A. Danchin and F. O’Gara,J. Bact. 172,2614 (1990). 88. M. O’Regan, B. Kiely and F. O’Gara, Gene 83, 243 (1989). 89. M. L. Guerinot and B. K. Chelm, J. %act. 159, 1068 (1984). 90. M. J.D. San Francisco and G. R. Jacobson, FEMS Microbiol. Lett. 35, 71 (1986). 91. C. A. Catanese, D. W. Emerich and W. L. Zahler, J. Bact. 171, 4531 (1989). 92. A. R. Glenn, R. Arwas, I. A. McKay and M. J. Dilworth, J. Gen. Microbiol. 130, 231 (1984). 93. W. R. Pearson and D. J. Lipman, PNAS 85, 2444 (1988): 94. G. E. Schulz, M. Elzinga, F. Marx and R. H. Schirmer, Nature 250, 120 (1974). 95. D. C. Fry, S. A. Kuby and A. S. Mildvan, Bchem 24, 4680 (1985). 96. T. Yagami, M. Tagaya and T. Fukui, FEBS Lett. 229, 261 (1988). 97. D. Dreusicke and G. E. Schulz, FEBS Lett. 208, 301 (1986). 98. M. Tagaya, T. Yagami, T. Noumi, M. Futai, F. Kishi, A. Nakazawa and T. Fukui, JBC 264, 990 (1989). 99. T. M. Duncan, D. Parsonage and A. E. Senior, FEBS Lett. 208, l(1986). 100. D. T. Chin, S. A. Goff, T.Webster, T. Smith and A. L. Goldberg, JBC 263, 11718 (1988). 101. R. Rao, J. Pagan and A. E. Senior, JBC 263, 15957 (1988). 102. J. Devereux, P. Haeberli and 0. Smithies, NARes 12, 387 (1984). 103. D. Young, M. Riggs, J. Field, A. Vojtek, D. Broek and M. Wigler, PNAS 86,7989 (1989). 104. J. Krupinski, F. Coussen, H. A. Bakalyar, W.-J. Tang, P. G. Feinstein, K. Orth, C. Slaughter, R. R. Reed and A. G. Gilman, Science 244, 1558 (1989). 105. W.-J. Tang, J. Krupinski and A. G. Gilman, JBC 266, 8595 (1991). 106. S. Shah and A. Peterkofsky, J . Bact. 173, 3238 (1991). 107. M. M. Holland, T. K. Leib and J. A. Gerlt, JBC 263, 14661 (1988). 108. A. Danchin and G. Lenzen, Second Messengers and Phosphoproteins 12, 7 (1988). 109. M. Mock, M. Crasnier, E. Duflot, V. Dumay and A. Danchin, J . Bact. 173, 6265 (1991). 110. E. P. Peters, A. F. Wilderspin, S. P. Wood, M. J. J.M. Zvelebil, 0. Sezer and A. Danchin, Mol. Microbial. 5, 1175 (1991). 1 1 1 . T. Kataoka, D. Broek and M. Wigler, Cell 43, 493 (1985). 112. H. A. Bakalyar and R. R. Reed, Science 250, 1403 (1990). 113. B. Gao and A. G. Gilman, PNAS 88, 10178 (1991). 114. P. G. Feinstein, K. A. Schrader, H. A. Bakalyar, W. Tang, J. Krupinski, A. G. Gilmanand R. R. Reed, PNAS 88, 10173 (1991). 115. A. Roy, A. Danchin, E. Joseph and A. Ullmann, JMB 165, 197 (1983).

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hitiation of Transcription by RNA Polymerase II: A Multi-step Process LEIGHZAWEL AND DANNYREINBERG' Department of Biochemistry Robert WoodIohnson Medical School University of Medicine and Dentistry of New Iersey Piscataway, New Iersey 08854

I. The Structure of Class I1 Promoters ............................... 11. RNA Polymerase I1 . . . . . . . A. The C-terminal Domain o B. Carboxy-terminal Domain Kinases ............................. 111. Transcription Factors and Systems ................................. A. Transcription Factor IID ..................... B. Transcription Factor IIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Transcription Factor IIF D. Transcription Factor IIE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Transcription Factor IIH ............................. F. Transcription Factor IIA ...................................... G. Other Transcription Systems . . . . . IV. Preinitiation and Initiation Complexes and Motifs . . . . . . . . . . . . . . . . . . . A. Complexes on TATA-containing Promoters ....................... B. Complexes on TATA-less Promoters ............................ C. Insights Regarding Initiator-mediated Initiation D. Cooperation between TATA and Initiator Motifs . . . . . . . . . . . . . . . . . . V. Activation and the General Transcription Factors .................... VI. Repression of Class I1 Gene Transcription .......................... References .....................................................

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The last decade has witnessed an explosion of information pertaining to gene regulation in higher eukaryotes. This has led to discoveries in gene splicing, the development of systems that can mimic specific initiation of transcription in uitro, and the discovery that some oncogenes can directly affect transcription of specific genes, among others. This review focuses primarily on studies, some completed but most still in progress, on the initiation of transcription of protein-coding genes. In the 1960s and 1970s, we learned a great deal about transcription in prokaryotes. Studies revealed the presence of an activity in bacterial cells 1To whom correspondence may be addressed. 67 Progress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright Q 1993 by Academic Press. Iuc. All rights of reproduction in any form reserved.

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that could, in a DNA-dependent fashion, catalyze the synthesis of all RNA molecules. This is in contrast to eukaryotic cells, which contain three distinct RNA polymerases, each containing from 8 to 14 polypeptides and responsible for transcribing its own set of genes: RNA polymerase I (RNAPI), which transcribes ribosomal RNA; RNAPII, the RNA polymerase of protein-coding, or class 11, genes; and RNAPIII, which transcribes 5-S rRNA and tKNA genes. In keeping with the theme of this review, only RNAPII is discussed here, and only as it pertains to transcription initiation. [For a more comprehensive overview of RNAPII structure and function, see Young (1). For RNAPII-catalyzed elongation, see Spencer and Groudine (2) and Kerppola and Kane (2a).] Bacterial sigma (cr) factors, which are integral components of bacterial RNAP holoenzyme, are essential to the enzyme’s function. Sigma factors recognize promoter sequences and position the core component of RNA polymerase at transcriptional start-sites. Following start-site selection and initiation, sigma factors dissociate from the core enzyme, which then proceeds to catalyze RNA synthesis. In contrast to bacterial RNAP, mammalian RNAPII cannot specifically2 initiate transcription on its own. In order to fimction efficiently, mammalian RNAPII requires a set of auxiliary factors, otherwise known as “general transcription factors” (GTFs), present in crude cell extracts (3, 4). The last decade has seen substantial progress in the purification and characterization of the GTFs such that present day in vitro transcription systems are reconstituted with highly purified components. To date, seven GTFs have been identified and extensively purified, and four have been cloned (IID*, IIA, IIB*, IIF*, IIE*, IIH, and IIJ, where the asterisk denotes factors whose cDNAs have been cloned).

I. The Structure of Class II Promoters The transcription research field blossomed some 13 years ago when transcription processes were first duplicated in a cell-free system reconstituted with crude cellular extracts (3).In this work and in subsequently developed systems, it became clear that accurate transcription by RNA polymerase I1 is entirely dependent on the presence of promoter-containing eukaryotic DNA (3, 4). It has since become clear that sequences of the DNA template act as signals that direct transcription factors and RNA polymerase to the initiation ‘Abbreviations: RNAP, RNA polymerase; TF, transcription factor; CTF, General transcription factor; Inr, initiator; CTD, carboxy-terminal domain; Ad-MLP, adenovirus major late promoter; TBP, TATA binding protein; TAF, TBP-associated factor; RAP, RNAP-associated protein. %Specific, or accurate, initiation refers to transcription initiation that occurs at discrete start-sites in the promoter region, the same sites utilized in uiuo. If only purified RNAPII is incubated with promoter containing DNA under in oitro transcription conditions, transcripts are svnthesized nonspecifically at spurious locations along the template.

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site. Four classes of cis elements have been identified in the promoters of class two genes. The TATA box and the initiator (Inr) region constitute the first two of these classes and are considered minimal promoter elements. One or both of them appear to be present in all protein-coding genes, and each is independently capable of directing the formation of a transcriptioncompetent complex. RNA polymerase I1 and the general transcription factors, collectively known as the basic transcription machinery, function through these minimal promoter elements. The other two classes, consensus sequence elements (which are recognized and bound by specific DNA binding proteins) and enhancers, are considered variable elements, because their presence or absence and the particular order of arrangement in which they occur are gene-specific. It is the combination of all these cis elements that gives a promoter its characteristic strength.

II. RNA Polymerase II The RNAPII of the yeast Saccharomyces cerevisiae has been a useful prototype in the study of eukaryotic RNAPII, as features such as hnction and subunit structure have been highly conserved. The yeast RNAPII is composed of 11 polypeptides with apparent masses ranging from 220 to 10 kDa. HeLa cell RNAPII contains 10 subunits ranging from 240 to 10 kDa (Fig. 1).The genes encoding all 11 yeast subunits have been cloned and shown to be essential for wild-type growth. Unlike the bacterial core enzyme, RNA polymerase I1 activity has not been reconstituted from purified subunits; it is thus unclear whether all of the polypeptides are genuine subunits as opposed to associated factors. An interesting finding that shed light on the possible function of the two largest RNAPII subunits is that significant amino-acid sequence homology exists among the two largest subunits of yeast and Drosophila RNAPII, yeast RNAPI and RNAPIII, and the p and p' subunits of Escherichia coli RNAP. These subunits are thought to be involved in DNA and nucleotide binding (5, 6). Biochemical and genetic (7, 8) experiments have implicated the yeast 32and 16.9kDa subunits as components important to promoter recognition. Mutant forms of RNAPII devoid of these two subunits behave like wild-type forms with respect to promoter-independent initiation, chain elongation, and recognition of pause sites. However, these mutants are inactive with respect to promoter-directed initiation in vitro.

A. The C-terminal Domain of RNA Polymerase II The largest subunit of eukaryotic RNAPII contains an unusual C-terminal domain (CTD) consisting of multiple repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Such a domain is not present in prokaryotic

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FIG. 1. Polypeptide composition of phosphorylated (110) and nonphosphorylated (IIA) forms of human RNAPII. Silver staining ofa 5-17% SDS-polyacrylamide gel on which purified human RNAPII was electrophoresed, showing the polypeptide composition of (A) the 110 and (B) the IIA forms of RNAPII. Migration of molecular-weight protein standards are indicated to the left of A and to the right of B. Subunit composition of RNAPII is indicated to the right of A and to the left of B.

RNAP \ r in eukaryotic RNAPI and RNAPIII. The length of the repeat appears to correlate with the genomic complexity of the organism. For example, the heptapeptide sequence is repeated, with some degeneracy, 26-27 times in yeast, 42-44 times in Drosophih, and 52 times in mouse and man (Fig. 2). Owing to a high content of serine and threonine residues in the

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muse Drosophila Yeast 1 YSPTSPA FCVSSPG YSPTSPN 2 YEPRSPGG YTASSPG FSPT SPT 3 YTPQSPS YSPTSPA GASPN 4 YSPTSPS YSPTSPS YSPSSPN 5 YSPTSPS YSPTSPS YSPTSPL 6 YSPTSPN YSPTSPS YA SPR 7 YSPTSPS YSPTSPS YASTTPN 8 YSPTSPS YSPTSPS FNPNSTG 9 YSPTSPS YSPTSPS YSPSSSG 10 YSPTSPS YSPTSPS YSPTSPV 11 YSPTSPS YSPTSPS YSPTVQ 12 YSPTSPS YSPTSPS FGSSPS 13 YSPTSPS YSPTSPS FAGSCSN I 14 YSPTSPS YSPTSPS YSPGN A 1 5 YSPTSPS YSPTSPS YSPSSSN 16 YSPTSPS YSPTSPS YSPNSPS 1 7 YSPTSPS YSPTSPS YSPTSPS 18 YSPTSPS YSPTSPA YSPSSPS 19 YSPTSPS YSPTSPS YSPTSPC 2 0 YSPTSPS YSPTSPS YSPTSPS 2 1 YSPTSPS YSPTSPS YSPTSPN 2 2 YSPTSPN YSPTSPS YTPVTPS 2 3 YSPTSPN YSPTSPN YSPTSPN 24 YTPTSPS YSPTSPS YS ASPQ 2 5 YSPTSPS YSPTSPG YSPASPA 2 6 YSPTSPN YSPGSPA YSQTGVK 2 7 YTPTSPN YSPKQOEQKHNENENSR YSPTSPT 2 8 YSPTSPS YSPPSPSDG 2 9 YSPTSPS YSPGSPQ 3 0 YSPTSPS YTPGSPQ 3 1 YSPSSPR YSPASPK 32 YTPQSPT YSPTSPL 3 3 YTPSSPS YSPSSPQ 34 YSPSSPS HSPS SQ 35 YSPTSPK YSPTGST 3 6 YTPTSPS YSPTSPR 3 1 YSPSSPE YSPNClsI 3 8 YTPASPK YSPSSTK 3 9 YSPTSPK YSPTSPT 4 0 YSPTSPK YTPTARN 4 1 YSPTSPT YSPTSPM 4 2 YSPRPK YSPTAPSH 4 3 YSPTSPT YSPTSPA 4 4 YSPTSPV YSPSSPTFEESED 4 5 YTPTSPK 4 6 YSPTSPT 47 YSPTSPU 18 YSPTSPT 4 9 Y SPTSPKGST 50 YSPTSPG 5 1 YSPTSPT 52 YSLTSPAISPDDSDEEN

FIG.2. The C-terminal domain of RNAPII contains a heptapeptide repeat conserved through evolution. Sequences of the heptapeptide repeats from the CTD of yeast, mouse, and Drosophih RNAPII are indicated. Note the limited degeneracy in primary amino-acid sequence and the divergence with respect to the total number of repeats observed in different species. [Adapted from Young (I).]

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heptapeptide, the CTD can be highly phosphorylated (phosphotyrosine has not been detected). As a result of this phosphorylation, the largest subunit of RNAPII, which contains the CTD, can be resolved into two major forms on SDS-polyacrylamide gels. The form called 110 has the lowest mobility at 240 kDa (Fig. 1A) and contains the CTD in its most highly phosphorylated state. The IIA form (Fig. 1B) is 215 kDa, unphosphorylated, and probably a primary translation product in oiuo. A third form, IIB, is 180 kDa and lacks most or all of the CTD. From IIB is observed only in oitro and is thought to be a proteolytic artifact of purification. WHATIs

THE

FUNCTION OF THE C-TERMINAL DOMAIN?

Deletion mutants that result in the loss of more than half of the repeats in mouse, Drosophila, and yeast are lethal, suggesting that this domain has an essential role in uioo (Ba, 9, 9a, 9b). Although the heptapeptide repeats are highly conserved among eukaryotes, significant deviations from the consensus sequence and in the overall length of the CTD occur in Drosophila, yeast, and hamster. To assess the significance of these species-specific differences, Allison et al. (9) made yeast RNAPII fusions by replacing the yeast CTD with that from Drosophila and hamster. The hamster fusion-containing yeasts were viable, whereas the Drosophila fusion mutations were lethal. Interesting, the Drosophila CTD, though it has 44 repeats, is much more degenerate in sequence than the hamster or the yeast. The hamster CTD bears 52 repeats, which are more homologous to the yeast CTD and may be functionally redundant. It was postulated that these repeats may provide sites for protein-protein contacts regulating transcription. One possible function of the CTD may be to mediate transcription activation by upstream regulators. Saccharomyces cerevisiue strains harboring a wild-type CTD (26 heptatpeptide repeats), a CTD with 13 repeats, or a CTD with 38 repeats are equally capable of mediating activation by the strong acidic activator Gal4 (10).However, when a battery of Gal4 deletion mutants containing activating region deletions were assayed for transcription activation, in all cases, the shorter CTD suppressed activation whereas the longer CTD enhanced activation relative to the wild-type CTD. Thus a longer CTD seems to complement Gal4 deletion mutations. It was postulated that the heptapeptide repeat is a functionally redundant motif that can interact with the activation domains of some regulatory proteins. In this manner, RNAPII could be recruited to promoters where further contacts between RNAPII and the GTFs position the enzyme, defining transcriptional start sites. Though from the data it is unclear whether such CTDIactivator contacts are direct or indirect, the model is plausible and warrants further analyses. In collaboration with Y.Aloni (The Wiezmann Institute), we have demonstrated

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that the CTD specifically interacts with TFIID (IOU), a finding that strengthens this particular model. Alternatively, others have shown that the CTD is unnecessary for Spl-(II) and MLTF-mediated (12) activation in uitro. Perhaps the CTD mediates stimulation of only a subset of activators, or only in the context of a subset of specific promoters. Interestingly, the IIB form of RNAPII, which contains no CTD, can accurately transcribe the actin 5c and Ad-ML promgters in uitro (9b, 13). A requirement for the CTD in uitro is observed with the murine DHFR promoter, a TATA-less promoter (14). While these observations may suggest a requirement for the CTD with TATA-less promoters, preliminary data from our laboratory and from P. Farnham et at. (Unpublished) indicate that the requirement for the CTD in basal transcription may be determined by the promoter utilized, and more specifically, by the class of initiator motif present therein. Another viewpoint is that the CTD may act to remove, or to overcome the effects of, factors that negatively regulate transcription. This is suggested by the observation that wild-type viability is restored to yeast strains containing CTD truncations upon deletion of the repressor, SIN1 (144. There is physical evidence suggesting that the CTD can intercalate into DNA nonspecifically (15).Such an activity could strengthen RNAPII binding and/or initiation complex stability. Perhaps CTD phosphorylation releases the CTD/DNA interaction, thereby facilitating the transition from initiation to elongation (16). To investigate the functional significance of CTD phosphorylation, Dahmus and Laybourn (17) followed changes in the CTD phosphorylation state through the transcription cycle. They found that (1)the nonphosphorylated IIA form stably associates with the preinitiation complex, (2) the I10 form can be isolated from actively elongating complexes, and (3) the conversion of RNAPIIA to RNAPIIO occurs prior to the formation of the first phosphodiester bond. A model was presented in which the unphosphorylated polymerase preferentially associates with the promoter-bound initiation complex, and the subsequent phosphorylation of the CTD potentiates the transition from transcription initiation to elongation (Fig. 3). Several additional lines of evidence further support this model. (1) Crosslinking experiments demonstrated that the RNAPII, which was actively elongating, was in the I10 form (18, 19). (2) Antibodies with a preference for IIA preferentially inhibit initiation over elongation (20, 21). (3) Lu et al. (22) have recently developed a method to purify the RNAPIIA and RNAPIIO forms to apparent homogeneity from HeLa cells. Using pure forms of the enzyme, it was shown by gel shift assay that, in agreement with Dahmus’ model, the IIA form preferentially associates with the assembling initiation complex. Furthermore, the efficiency with which the I10 form of the polymerase associ-

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Elonaation:

DNA GTFs

CTD Kinase(s)

/

Phosphoprotein

FIG.3. Phosphorylation of RNAPII modulates the transition from transcription initiation to elongation. The IIA form of RNAPII associates with the assembling preinitiation complex. One or more protein kinases trigger the elongation phase by phosphorylating the CTD. The action of a phosphatase would reset the cycle. [Adapted from Buratowski and Sharp (12).]

ates with the complex can be increased substantially by subjecting the I10 polymerase to phosphatase treatment.

B. Carboxy-terminal Domain Kinases A great deal of work has gone into identifying the cellular protein kinase(s) that modify the CTD. Dahmus and Laybourn proposed that the CTD kinase may be one of the general transcription factors present in the preinitiation complex (17). TFIIH was recently found to contain a specific CTD kinase activity drastically stimulated by factors that direct the polymerase and TFIIH to the promoter (224. Dynan and co-workers have isolated a kinase with specificity for the CTD that depends on DNA for phosphate transfer for catalysis (23). The TFIIH kinase and the kinase reported by Dynan can be distinguished by their DNA requirements. Whereas the TFIIH kinase is stimulated only by DNA elements that can direct the formation of a transcription complex, the other is nonspecific in this respect. Corden and Cisek (24) purified a murine kinase as a heterodimer of 58and 34-kDa moieties that had specificity for the CTD. Interestingly, the smaller subunit of the heterodimer has been identified as the cdc2 gene product, a protein involved in cell-cycle control and previously shown to be a component of the M-phase-promoting factor (MPF), an M-phase-specific histone H1 kinase. This protein is a homologue of the Saccharmyces pombe c&2-encoded protein and the S. cerevkiae cdc28-encoded protein, both of which are known to be cell-cycle regulators (25). It will be of interest to determine if CTD phosphorylation changes with the cell cycle. A yeast kinase with specificity for the CTD, purified to near homogeneity (26), contained three subunits of 58, 38, and 32 kDa. Since none of these

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subunits cross-reacts with anti-cdc2 antibodies, this kinase is immunologically distinct from MPF kinase. The gene encoding the largest subunit, CTK1, was cloned and found to contain a protein kinase catalytic subunit motif(27). CTK1- mutants are viable but grow at reduced rates, indicating that this particular kinase is not essential. Interestingly, the RNAPII isolated from extracts of CTK1- cells was phosphorylated but not to the extent observed in wild-type cells. Thus, at least two CTD kinases are thought to exist in S. cereuisiae. One is the CTKl kinase, which phosphorylates RNAPII, converting RNAPIIA to RNAPIIO. The other kinase phosphorylates the yeast CTD, but not to the extent that the RNAPIIA form is converted to the RNAPIIO form. A similar situation has been observed in Aspergillus niduluns, where at least two CTD kinases have been described (27a). Kinase I, a serine kinase contained in a single polypeptide of 57 kDa, can phosphorylate RNAPII in solution, but cannot convert IIA to I10 unless it is contained within a preinitiation complex. Kinase 111, on the other hand, preferentially phosphorylates RNAPII in solution and cannot phosphorylate RNAPII when it is associated with the preinitiation complex. By converting free RNAPIIA to RNAPIIO, kinase I11 essentially limits the pool of RNAPII that can effectively enter into the preinitiation complex. Thus, in Aspergillus, the function of kinase 111 may be to regulate overall levels of class-I1 gene transcription. We have identified two CTD kinases from HeLa cell extracts (22). Both kinases behave similarly to Aspergillus kinase I with respect to their preference for complex-associated RNAPII. One of the kinases was identified as the MPF kinase. The other kinase activity is TFIIH.

I11. Transcription Factors and Systems As discussed in the introduction, eukaryotic RNAPII is unable to catalyze accurate transcription without the assistance of auxiliary factors. To date, seven required activities have been characterized. They are referred to as general transcription factors (GTFs) and recent years have witnessed much progress in the purification of these factors from HeLa cells (Fig. 4).Of these seven factors, only one, TFIID, contains a DNA binding activity with specificity for the TATA box. The other factors and RNAPII enter into the transcription cycle by protein-protein interactions.

A. Transcription Factor IID TFIID was first identified as an activity in Drosopkilu nuclear extracts that can specifically bind TATA-containing promoter regions of class I1 genes (28).Though this observation was made in 1984, further characterization of

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FIG.4. Scheme of the purification of the human class41 general transcription factors. [For details, see text and Flores et al. (51).]

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TFIID has been slow, due to difficulties encountered in purifying the activity. However, using a partially purified factor, it was demonstrated that TFIID is the first factor to enter into the transcription cycle; it provides a foundation for the association of the other GTFs and RNAPII (28u-28c). A breakthrough was the discovery in 1988 that an activity in yeast can functionally replace human TFIID in a reconstituted system and support basal transcription levels (29, 30). The yeast protein has an apparent mass of 27 kDa, and, much like the human factor, exhibits DNA binding activity specific for the TATA box. Yeast TFIID protects a 20-nucleotide region centered around the TATA motif from DNase-I cleavage. The cDNA encoding yeast TFIID was cloned by several groups 1year later using reverse genetics (31-34). This represented the first GTF to be characterized at the sequence level. Significantly, the gene encoding yeast TFIID had been described previously as the SPTIS gene, a Tyl mutation suppressor, the absence of which results in cell death (35, 36). Not long after the yeast sequence was determined, cDNAs encoding Arubidopsis, Drosophila, and human TFIIDs were cloned by homology using degenerate primers and PCR (37-41). For a long time it was thought, due to the degeneracy of the TATA motif and the fact that specific responses were mediated through only certain TATA sequences (41u, 41b), that there are multiple TFIID genes encoding proteins whose binding specificities varied with different TATA sequences. With the exception of Arubidopsis, where two genes encoding TFIID were found (41), only one TFIID gene appears to exist in most eukaryotes. Thus, it seems that one molecule can recognize variant TATA motifs with different afhities and mediate different responses to defined stimuli. This versatility might be facilitated by the association of the TATA binding protein with different factors. The cloning of TFIID from a variety of species was facilitated by the highly conserved nature of TFIID, particularly in the carboxy-terminal half of the protein. The C-terminal180 amino acids of the human and Drosophila clones are 88% identical, while the yeast sequence shares 80% with either the human or Drosophila proteins (Fig. 5). In contrast, the N-terminal domain of TFIID is highly divergent across different species. The conserved C-terminal region of TFIID contains the following noteworthy structural motifs. (1)An imperfect 78-aminoacid direct repeat; these repeats may provide the molecule with an element of symmetry. Such a design may be important in recognizing a “directionless” TATA. The area between these repeats has been postulated to be involved in contacting TFIIA (41c).(2) A central basic core with an abundance of lysines (120-156); this region has the potential to form an a-helix with all basic residues oriented on one side, suggestive of a DNA binding or protein-protein interaction role. Individual basic residues within this region do not seem to be

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LEIGH ZAWEL AND DANNY REINBERG

Conserved Core Domain

Basic Region

(T

Homology

FIG,5 . TFIID structure is highly conserved in evolution. Schematics of the structure of TFIID from man, Ihosophila, Aruhidopsis, and yeast are shown. The shaded area extending from residues 155 to 355, with respect to human TFIID, denotes a region highly conserved among the different TATA binding proteins. Percentages indicate degree of sequence identity in the cnre domain relative to human TATA binding proteins. Horizontal arrows indicate direct repeats. The lightly shaded region indicates a highly basic region. The darker shaded area bears homology to prokaryotic u factors. In the N-terminal domain, black bars represent regions rich in serine, threonine, and proline. The shaded regions in the N-terminal domain of the human and Drosuphilu proteins signib Q-runs. [Adapted from Hoffmann et 02. (39).]

important for DNA binding or basal transcription (39). Perhaps this region is important to activation. (3) Sigma homology (197-240); this region bears sequence similarity to the portion of bacterial sigma factors known to interact directly with the - 10 element of bacterial promoters. Although the C-terminal domains from yeast and human TFIID are highly homologous and equally capable of supporting basal transcription in uitro, human TFIID is unable functionally to replace endogenous yeast TFIID in uico. This was not due to differences in the N-terminal domain because when this portion of the molecule was deleted, no detrimental effect on cell viability was observed (41d,41e). Analyses using humadyeast hybrid TFIIDs indicate that no single region within the conserved domain is responsible for the functional differences between the two proteins. Hather, a number of subtle polymorphisms throughout the entire conserved domain may cumulatively predispose a TFIID molecule to being functional in one species but not another. In striking contrast to the C-terminal domain, the TFIID N-terminal domain is highly divergent. In yeast, the TFIIDs N-terminal domain is only SO-60 residues (N-terminal to the N-terminal direct repeat), whereas in Drosophila and man it contains 173 and 155 residues, respectively, which

RNA POLYMERASE I1 TRANSCRIPTION INITIATION

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share only 24% identity. Overall, this portion of the molecule contains very few charged residues. Two motifs stand out, but in keeping with the overall divergence of the N-terminus, they are not strongly conserved across species: (1)The most notable feature is a stretch of 38 consecutive glutamines occurring in the central portion of this domain in humans. In Drosophilu there is a run of 6 and 8 glutamines (Q-runs) in the same respective position, while in the yeast protein there is no such motif. The function of these Qruns is unknown, but it is interesting to consider that the transcriptional activator S p l also contains them. (2) A Pro-Met-Thr tripeptide is repeated five times in human and four times in Drosophilu (it is absent from yeast) near the junction between the amino-terminal domain and the basic core. Consistent with the TFIID bipartite structure, biochemical studies indicate that the conserved domain can function somewhat independently of the amino-terminal domain. Mutational analyses demonstrate that in the yeast, Drosophila, and human clones, the highly conserved C-terminal domain is sufficient to provide DNA binding to the TATA motif and basal transcription activities (33u, 37, 40).Apparently, this portion of the protein is fully sufficient to participate in the formation of an active transcription complex. What then is the function of the divergent N-terminal domain? Recombinant TFIID is capable of supporting basal level transcription, but is unable to respond to activators (37, 42). Transcriptional activation as mediated by MLTF (39) or S p l (37) requires native human or Drosophilu TFIID. As discussed below in further detail, this apparently reflects the requirement for a novel class of “adaptor” molecules, termed coactivators, which are thought to bridge TFIID, or perhaps other GTFs, to transcriptional activators such as Spl. Mutant Drosophilu TFIID molecules lacking the Nterminal domain are incapable of mediating an S p l response (42),suggesting that the divergent N-terminal region may mediate interactions with coactivators. If this were the case, one would expect that interactions between TFIID and coactivators would be species-specific, since this region of the protein is highly divergent across different species. In fact, recombinant human or yeast TFIID is incapable of modulating activation mediated by Drosophila coactivators and S p l (42). Compelling in viuo evidence in support of this model has been presented by Zhou et ul. (43).By complementing an S . cereuisiae TFIID-deficient yeast strain with amino-terminal deletion mutants they demonstrated that (1)the conserved carboxy-terminal domain of yeast TFIID is sufficient for cell viability, but that (2) an acidic region (residues 48-57), just amino-terminal to the conserved domain, is required for a transcriptional response to upstream factor stimulation and normal cell growth. Biochemical studies using recombinant TFIID support earlier observations suggesting that, in viuo, human TFIID is part of a large protein com-

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LEIGH ZAWEL AND DANNY REINBERC

plex (28~).When Drosophila, yeast, or human cDNAs encoding TFIID are used to overexpress the protein in E . coli, or in insect cells, the purified protein not only binds specifically to the TATA box but also protects, from DNase-I cleavage, a 20-nucleotide region centered around this motif (37, 38, 40). This protection is in stark contrast to the protected region observed with partially purified native mammalian TFTID, which protects a region encompassing 75 nucleotides centered around + 1 on the adenovirus major late promoter (Ad-MLP). A similar discrepancy is found when apparent mass is considered. Endogenous human TFIID activity elutes from a sizing column with an apparent mass of over 100kDA, yet the cloned protein’s 339 amino acids comprise a 38-kDa polypeptide. Glycerol gradient sedimentation and immunoprecipitation analyses indicate that TFIID exists in the cell in a multiprotein complex with at least six polypeptides ranging in mass from 32 to 150 kDa (44). Because the protein-protein interactions between TBP and the TBP-associated factors (TAFs) are extremely tight, it has been difficult to resolve and characterize the components of this complex. By performing conventional chromatography in the presence of the denaturant urea, however, TBP can be separated from the TAFs. Interestingly, while urea-purified TBP is unable to support activated transcription, addition of the urea-purified TAFs fully restores an activation response. This, together with the fact that the TAFs are dispensable for basal transcription, suggests that one or several of these TAFs function as coactivators. In initiation complex assembly, the binding of TFIID to the TATA motif appears to be the first step. The TFIID-DNA complex provides a recognition site for the association of the other GTFs and RNAPII (45, 46). As discussed above, the TATA binding protein (TBP) appears to have the ability to interact with many factors. Two factors that effect basal transcription, TFIIA and TFIIB, probably interact with the conserved domain of TFIID, since both yeast and human TFIID can interact with mammalian TFIIA and TFIIB proteins. Association of TFIIA with TBP can occur in solution, in the absence of DNA, as well as with a TFIID molecule bound to the TATA motif (Fig. 6) ( 4 6 ~ )TFIIB . can also interact with TFIID. It is unknown whether this interaction occurs in the absence of DNA. However, when TFIID is bound to the TATA motif, TFIIB can stably associate, producing the DB complex (Fig. 6). The association of TFIIA with the TFIID-DNA complex is not required for TFIIB binding nor does it preclude the association of TFIIB with the TFIID-DNA complex. Rather, the presence of TFIIA appears to increase the overall stability of the resultant DAB complex. The association of TFIIB with a bound TFIID is required for the association of the other GTFs and RNAPII with promoter sequences.

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TATAH Inr

transciiption complexes FIG.6. Formation of the DA and DB complexes. TFIID recognizes and binds to the TATA motif. This is the first factor to associate with the promoter. TFIIA and TFIIB are equally capable of binding to the TFIID-DNA complex. If TFIIA binds before TFIIB, TFIIB can bind the DA-DNA complex, generatingthe DAB complex. The presence of TFIIB (in either a DB or a DAB complex) is necessary for the association of RNAPII and the other GTFs. Inr, Initiator.

B. Transcription Factor IIB TFIIB was first described as an activity present in phosphocellulose 0.5 M KC1 washes, which was absolutely required for transcription from class-I1 promoters and which copurified with a protein of 30 kDa (47). It was thought to participate in the formation of stable preinitiation complexes, as it was required to establish heparin-resistant transcription. Consistent with such a role, TFIIB recognizes the D-DNA or the DA-DNA complex and binds to it, generating the DB or the DAB-DNA complex in gel mobility shift assays (45, 46). At the time of its initial characterization, it was observed that free TFIIB could associate with crude TFIIE, a preparation that contained TFIIF and TFIIH, but could not directly associate with RNAPII in solution (47). Also around this time, BTF3, a 27-kDa transcription factor that behaved chromatographically like TFIIB, was reported (48). Even though BTFS was known to form a stable complex with RNAPII and TFIIB did not, there were many who believed that these two proteins were the same. It was not until both BTFS (49) and TFIIB (50) were cloned and shown to be distinct based on primary sequence that this controversy was resolved. The role of BTF3 in transcription, if any, is currently unknown (51). Reverse genetics has been used to obtain the cDNA clone encoding TFIIB (50). The polypeptide exhibits a molecular mass of 33 kDa. The nucleotide sequence of human TFIIB predicts an open reading frame encoding a polypeptide of 316 amino acids with a calculated molecular mass of

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LEIGH ZAWEL AND DANNY REINBERG

34.8 kDa. TFIIB overexpressed in E. coli (IIBr) is indistinguishable from purified native TFIIB with respect to (1)gel mobility shift assay, i.e., TFIIB can recognize and bind to the DA complex forming the DAB complex; the DABr-DNA complex is recognized by RNAPIIIIIF and subsequently assembling components; (2) basal transcription, i. e., reconstituted transcription reactions including IIBr in place of native TFIIB display similar or identical levels of transcription; and (3) activated transcription, i.e., in contrast to cloned TFIID, IIBr is capable of supporting stimulated transcription as mediated by S p l and MLTF (50). Recently emerging has been the model that TFIIB may be critical to interactions between the initiation complex and upstream activators. There is evidence suggesting that the synthetic acidic activator Gal4-AH (51a) stimulates transcription by recruiting TFIIB to the assembling complex (52) and that TFIIB association with the assembling preinitiation complex is the rate-limiting step in initiation. The acidic activator may stimulate transcription levels by recruiting and maintaining TFIIB in the promoter region. Consistent with this model, it was shown that a direct and specific interaction occurs between TFIIB, native or recombinant, and the VP16 activating region. This interaction appears to be important for activation because a mutant VP16, which is unable to support activation, also failed to interact with TFIIB (53) (see also Section V). Southern and Northern blot analyses indicate only one human gene encoding TFIIB. Computer searches fail to detect any genes with sequences substantially similar to TFIIB. An imperfectly repeated motif of 76 amino acids is present in the carboxy-terminal half of the protein (Fig. 7). That a structurally similar motif is also present in TFIID suggests these repeats may be functionally important. Deletion analyses indicate that these repeats are required for TFIIB to associate with the DA complex (53a).These mutations, as well as other deletions located anywhere in the protein, result in a TFIIB that is transcriptionally inactive, suggesting that the protein is overall very compact. In the 20 or so amino acids separating these repeats, there is a region bearing some similarity to prokaryotic sigma factors. Also nested between these repeats is an -15 aminoacid domain with the potential to form an arnphipathic a-helix containing hydrophobic residues along one side and charged basic residues along the other (Fig. 7). We suspect that this region may contain residues that contact VP16. Single point mutations in which a basic residue along the charged side of the putative amphipathic a helix is changed to a neutral residue behave like wild-type forms with respect to VP16 binding, but double point mutations in this area exhibit reduced or no binding to VP16 columns. If this entire motif is deleted, VP16 binding is abolished ( 5 3 ~ ) .

RNA POLYMERASE I1 TRANSCRIPTION INITIATION

83

FIG.7. Structural features of human TFIIB. Top: schematic of TFIIB polypeptide. Arrows in shaded regions denote imperfect direct repeats. The region bearing homology to prokaryotic u factors is located between repeats and overlaps slightly with the N-terminal repeat, as indicated; + + + +, depicts putative amphipathic a-helix with cluster of charged basic residues oriented along one side. Bottom: helical wheel depiction of putative amphipathic a-helix (residues 185-202). Shaded region emphasizes cluster of charged basic residues, which may represent contact region with Ga14-VP16.

+

C. Transcription Factor IIF In early reconstituted transcription systems, TFIIF was present in crude TFIIE preparations (54). This fraction was interesting, not only because it was absolutely required for transcription, but also because it was shown, using glycerol gradient analyses, that it bound tightly to RNAPII in solution (47). The 30- and 74-kDa polypeptides that make up TFIIF were first isolated by exploiting this particular property. When Greenblatt and colleagues fractionated calf thymus or HeLa cell-derived extracts over columns containing immobilized RNAPII, they isolated three major RNAPII-associating proteins, RAP30, RAP74, and RAP38 (55). RAP38 is equivalent to TFIIS, also known as SII, an elongation factor that affects the efficiency by which RNAPII passes through pausing sites (56, 57, 57a). TFIIF was purified to homogeneity from nuclear extracts using a functional transcription assay, and

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LEIGH ZAWEL AND DANNY REINBERG

the 30- and 74-kDa polypeptides were immunologically identical to RAP30 and RAP74 (58,59). TFIIF activity elutes from a gel filtration column with an apparent molecular mass of 220 kDa, suggesting that it exists in solution as an a2Pzstructure. In addition to being essential for transcription initiation, TFIIF has the ability to stimulate transcription elongation (60). The mammalian cDNAs encoding RAP30 (61)and RAP74 (61a, 61b)have been cloned. The sequence of RAP30 is rich in basic residues and contains several interesting motifs. (1)The central portion of the RAP30 249 amino acids (residues 93- 165) is homologous to two noncontiguous regions of E. coli u70 (Fig. 8). These two regions are the only regions conserved among all bacterial and bacteriophage u factors. They are postulated to contain contact sites with the core component of bacterial RNAP. Consistent with this proposed function, TFIIF can bind to E . coli RNAP and subsequently be displaced by bacterial u70 (62). (2) The amino-terminal portion of the protein (residues 36-42) features a consensus nucleotide binding motif. (3) Just C terminal to the cr70 homology domain (residues 164-174), a region of homology exists with the proposed DNA binding region of CREB (Fig. 8). Curiously, the calculated molecular mass of the polypeptide encoded by

RAP 30 Similaity to 070 4

C /-

-\

/Nucleotide\, Bindina Motil-

/-

-\

Homology o t' \ CREE DNA Binding Domain

RAP 74 428 437 450

+-+-+-+-

517

.' ..'

' ,

Charge Clusters 0

'Nucleotide Binding / Motil Homology

/'.\

/

\

\

'

RNAPI'

\

\ Homology

FIG. 8. Structural features ofthe srnall and large sullunits of human TFIIF. Top: schematic of KAPJO, the small subunit of TFIIF. Residues 36-42 contain a nticleotide-binding motif represented b y horizontal bars. The shaded region in the center of the protein bears sequence similarity to prokaryotic u factors. Blackened region (residues 165-174)bears homology to the D N A binding doniain of CREB. Bottom: scheniiltic of RAP7.1, the large stibunit of TFTIF. The centcr of the protein contains a region rich in charged amino acids. shown here as charge cliisters. Tlie C-terminal domain cwntairis a region with homology to a 1iiicleotide-lindirig motif idiagonal lines) and a region with homology to RNAPI (shaded area).

RNA POLYMERASE I1 TRANSCRIPTION INITIATION

85

RAP74 cDNA is 58.2 kDa. Both subunits ofTFIIF are phosphorylatable and the largest subunit is extensively phosphorylated in vivo. RAP74 contains a globular N-terminal domain, a highly charged central domain containing clusters of acidic and basic residues, and a globular C-terminal domain, which includes two interesting motifs (Fig. 8). The first is a weak region of homology with a subdomain (region IV) present in yLast RNAPI and other eukaryotic RNAPs. The function of this region is unknown. The other motif is a region of homology to the phosphate binding loop of thymidine kinase. This is intriguing in light of the fact that the large subunit of TFIIF has been proposed to contain an ATP-dependent DNA helicase activity, the probable function of which is to melt the DNA during initiation of transcription (61). Recombinant RAP74 does not, however, contain intrinsic adenylation or helicase activity, be it in the presence or absence of RAP30. Also, highly purified, transcriptionally active native TFIIF is devoid of helicase, as well as any kinase or phosphatase activities (59). If TFIIF is involved in one or several of these enzymatic activities, it most likely requires additional polypeptides present within the preinitiation complex. Mechanistically, TFIIF appears to have the critical role of associating with RNAPII and delivering the enzyme to the assembling preinitiation complex on the DNA (Fig. 9). Buratowski et al. have proposed, based on gel shift experiments, that RNAPII can associate directly with the DAB complex (45). However, when highly purified or recombinant factors are used, neither TFIIF nor RNAPII can join the DAB complex alone (63). When TFIIF and

FIG.9. Model for the association of RNAPII with the promoter. RNAPII, in association with TFIIF, recognizes the DAB or the DB protein-DNA complex generating the DAB-PoIF or DB-PoIF complexes, respectively.

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LEIGH ZAWEL AND DANNY REINBERG

RNAPII are added together to the DAB complex, a slower migrating DABPolF complex is formed. This RNAPII-recruitment function is particularly in keeping with the u homology described in RAP30. Killeen and Greenblatt have observed (63a)that TFIIF or RAP30 can suppress nonspecific binding of RNAPII to DNA. Interestingly, the 30-kDa subunit can, independent of the large subunit, recruit RNAPII to the DAB complex. The DAB-PolRAP30 complex is, however, less stabile to electrophoresis than DAB-PolF, suggesting that RAP74 may stabilize the complex. Though RAP74 does not appear to be obligatory for the recruitment of RNAPII, studies in which binding complexes were disrupted and probed with antibodies indicated that both the 30- and the 74-kDa subunits are retained in the DAB-PolF complex (63). In reconstituted transcription systems, RAP30 is unable to replace TFIIF. The formation of the DB-PolF or DAB-PolF complex provides the foundation for the association of the remaining factors required for basal transcription, TFIIE, TFIIH, and TFIIJ.

D. Transcription Factor IIE TFIIE copurifies with TFIIF and TFIIH through phosphocellulose, DEAE-Sephacel, and gel-filtration chromatography; thus early TFIIE preparations contained a variety of activities, making its characterization a slow and difficult process. The eventual purification of TFIIE to apparent homogeneity resulted in the following observations: (1) TFIIE is essential for accurate in oitro transcription from class-I1 promoters; (2) activity copurifies as a heterodimer of 34 and 56 kDa, and the stoichiometry appears to be 1:1; (3)gel filtration analyses indicate that the factor appears to exist in solution as a tetramer, with a native apparent mass of 200 kDa (64,65). Assembly of TFIIE into the initiation complex occurs after the association of RNAPII/IIF with the DAB complex (65).In gel mobility shift assays, the association of TFIIE with the DAB.PolF complex produces a slower migrating DABSPolFE complex. The prerequisite that RNAPII must be stably bound prior to TFIIE association is in agreement with the observation that TFIIE and RNAPII cosediment in glycerol gradients (47, 6Sn) and suggests that the association of TFIIE with the complex may be mediated, in part, through an interaction with RANPII. Both subunits of TFIIE have been cloned from HeLa cDNA libraries using reverse genetics (66).The open reading frame of the p56 cDNA encodes a polypeptide of 493 amino acids with a predicted mass of 49.5 kDa. The 291 amino acids of p34 comprise a polypeptide of 33 kDa. Both subunits have been cxpressed in E . coli. In gel mobility shift assays, rTFIIE behaves identically to native TFIIE. Recombinant TFIIE can replace native TFIIE in a reconstituted system and support basal, as well as activated, transcription. These experiments revealed that, in contrast to initial observations

RNA POLYMERASE I1 TRANSCRIPTION INITIATION

87

based on the renaturation of purified, native subunits, neither TFIIE subunit can functionally replace TFIIE independently. The factors used in the initial experiments were most likely contaminated with the small TFIIE subunit. The sequences of p34 and p56 are not closely related to any existing proteins in the data base. In both subunits, however, there are several interesting motifs that may provide some clues as to the functional role of TFIIE (Fig. 10). In stark contrast to one another, the 56-kDa subunit is highly acidic, with a PI of 4.5, whereas the 34-kDa subunit is highly basic, with a PI of 9.5. In particular, there is a stretch of acidic residues near the C-terminus of p56 and a stretch of basic residues in the corresponding region of p34 (Fig. 10A). The opposite charges probably contribute to the strong subunit interaction observed in gel filtration analyses. Consistent with observations suggesting an interaction of TFIIE with RNAPII and other GTFs is the presence in p56 of a sequence with the potential to form an amphipathic a-helix with a cluster of basic residues on one face and hydrophobic residues on the other. A cluster of cysteine residues arranged in the pattern CX,CX,,CX,C is contained in p56 (Fig. 10B). Similar zinc-finger patterns have been noted in a number of nucleic-acid binding proteins, including the steroid hormone family of receptors, Spl and others. This region shares considerable sequence homology with the zinc-finger motifs present in the UvrA and UvrB proteins of E . coZi. These proteins are components of the ABC excinuclease, a DNA-repair enzyme consisting of three subunits, UvrA, UvrB, and UvrC (67). Significantly, UvrA has DNA-binding activity, albeit with a preference for damaged DNA. As yet, TFIIE is not known to possess any independent DNA-binding activity and so the significance of this domain remains to be determined. As noted in Fig. lOC, p56 contains a region bearing homology to a consensus sequence present in the catalytic loop of several kinases, including RAFl kinase, protein kinase C, and Src kinase among others. This motif is especially similar to a domain common to the protein kinase C family. Though the 56-kDa subunit lacks an accompanying consensus sequence for nucleotide binding, there is such a motif in p34 (Fig. 10D). This region in p34 is similar to the nucleotide binding site present in the human multidrugresistance protein (68). Previous studies have suggested that TFIIE contains an ATPase activity (69).While potential kinase and nucleotide binding motifs are present in TFIIE, biochemical studies attempting to demonstrate kinase or ATP-binding activity using recombinant TFIIE have failed. Homogeneous preparations of native TFIIE that are transcriptionally active are devoid of ATPase, topoisomerase, and DNA helicase activities (65). The meaning of these motifs in TFIIE is unknown, but it is tempting to speculate that

88

LEIGH ZAWEL AND DANNY REINBERG

A 34K

26-42

122-135

SK

Nucleotide BS-2

I I@

Basic 9

56K a-Helix

Zinc finger

Kinase consensus

Acidic

B F

C

56K PKCp PKCG Raf 1 cdc2 Src MIW

MDR-1 pfMDR WHITE

T+

~ E DQ Y N v vII T N[M@

[ K P

I I I I V F R F

I I L

AR AR A S

E E E NUCLEOTIDE BINDING SITE 2

FIG.10. Structural features of human TFIIE. (A) Schematic of p34 and p56. In p34, S and K represent runs of serine and lysine residues, respectively. The shaded area in the center of the 34-kDa protein represents a region bearing homology to a nucleotide-binding motif. Sequence similarity of this region with MDR-1 nucleotide-binding motifs is further illustrated in D. Blackened area represents a region rich in basic residues. In p56, diagonal bars depict region predicted to form amphipathic a-helix. C-C--C-C represents a sequence that could form a zinc finger, also depicted in B. The crosshatched box indicates kinase consensus sequence; the similarity to other kinases is shown in C. The shaded area depicts region rich in acidic residues. (B) Schematic of the potential zinc-finger structure within p56. Circles indicate residues that are

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RNA POLYMERASE I1 TRANSCRIPTION INITIATION

TFIIE, in association with other GTFs, perhaps TFIIH (see Section II1,E) may constitute an ATPase or a kinase activity. The association of TFIIE with the preinitiation complex requires the DAB-PolF complex, and gives rise to the DAB-PolFE complex. This provides the foundation for the association of TFIIH.

E. Transcription Factor IIH When TFIIF preparations were purified by phenyl-Superose chromatography, a previously uncharacterized, essential basal transcription activity was discovered (51).The elution profile of this activity, named TFIIH, is distinct from TFIIF as determined by Western blot analyses using antibodies against RAP30 and RAP74. The use of TFIIH in this system does not overcome the requirement for any of the previously described GTFs. TFIIH is required for transcription from a variety of class-I1 promoters, including those for P-globin, Hsp70, Ad-MLP, and Ad-IVa2. TFIIH activity coelutes with five polypeptides that migrate on SDS-polyacrylamide gels with apparent masses of 90,60,43, 41 and 35 kDa (69a). The cDNA encoding the 60kDa subunit has been isolated (69b). TFIIH activity elutes from a' sizing column with an apparent mass of 230 kDa. TFIIH enters the preinitiation complex after TFIIE and before TFIIJ. We have recently detected a kinase activity associated with THIIH that can phosphorylate the CTD of RNAPII (22a).

F. Transcription Factor IIA Regarding its role, its requirement, and even its polypeptide composition, TFIIA is perhaps the most controversial of all the general transcription factors. Egly et al. purified a 43-kDa protein from HeLa cells to apparent homogeneity; this protein contained basal transcription stimulatory activity and appeared to act early in initiation complex formation (70). Surprisingly, it possessed a number of intriguing similarities to the filamentous structural protein, actin. In addition to having the same molecular weight as actin, this protein cross-reacted with anti-actin antibodies, could self-polymerize and -~ ~

~~

identical (unbroken) or similar (broken) to the first zinc finger of UvrA. Residues were found to be identical (asterisk) or similar (caret) to a region of the UvrB protein. (C) Comparison of the region of p56 containing a kinase consensus sequence with segments p and 6 of protein kinase C, the kinase-related transforming proteins Rafl and Src, and the yeast cell-division control proteins cdcz and MIW. Conserved residues are boxed and shaded. The region proposed to be the catalytic loop is underlined. (D) Comparison of the nucleotide-binding motif present in p34 with similar motifs in the human MDR-1 protein, the Plosmodiumfalciparum MDR protein (pfMDR), and the Drosophila white gene product (WHITE). Conserved residues are boxed and shaded. Residues thought to constitute the nucleotide-binding fold are underlined. [From Peterson et al. (66).]

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LEIGH ZAWEL AND DANNY REINBERG

depolymerize, and could bind and inactivate DNase I. Actin’s known localization in the nuclear matrix further fueled speculation that it might have a role in transcription. It seems likely that these observations (70)were due to trace, nondetectable amounts of TFIIA that copurified with actin. Subsequently, it was demonstrated that fractionation of TFIIA on H P E hydroxylapatite and TSK-phenyl columns separates TFIIA activity from actin (28c). Numerous actin-free, transcriptionallly active TFIIA preparations have subsequently been described. Samuels and Sharp isolated from calf thymus an activity that could functionally replace HeLa cell fractions containing TFIIA (71).This activity is associated with three polypeptides of 19.6, 19.1, and 12.8 kDa. The native molecular mass was estimated to be around 30 kDa based on gel filtration and sedimentation analyses. Reconstitution of TFIIA activity from individual polypeptides was not attempted; thus, the polypeptide composition of TFIIA was not conclusively established. Reinberg and Roeder described HeLa TFIIA as a factor required for specific initiation of transcription; it was eluted from phosphocellulose by 0.1 M KC1 (28c). Curiously, there is a variable requirement for TFIIA in a reconstituted system that depends on the purity of the TFIID used. The most crude TFIID preparation, that derived from the 1.0 M KCI wash of phosphocellulose, abolished the requirement for TFIIA. Functional analyses suggested that a specific interaction occurred between TFIID and TFIIA in the absence of DNA (28c).The mass of TFIIA was estimated to be 84 kDa. Later, using TFIID &nity chromatography, TFIIA was isolated as a heterotrimer of 34, 19, and 14 kDa (46a). Usuda et al. (72)recently reported that TFIIA activity from HeLa cells is contained in a single polypeptide of 38 kDa with a native mass ranging from 90 to 160 kDa. Purified preparations of p38 stimulated a reconstituted transcription reaction; however, renaturation of p38 from protein gels was not attempted. It thus remains unclear whether TFIIA activity is contained in the single polypeptide. TFIIA activity from wheat germ extracts was also reported to be contained in a single polypeptide of 35 kDa (73).This protein could also replace human TFIIA in a reconstituted transcription system. Significantly, Burke et al. (73) renatured the 35-kDa protein from protein gels and recovered transcription activity. This finding is particularly surprising in view of the multisubunit composition reported for TFIIA from HeLa cells (46a, 72), calf thymus (71),and yeast (74).Perhaps in the experiments using renatured protein, one or more TFIIA subunits cross-contaminate other transcription factor preparations used in the system. TFIIA has recently been purified as a heterodimer of 32 and 13.5 kDa from S . cereuisiae (74).The genes encoding each subunit have been cloned. Yeast TFIIA expressed in E . coli is functional in a yeast transcription system

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and can functionally replace human TFIIA in a reconstituted HeLa cell system. Both subunits are necessary for yeast TFIIA activity, and more importantly, the genes encoding both subunits are essential (744. Further complicating the TFIIA picture is a recent report introducing TFIIG as an essential specific initiation factor that can partially be replaced by TFIIA (75). TFIIG, which elutes from P11 with TFIID, is required to reconstitute transcription in the absence of TFIIA; the converse holds true as well. The addition of TFIIG to transcription reactions saturated with TFIIA results in a two- to threefold stimulation, suggesting that the two factors are not functionally equivalent. No polypeptide composition was reported for TFIIG. Recent findings from our laboratory can perhaps reconcile several years of conflicting observations regarding the role and requirement of TFIIA. In the course of extensively purifying HeLa cell TFIIA, Cortes et al. (46a) found that the factor can be separated into two distinct activities, termed TFIIA and TFIIJ. TFIIA purified by yeast TFIID &nity chromatography is composed of three polypeptides of 34, 19, and 14 kDa. TFIIA stimulates basal transcription when native HeLa cell TFIID is used, but is without effect in a system reconstituted with bacterially produced TFIID. A model is proposed in which one of the polypeptides that tightly associates with native TFIID negatively affects TFIID activity. TFIIA is thought either to force the dissociation of this negative component, NC, or to effect an alteration in TFIID conformation such that the influence of NC is removed (Fig. 11). This model is consistent with recent observations (76) regarding a fraction called USA, isolated from the 0.8-1.0 M KCI wash. USA has the following properties: (1) it represses basal transcription (as a result of components named NC1 and NC2) and (2) it enhances MLTF- and Spl-mediated activation (due to another component, PC1). If TFIID and TFIIA fractions are incubated with DNA prior to the addition of USA, then the effects of USA are drastically reduced. What we have described as NC displays the same characteristics as the NC1 component of USA, namely, it is a repressor of basal transcription, the effect of which can be removed by TFIIA. NCl can specifically alter the mobility of the TFIID*DNA complex and is competed for this binding by TFIIA. In our laboratory an activity (called Dr-2) was isolated that may be equivalent to NC1 and to the negative component that copurifies with native TFIID (see also Section VII). TFIIJ, which is separated from TFIIA during hydroxylapatite chromatography, is required for transcription when bacterially produced TFIID is used, but has only a modest effect with native TFIID. The chromatographic behavior of TFIIJ helps to explain its variable requirement. TFIIJ was originally purified from crude TFIIA preparations derived from

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FIG. 11. TFIIA removes a negative component from native TFIID. Model of the possible function of TFIIA in transcription. TBP represents the TATA binding protein, TFIID. (A) Recombinant TBP is devoid of TBP associating factors (TAFs), including the negative cofactor (NC), a component that inhibits TFIID activity. Thus, the activity of recombinant TFIID is high and the association of TFIIA with TBP is without an effect. (B) Native TFIID is shown to be complexed with several TAFs, including NC. In the absence of TFIIA, native TFIID activity is lower than that observed with bacterially produced TFIID. The association ofTFIIA with native TFIID is thought either to induce a conformational change in the TFIID complex or to displace NC directly and, as a result, stimulate TFIID activity.

the 0.1 M KCl phosphocellulose fraction. We have determined that TFIIJ activity can also be recovered from the 0.8-1.0 M KCl phosphocellulose fraction, the same fraction that contains TFIID and TFIIG. Thus, native TFIID preparations are likely already to contain TFIIJ. Complementation analyses with TFIIH indicate that this activity is also present in the TFIIG fraction (51, 75). Thus, TFIIG seems to be a combination of TFIIH and TFIIJ (see Fig. 4). In complex formation, TFIIA can associate with the TFIID-DNA com-

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plex. Furthermore, TFIIA can associate with all the preinitiation complex intermediates thus far characterized: DB, DB-PolF, DB-PolFE, etc. These observations, together with the finding that TFIIA is not required for transcription when recombinant TBP is used in place of native TFIID, suggest that TFIIA association with the promoter region is not necessary for the formation of a transcription-competent complex. TFIIJ, on the other hand, is required (when TBP is used) and is the last factor to enter into the preinitiation complex. TFIIJ recognizes and associates only with the DB-PolFEH complex, resulting in the formation of the DB-PolFEHJ complex.

G. Other Transcription Systems In addition to the set of HeLa cell transcription factors defined initially by Roeder et al., a number of other systems have been developed. Fractionation of yeast nuclear extracts has resulted in the identification of at least five fractions that are required, in addition to RNAPII, for efficient class two transcription ( 7 6 ~ )These . activities have been designated factors a, b, c, d, and e. Factor d is homologous to TFIID, and factor a may correspond to TFIIE. Factor b has been purified to homogeneity, is comprised of 87, 75and 50-kDa polypeptides (76b), and has recently been shown to contain a kinase specific for the CTD of RNAPII. We believe that factor b is homologous to TFIIH in our system. Factor e is equivalent to human TFIIB (unpublished observations). Further purification of factors a, b, d, and e reveals that factor c is not required for transcription (76b). Seven factors (designated factors 1-7) have been fractionated from Drosophila Kc cell extracts (76c). Of these seven, only one, factor 5, is clearly homologous to a component of our system. Like TFIIF, factor 5 is a heterodimer of comparable size (34 and 86 kDa); it stimulates the elongation rate of RNAPII, and associates with RNAPII in solution (764. A basal transcription system using factors purified from Drosophilu embryos has yielded three main fractions in addition to RNAPII that are required for basal transcription: TFIID, TFIIB, and TFIIE/F (76e). TFIID (40, 40a) and TFIIB (J. Kadonaga, personal communication) have been cloned from Drosophila. Drosophila RNAPII, TFIIB, and TFIIEIF are functional in the context of a HeLa cell reconstituted system. Native DrosophiZa TFIID is a highly complex fraction that, as suggested by the following observation, may contain other basal factors. A reconstituted transcription system in which recombinant TFIID is substituted for native TFIID cannot mediate transcription from the Kruppel gene promoter. This promoter, but not the alcohol dehydrogenase promoter, also requires TFIIZ, an activity that copurifies with TFIID. TFIIA cannot substitute for TFIIZ, and it does not appear to be required in this system (J. Kadonaga, personal communication). A set of five initiation factors, designated a,P-y, 6 , E, and T, have been

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fractionated from rat liver extracts. Factors a,P-y, and E have been purified to homogeneity. The a activity is contained in a single 35-kDa polypeptide and most closely resembles TFIIB (76f).Factor P-y is a heterodimer of 30 and 70 kDa that shares immunological identity with TFIIF (76g). Factor E is composed of 34- and 58-kDa polypeptides (76h) and may be equivalent to TFIIE. Using a functional transcription assay, T was shown to be the rat liver homologue of TFIID (76h). The exact polypeptide composition of 6 has not been determined. Interestingly, 6 has been reported to contain a closely associated DNA-dependent ATPase activity (76i), the proposed function of which is to participate in activation of the preinitiation complex (see Section IV). Chambon and colleagues and Weissman and colleagues have independently developed HeLa cell-derived systems. The latter group identified a set of six factors, designated FA, FB, FC, FD, FE, and FF (76j).Factor FC seems to be equivalent to TFIIF, based on its polypeptide composition and its ability to associate in solution with RNAPII (76k).Factors FA and FE have been purified to near homogeneity; both are 33-kDa polypeptides. Factor FA may be equivalent to TFIIB (764, however, the identity of the remaining factors with respect to our system is unclear. Similarly, the Chambon group (76rn, 76n)has isolated several factors that are clearly homologous to components of our system, and others that are not as easily reconciled. Their BTFl and STF appear to be equivalent to TFIID and TFIIA, respectively. BTFS has no clear homologue in our system (see also Section 111).We believe that BTFS is equivalent to TFIIH based on polypeptide composition ( 6 9 ~ )BTF4 . seems to be equivalent to TFIIF (76n).

IV. Preinitiation and Initiation Complexes and Motifs A. Complexes on TATA-containing Promoters At least seven different protein factors, in addition to RNAPII, operate via the TATA element to modulate basal transcription: IID, IIA, IIB, IIF, IIE, IIH, and IIJ. Transcription is preceded by the assembly, on promotercontaining DNA, of all the factors in a highly ordered fashion (Fig. 12). Much of what is known about this assembly process has come from template competition studies (28u, 28b), kinetic analyses on the association of factors (28c), and DNA binding assays in which native gels are used to resolve, electrophoretically, complexes formed between labeled promoter-containing DNA and purified GTFs (45, 63). TFIID is the first GTF to associate at the promoter. As described earlier, it is the only factor possessing specific DNA binding activity. DNase-I footprinting studies using cloned human TFIID indicate that, when bound

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FIG. 12. Preinitiation complex formation on TATA-containing promoters. Model depicts the order of assembly of the GTFs and the transition from initiation to elongation as mediated by a CTD kinase. The formation of the DAB and of DAB-PolF complexes is illustrated in Figs. 6 and 9, respectively. TFIIE, followed by TFIIH, and then TFIIJ, are next to assemble. As also depicted in Fig. 3, this model indicates that the nonphosphorylated IIA form of RNAPII associates with the preinitiation complex. The action of a CTD kinase that converts RNAPIIA to RNAPIIO is thought to be at least one event required to activate the complex. Transcription begins when nucleotides (NTPs) are supplied to an activated complex. The action of a phosphatase that converts RNAPIIO to RNAPIIA is required for RNAPII to reenter the cycle.

at the TATA, TFIID protects a 20-nucleotide region centered around the TATA motif from -36 to -17 on the Ad-MLP (37). Though TFIIA can associate with the TFIID-DNA complex, as well as with all of the subsequently formed intermediates, it is dispensable for transcription, provided that bacterially produced TFIID is used (see Section 111,F). Formation of the DA complex is characterized by an increase in the nucleotide region protected from DNase-I or chemical cleavage. These nucleotides map upstream of the TATA motif with respect to the start site. This footprint is not characterized by any sequence specificity since the binding of TFIIA is mediated entirely through its interaction with TFIID. TFIIB associates with the DAaDNA complex, resulting in the formation of the DAB complex (45, 46). Early observations from the Sharp laboratory (45) using a heterologous system that included native yeast TFIID and mam-

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malian factors indicated that the association of TFIIB with the DA complex resulted in protection of nucleotides around the transcription start site from DNase-I cleavage (Fig. 6). Similar results were obtained using all human factors (46). Interestingly, when recombinant TFIID, either human or yeast, was used to form the DAB complex, it was found that association of TFIIB required sequences downstream of the TATA motif, but resulted in protection only of the TATA motif and not of the transcription start site (46). While this observation has been overlooked by many in the field, it is possible that TFIIB-induced protection over the transcription start site is not due directly to TFIIB, but rather to a factor that exists in association with native human or yeast TFIID. RNAPII is next delivered to the DAB-promoter complex by TFIIF, thereby creating the DAB-PolF complex (63). The studies of Flores et al. (63)demonstrated that the association of RNAPII with the preinitiation complex is strictly dependent on TFIIF. This is in contrast to the findings of Conaway et al. (76g),who proposed that a and Py, the rat liver homologues of TFIIB and TFIIF, respectively, act in combination to promote binding of RNAPII at the TFIID-DNA complex. Consistent with models proposed by Dahrnus and others (19, 78), the IIA (nonphosphorylated) form of RNAPII is more efficiently incorporated into the assembling complex than is the phosphorylated, I10 form (22). The subsequent phosphorylation of the CTD is thought to be a key step in the transition from initiation to elongation. The association of TFIIF with RNAPIIA significantly extends the DNase-I-protected region of the Ad-MLP toward and beyond the transcription start site, from -42 to +17 (63). Gel mobility shift assay has shown that TFIIE, followed by TFIIH, recognizes and associates with DAB-PolF forming the DABSPolFE and DAB-PolFEH complexes, respectively (51, 65). Experiments in which the amounts of TFIIE and TFIIH are varied indicate that these two factors may bind cooperatively. Finally, TFIIJ binds, completing assembly of the transcription-competent preinitiation complex (Fig. 12). Preliminary DNase-I footprinting experiments from our laboratory indicate that the association of TFIIE, TFIIH, and TFIIJ does not significantly extend the protected region beyond what is observed with DAB-PolF (-42 to + 17). Currently, chemical footprinting methodologies are being employed to further characterize the DNA contact regions of these, as well as elongated, complexes. It has long been known that ATP hydrolysis between the P and y phosphates is required for accurate initiation of transcription to occur (79). This hydrolysis seems to occur subsequent to the complete assembly of the initiation complex but prior to the formation of the first phosphodiester bond. The role of ATP hydrolysis is unknown but some of the more popular speculations

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include (1) to phosphorylate one or several of the GTFs such that the complex becomes activated, (2) to facilitate the conversion of RNAPIIA to RNAPIIO, and (3) to provide energy for the formation of the first phosphodiester bond. The events that establish the open complex, a transcription-ready intermediate in which the template DNA has been at least partially unwound, are poorly understood. Once this is established, all that is needed for transcription to ensue is ribonucleoside triphosphates. If nucleotides are present, promoter clearance, the process by which the activated complex beings to move down the DNA in the elongation stage, will follow. It is believed that some of the GTFs remain at the promoter and are available for reinitiation, while others proceed along with the elongating complex and may eventually recycle back to the promoter. Factor u homologies have been found in TFIID, TFIIB, and TFIIF, and may exist in still other GTFs not yet cloned. In eukaryotes, it appears that the multiple roles of the bacterial u factors have been distributed among several of the mammalian GTFs. For example, TFIID has replaced the promoter binding function and IIF has replaced the polymerase delivery function. This dispersion of function allows for the greater regulatory complexity typical of eukaryotic gene expression.

B. Complexes of TATA-less Promoters Functional analyses (28a, 28b, 28c) indicate that the binding of TFIID to the TATA motif is the first step in the formation of a transcription-competent complex and provides a nucleation site for the association of the other GTFs and RNAPII. This has also been confirmed by DNA binding studies (45,46, 28a). It is now known that a large number of class-I1genes contain promoters that lack any recognizable TATA element. Most of these are “housekeeping” genes, genes active in all cells and transcribed at a reduced rate. Though these promoters are generally not as strong as TATA-containing promoters, they can, to a somewhat lesser extent, modulate accurate transcription initiation. In uitro reconstitution experiments using TATA-less promoters indicate that transcription requires all the GTFs, including TFIID, the TATA-binding protein (80-82). The question then becomes, in the absence of a TATA motif-an element known to play an important role in start site positioningwhat provides the entry for RNAPII and the other GTFs, and how is accurate initiation maintained? In an effort to understand what alternative control elements work to direct transcription in such promoters, Smale and Baltimore made deletion mutants in the TATA-less terminal deoxynucleotidyltransferase (TdT) gene promoter and assayed for specific transcription in vivo. They demonstrated that 17 nucleotides surrounding the transcription start site contained the

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information necessary to direct transcription initiation independently (80). This element was termed the initiator (Inr). Recently, many TATA-less promoters have been carefully scrutinized and it appears that Inr elements can be grouped into families based on their nucleotide sequences. The Inr present in the TdT is composed of 11nucleotides, 5‘-GCCCTCATTCT-3’, with the A residue serving as the transcriptional start site. The Inr present in the TdT promoter is similar to that present in the adenovirus-endcoded major late promoter, a TATA sequence containing promoter, and the adenovirus IVa2 promoter, a promoter containing an unusual functional TATA motif located downstream from the transcription start site (the boldface sequences denote nucleotides conserved among these three Inrs) (81). Our studies demonstrate that the Inr present in the Ad-ML and Ad-IVa2 promoters is weakly recognized by RNA polymerase I1 (82). Another class of Inr is that present in the TATA-less promoter of the dihydrofolate reductase (dhfr)gene, which shows no sequence similarity to the TdT-Inr, but does contain a recognition site for a specific DNA binding protein (83,84). A third distinct class of Inr is located within the TATA-less porphobilinogen deaminase (PBGD) promoter. Like the TdT-Inr, the conserved 5’-GxxCTCAxxxT-3’ motif is present in the PBGD promoter, with the A residue representing the transcriptional start site (85). The PBGD-Inr also contains a binding site for a specific DNA binding protein immediately 3’ to the transcriptional start site (+3 to +12). Mutations in the protein binding site or at - 1 and + 1 abolish transcription (85). Interestingly, transcription from the PBGD promoter initiates at a precise site and appears to be independent of TFIID, the TATA binding protein (85). Another distinct class of Inr is that present in the promoter of the ribosomal protein S16 (rpS16) gene. The initiation of transcription in the rpS16 gene is defined by and within a polypyrimidine tract (86). An element distinct from the TATA motif is located in the -30 region, which does not fix + 1 initiation but affects the levels of transcription and can be substituted by a TATA motif (86). Thus, it appears that there are different classes of Inr elements, and that this DNA element is present in all promoters regardless of the presence (AdML) or absence (TdT, dhfi, PBGD, rpS16) of the TATA motif.

C. Insights Regarding Initiation-mediated Initiation The fact that most TATA-less promoters still require all the GTFs, including TFIID, suggests that, with the exception of the TATA-recognition event, the overall initiation mechanism may not be that different from what is thought to occur in TATA-containing promoters. Consistent with this idea, Carcamo et al. (82) demonstrated, using functional transcription assays and gel mobility shift assays, that factors TFIID, TFIIB, TFIIF, and RNAPII are all required for transcription from a TATA-less promoter and can form a

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specific complex on a DNA fragment containing an Inr motif. This complex was specifically competed by an oligonucleotide containing a wild-type TdT Inr motif but not by oligonucleotides containing mutations in the nucleotides conserved among the TdT, MLP, and IVa2 Inrs. The involvement of TFIID was substantiated by the observation that oligonucleotides containing a TATA motif effectively competed DNA-protein complexes founded on Inrcontaining DNA fragments. We have demonstrated that RNAPII weakly but specifically recognizes the Ad-MLP and IVa2 Inrs, and in so doing can provide a foundation for the association of the other GTFs. The conserved nucleotides in the Inr were required for recognition by RNA polymerase I1 (82). By this model, RNAPII provides the entry for the rest of the basal machinery in TATA-less promoters (Fig. 13). In TATA-less promoters, which contain Inrs distinct from that present in Ad-MLP, TdT, and IVa2, a specific factor may bind to the Inr,

FIG.13 Preinitiation complex formation on TATA-less promoters. Schematics compare pathways of preinitiation complex formation on a TATA-containing promoter (left) and a TATAless promoter (right). In the absence of a TATA motif, the initiator (Inr) is thought to direct the formation of the transcription complex. As shown on the right, RNAPII recognizes and binds weakly to the initiator. An Inr-bound RNAPII provides a nucleation site for the association of the other general transcription factors. Subsequent complex assembly stabilizes the association of RNAPII with the initiator. Note that data to support this model have been obtained only for the initiator present in the Ad-MLP, TdT, and IVa2 promoters, which contain the GxxCTCAxxxT motif. TBB, TATA binding protein; NC, negative cofactor.

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which can interact with RNAPII and or one or more of the GTFs. The Inrprotein complex could provide a nucleation site for the association of the other GTFs and RNAPII. Recent studies indicate that, with a construct containing no TATA and multiple S p l sites upstream from the Inr, an additional factor-the tethering factor-is required for optimal Spl-mediated activation. This factor appears to have no role on promoters containing the TATA motif (86~).

D. Cooperation Between TATA and lnitiatior Motifs Various lines of evidence suggest that, when present simultaneously, the TATA and Inr function cooperatively to interact with the transcription machinery to ensure specific initiation. (1) Significantly, double mutants containing base changes in the TATA and Inr are transcriptionally nonfunctional, a drastic effect not observed when either element is singularly mutated (87). (2) When the TATA motif of the Ad-MLP is replaced with random sequence, resultant null mutants exhibit greatly decreased transcription levels but accurate initiation is maintained both in vitro and in vivo (80, 82). We believe that transcription-competent complexes can form on either TATA or Inr motifs, but that, due to steric constraints, there is mutual exclusivity. A complex built solely on an Inr element has only one anchorage point. As a result, it slides, and multiple start sites, though all in the vicinity of the CAP site, are observed. The presence upstream or downstream of a second element recognized by a component of the preinitiation complex (such as the TATA, which is recognized by TFIID, or the Inr, which can be bound by RNAPII), or a site recognized by a specific transcription factor, such as an Inr-binding protein, imparts upon the complex a second anchorage point, greater stability, and the capacity to initiate transcription from a discrete nucleotide.

V. Activation and the General Transcription Factors Transcription of protein-coding genes can be stimulated by a large array of DNA-binding proteins otherwise known as sequence-specific transcription factors or transcriptional activators. The classical transcriptional activator protein is most simply characterized as having two main domains: (1) a sequence-specific DNA-binding domain that recognizes modular sequences occurring in a promoter one, two, three, etc. time(s), depending on the gene, and (2) an activation domain, the portion of the molecule genuinely responsible for stimulating transcription. Activator proteins have been loosely classified into groups based on the properties of their activation domains. For example, the yeast activator Gal4 and herpes simplex virion protein VP16 are thought of as acidic activators because their activation

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domains are rich in aspartate and glutamate residues. Alternatively, the activation domains of Spl and CTF are rich in glutamine and proline residues, respectively. The mechanism by which these proteins stimulate transcription is unclear. Two lines of evidence suggest that the mechanism exploits a theme common to all class-I1 promoters: (1)any one activator can stimulate transcription in a wide variety of different promoters; (2) any one activator functions cooperatively and synergistically in combination with other activators, be they of the same or of a different activator class. Transcription initiation is thought to be the target of these regulatory proteins. The multistep, highly ordered assembly process through which the GTFs and RNAPII associate into a transcription-competent complex provides a multitude of steps that can be regulated. Insight into which steps or, more specifically, which factors are the targets of interactions with upstream activators has come from studies using affinity columns containing immobilized activator molecules such as VP16 or just the activating domain coupled to a carrier such as protein A or glutathione S-transferase (GST). TFIID is bound tightly and selectively by VP16 in this manner (88). Importantly, when control columns containing mutant forms of VP16 were tested, there was a correlation between the strength of the activator (in terms of its potential to stimulate transcription) and its ability to bind TFIID (89). In addition to this work, there is some indirect evidence suggesting that TFIID is a target for activation. The binding of MLTF within the AD-MLP (90) and the binding of Gal4 (91) and the EZV promoter upstream factor (92) to their respective binding sites affects the binding of TFIID to the TATA motif. As discussed in Sections III,A and IV,A, TFIID binding to the TATA motif represents the first step in the formation of the preinitiation complex. This bi,nding, which is slow (28c, 93, 94), may be facilitated by upstream factors such as VP16 and MLTF. In addition to TFIID, TFIIB is selectively bound by the activating region of VP16 (52). A mutation in the activating region of VP16 that reduced the activation potential of the protein but not the overall net negative charge eliminated the interaction with TFIIB, suggesting that the interaction is specific. This specific interaction was also observed with recombinant TFIIB (53).Indirect evidence implicating TFIIB as a target for acidic activators was also obtained using a functional transcription assay in which the DNA template was immobilized on agarose beads (52). In this system, transcription complexes formed on the template are stable to washing, and complete RNA synthesis can occur. Complex formation stalled at the point where TFIIB enters, indicating that TFIIB binding was the rate-limiting step. The inclusion of the synthetic acidic activator Gal4-AH (514 in preincubations, prior to washing, resulted in more efficient binding of TFIIB. It was postulated that Gal4-AH stimulates transcription by accelerating TFIIB recruitment to

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the promoter and perhaps by maintaining the factor there through multiple rounds of initiation. Through independent collaborations with M. Green and J. Greenblatt, we have recently learned that TFIIH can also interact with VP16 (unpublished observations). Thus it seems that transcription activation may happen at many levels, as a result of interactions between an activator molecule and several of the initiation complex components. The fact that an activator may contact several GTFs helps to explain the phenomenon of synergy, whereby two activators stimulate transcription multiplicatively, as opposed to additively. It is important to stress that while specific interactions between an acidic activator and one or more of the GTFs can be demonstrated, this interaction is not necessarily sufficient for activation. For example, it was mentioned earlier that recombinant TFIID proteins cannot participate in activation, yet an interaction between the acidic activation domain of VP16 and recombinant TFIID proteins can be demonstrated. Furthermore, while an interaction between VP16 and three of the GTFs has been observed, addition of Gal4-AH or Ga14-VP16 to a highly purified reconstituted transcription system containing native TFIID resulted in no activation (A. Merino and D. Reinberg, unpublished observations). Ongoing studies have resulted in the isolation of a protein fraction that is necessary, in addition to the GTFs and native TFIID, for activation. The further fractionation of this material has resulted in separation of at least three components, two of which, Dr-1 and Dr-2, interact with TFIID. Surprisingly, addition of these two factors to reconstituted transcription systems results in repression of basal transcription. Thus it is possible that activation of transcription, as defined by in uitro experiments, involves at least two separate processes: (1)the removal of factors that negatively effect transcription, i.e., antirepression (also see Section VI) and (2) true stimulation of transcription. Note that all the studies described here have used only the acidic activators. The mechanisms by which S p l and other activators stimulate transcription may be quite distinct and perhaps provide even greater complexity to transcriptional regulation. The mechanisms driving transcription activation are just starting to be understood. The coming years promise great advances in our understanding of this very important phenomenon.

VI. Repression of Class II Gene Transcription It is now becoming clear that just as a number of diverse mechanisms have evolved to stimulate class I1 gene transcription, a variety of mechanisms also exist that repress it.

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Recently, histone-mediated repression has received a great deal of attention. Histones were long thought to be simple, structural, nonspecific DNAbinding proteins abundant in chromatin. Since most experimental systems utilize purified, “naked DNA, the effects of histones in transcription have traditionally been overlooked. The picture now emerging is one in which the formation of functional transcription complexes on DNA is in direct competition with the assembly of the DNA into nucleosomes (95). This realization alters the way in which we think about activators. Activators are now thought to work at two levels: by contacting one or several of the GTFs and stabilizing or accelerating complex formation, and by binding DNA in the promoter and essentially freeing this from histone-mediated interference. This has been defined as antirepression (96). A very different repression mechanism involves Dr-1, a 19-kDa protein isolated by J. Inostroza and D. Reinberg (unpublished). Dr-1 was isolated from HeLa cell extracts as an activity that can form complexes with TFIID and that, when added to transcription assays, inhibits transcription. Dr inhibits transcription in two ways: (1)by binding to TFIID molecules bound at the TATA motif-TFIID-Dr complexes are not recognized by IIA, IIB, etc. (Fig. 14), and by the ability of (2) Dr to disrupt preformed initiation complexes such as DABePolF. Interestingly, EIa and SV40 large-T antigen can disrupt TFIID-Dr complexes, indicating that Dr function may be tightly regulated, particularly within the context of cellular growth-signaling pathways (V. Kraus, J. Inostroza, J. Nevins, and D. Reinberg, unpublished observations). NC1 (negative cofactor l), which was introduced in Section III,F, represents yet another repressor of basal transcription (76). NC1 consists of one or more polypeptides in the size range of22-28 kDa that can associate with and mod+ the mobility of TFIID-DNA complexes in gel mobility shift assays. It is this interaction with TFIID that is thought to result in down-regulation of basal promoter activity. Increasing concentrations of TFIIA can displace NC1 and compete for TFIID binding. We have isolated a protein fraction, Dr-2, which may be equivalent to NC1. Like NC1, Dr-2 represses transcription by binding to TFIID and its effect can be overcome by TFIIA and or by an acidic activator (A. Merino and D. Reinberg, unpublished observations). As discussed in Section III,F, we suspect that Dr-2 is a negative regulator that associates with native TFIID and that is displaced by the association of TFIIA with TFIID. All the repressors described here inhibit basal transcription through protein-protein interactions. There have been numerous reports of specific gene transcription being repressed as a result of protein-protein interactions. The Id protein is a helix-loop-helix (HLH) protein lacking a basic, DNA-binding region (97). When it dimerizes with other HLH proteins, they

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FIG. 14. Dr is a repressor of basal transcription. Model illustrates the ability of Dr to associate with TFIID molecule bound to the TATA motif. This association prevents the further assembly of the preinitiation complex. Though not indicated here, Dr is also capable of disrupting preformed preinitiation complexes and of associating with TFIID in the absence of DNA.

cannot bind to their recognition sites, which are present in different enhancers, and myogenesis, or Ig gene expression, is inhibited. Another specific inhibitor of transcription is IKB, a factor interacting with NF-KB in the cytosol, which inhibits the translocation of NF-KB to the nucleus, thereby repressing transcription of NF-KB-responsive genes (98-100). This should not imply that protein-protein interaction is the only repression mechanism. Examples exist in which repression is the direct result of a protein binding to DNA. Drosophilu P-element transposase binds to the TATA region of the P-element promoter and blocks association of RNAPII and the

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GTFs (101). Similarly, the homeodomain protein encoded by engruiled inhibits preinitiation complex formation by binding to the TATA box of the Drosophilu Hsp70 as well as other eukaryotic promoters (102). As we move nearer to a transcription system fully reconstituted with cloned proteins, the identification of specific repressors and activators of basal transcription from cell extracts probably will become more routine. Our understanding of how gene expression is regulated will remain incomplete until this family of factors is better defined. ACKNOWLEDGMENTS We thank our colleagues in the transcription field for providing information prior to publication. We extend sincere apologies to those who, due to the enormous amount of material that had to be condensed, we failed to acknowledge. We thank Roberto Weinmann Masayori Inouye, Michael Dahmus, Richard Young, David Arnesti, Robert Tjian, Osvaldo Flores, and Nancy Stone for comments on the manuscript and all members of the Reinberg laboratory for helpful discussions. L.Z. is supported by NIH Training Grant in Molecular and Cellular Biology GM 08360. D.R. is supported by the NIH, the National Science Foundation, the American Cancer Society, the New Jersey Commission on Cancer Research, the Foundation of the University of Medicine and Dentistry of New Jersey, and Robert Wood Johnson Medical School; he is the recipient of an American Cancer Society Faculty Research Award. REFERENCES 1. R. A. Young, ARB 60,689 (1991). 2. C. A. Spencer and M. Groudine, Oncogene 5, 777 (1990). 2a. T. Kerppola and C. M. Kane, FASEB J . 5, 2833 (1991). 3. P. A. Weil, D. S. Luse, J. Segall and R. Roeder, Cell 18, 469 (1979). 4. J. L. Manley, A. Fire, A. Cano, P. Sharp and M. Gefter, PNAS 77, 3855 (1980). 5. L. A. Allison, M. Moyle, M. Shales and C. J. Ingles, Cell 42, 599 (1985). 6. J. Biggs, L. L. Searles and A. Greenleaf, Cell 42, 611 (1985). 7. A. Ruet, A. Sentenac, P. Fromageot, B. Winsor and F. Lacroute, JBC 255, 6450 (1980). 8 . A. M. Edwards, C. M. Kane, R. A. Young and R. D. Kornberg, JBC 266, 71 (1991). 8a. M. Nonet, D. Sweetser and R. Young, Cell 50, 909 (1987). 9 . L. A. Allison, J. K. Wong, D. Fitzpatrick, M. Moyle and J. C. Ingles, MCB 8,321 (1988). 9a. M. S. Bartolomei, N. F. Halden, C. R. Cullen and J. L. Corden, MCBiol8, 330 (1988). 9b. W. A. Zehring, J. M. Lee, J. R. Weeks, R. S. Jokerst and A. L. Greenleaf, PNAS 85,3698 (1988). 10. L. A. Allison and C. J. Ingles, PNAS 86, 2794 (1989). 10a. A. Usheva, E. Maldonado, A. Goldring, H. Lu, C. Honbavi, D. Reinberg and Y. Aloni, Cell 69, 871 (1992). 1 1 . W. A. Zehring and A. Greenleaf, JBC 265, 8351 (1990). 12. S. Buratowski and P. A. Sharp, MCBiol 10, 5562 (1990). 13. W. Y. Kim and M. E. Dahmus, JBC 264, 3169 (1989). 14. N. E. Thompson, T. H. Steinberg, D. A. Aronson and R. R. Burgess, JBC 264, 11511 (1989). 14a. C. L. Peterson, W. Kruger and I. Herskowitz, Cell 64, 1135 (1991). 15. M. Suzuki, Nature 344, 562 (1990). 16. J. L. Corden, Trends Biochem. Sci 15, 383 (1990).

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Regulation of Repair of Alkylation Damage in Mammalian Genomes SANKARMITRA*,~AND BEFWD K A I N A ~ *Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37831 fznstitut fur Genetik und Toxikologie Kemforschungszentnm, Karlsruhe 0-7500 Karlsruhe, Germany I. 11. 111. IV. V.

Historical Perspective ........................................... Unusual Repair of 06-Alkylguanine ............. Multistep Repair of N-Alkylpurines ................................ Properties of Mammalian 06-Methylguanine-DNA Methyltransferases . . Cloning of Mammalian Alkylation Repair Genes by Phenotypic Rescue

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1. Historical Perspective DNA repair is a universal and ubiquitous process that is essential for survival. Lethal and mutagenic damages in DNA result not only from exposure to external chemical and physical agents but also from spontaneous chemical reactions, in particular, deamination of cytosine (and S-methylcytosine) to uracil (and thymine) and spontaneous loss of purines (1).Such alterations, if left unrepaired, would result in C * T transition mutations and in apurinic/apyrimidinc (AP)2 sites, which usually block replication (2-4). 1 To whom correspondence may be addressed. Present address: Sealy Center for Molecular Science, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch H-52, Galveston, Texas 77555. 2 Abbreviations: Ada, product of Escherichia coli adu gene regulating adaptive response; A M , product of E. coli alkA gene, a component of ada regulon; AP, apurinic/apyrimidinic CHO, Chinese hamster ovary; CNU, N sites; BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea; chloroethyl-N-nitrosourea; CREB, CAMP response element binding protein; DM, double minute chromosome; DMS, dimethyl sulfate; DHFR, dihydrofolate reductase; DTIC, dacarba-

109 Progress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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A wide variety of damages can be induced in DNA in uiuo by physical agents, such as ultraviolet light and ionizing radiation, and by a plethora of chemical agents, some of which are as simple in structure as vinyl chloride or ethylene oxide, or as complex as the polynuclear aromatic hydrocarbons. With all chemical mutagens, the reactive species is an electrophile that attacks various nucleophilic targets in the bases and phosphates of DNA (5, 6). In most cases, the chemical agent is not itself reactive, but is activated via oxidative metabolism in detoxification pathways (7). Simple alkylating agents, such as N-alkylnitrosamines, include many known carcinogens, mutagens, and toxic agents. Some of them, e.g., methyl methanesulfonate (MMS), alkylate directly in an S,2 reaction. Others, particularly those requiring metabolic activation, e.g., N-alkylnitrosamines, generate a reactive alkylcarbonium ion as the intermediate (6). The hallmark of these simple aliphatic chain-containing agents is their reaction with a number of sites in DNA. The distribution of the adducts at various sites depends not only on the chemical structure of the alkylating agent but also on the alkyl group. For example, ethyl methanesulfonate (EMS) alkylates oxygen sites at a much higher proportion than its congener, methyl methanesulfonate (8). Most simple alkyl adducts of DNA bases, unlike the bulky adducts, do not block DNA replication absolutely. However, they are often toxic and/or mutagenic (9). Since the original proposal of Loveless more than two decades ago, that among these adducts 06-alkylguanine is the critical mutagenic and carcinogenic lesion (lo),a variety of in uiuo and in uitro experiments have shown conclusively that 06-alkylguanine is indeed the primary mutagenic lesion (11-14). Organotropism studies of carcinogenesis induced by N-alkylnitrosamides, e.g., N-methyl-N-nitrosourea (MNU), and N-alkylnitrosamines also suggest a causal link of 06-alkylguanine and tumor Recent experiments on mammary tumor induction induction in rats (15,16). by MNU in rats causing activation of the Ha-ras oncogene are consistent with the notion that tumorigenesis resulting from G + A transition mutations in the oncogene is due to the formation of 06-alkylguanine, which zine; ERCC, human gene correcting excision repair defect by cross complementation; HeCNU, N-hydroxyethyl-N-chloroethyl-nitrosourea;HPRT, hypoxanthine phosphoribosyl phosphotransferase; HSR, homogeneous staining region in a metaphase chromosome; MAG, 3-methyladenine-DNA glycosylase; MDR, multiple drug resistance; EMS, ethyl methanesulfonate; Mer- , Mex- , 06-methylguanine repair defective; MGMT, 06-methylguanine-DNA methyltransferase; MMS, methyl methanesulfonate; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; MPG, N-methylpurine-DNA glycosylase (same as MAG for eukaryotes);Ogt, product of E . coli ogt gene for constitutive repair of 06-alkylguanine damage; PCR, polymerase chain reaction; RFLP, restriction fragment-length polymorphism; Tag, 3-methyladenine-DNA glycosylase (product of E . coli tug gene). UDS, unscheduled DNA synthesis, due to excision repair.

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behaves like adenine during DNA replication (14). While the N-alkylpurines have not been shown experimentally to be mutagenic, they can be indirectly mutagenic because their removal, either in spontaneous chemical reaction or during excision repair, results in the formation of AP sites. As already mentioned, AP sites normally prevent DNA replication. However, under special circumstances they can also lead to mutations (17). Even though the N-alkylpurines are not directly mutagenic, they are toxic as indicated by the fact that agents such as MMS and dimethylsulfate (DMS), which induce mostly N-methylpurines and methylphosphotriesters and only minute amounts of 06-methylguanine compared to N-nitrosamines and N-nitrosoamides, are quite cytotoxic in cultured cells, and it is believed that the methyl adducts, particularly N-methylpurines, contribute significantly to the overall toxicity (18-20). Hypersensitivity of N-methylpurine repair-deficient mutant strains of Escherichia coli to alkylating agents has directly confirmed the cytotoxic nature of 3-methyladenine in DNA (21, 22). N-Alkylpurines in DNA may also contribute to other biological effects, especially induction of chromosomal aberration. There is a correlation between N-alkylation level and clastogenic efficiency of various alkylating agents (23). Studies with mice exposed to MMS suggest that N-methylpurines in DNA cause both dominant lethal and somatic mutations as well as reciprocal heritable translocations in a stage-specific fashion during germ cell differentiation and embryonic development (24-26). Exposure to DMS and MMS caused induction of tumors of the central nervous system. Such data strongly argue for a carcinogenic role, direct or indirect, of the N alkylpurines in DNA (18, 27). N-Methylpurines in general, and 7-methylguanine in particular, have been implicated in the aging process on the basis of circumstantial evidence (28).Excision repair in response to exposure to alkylating agents is deficient in older animals (29, 30). At the same time, 7-methylguanine appears to accumulate in the DNA of aging mice (31). At the teleological level, the evolution of repair systems for alkyl adducts may be related to the fact that even in the absence of exposure to external alkylating agents, spontaneous methylation of various nucleophilic sites in DNA may occur in vivo by nonenzymatic reaction with S-adenosylmethionine (32, 33). This review is not intended to be a comprehensive compilation of the findings embodied in the vast literature on alkylation damage and DNA repair. Such information can be found in several recent reviews (21, 34-36). Our focus is a review of our current understanding of the molecular basis of the regulation of alkylation damage repair in mammals. Such molecular studies were not possible until the recent success in the cloning of the

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alkylation repair genes. By way of background, we first review the basic mechanisms of repair proteins.

II. Unusual Repair of 06-Alkylguanine Similar mechanisms are operative for the repair of alkyl adducts in the DNAs of all organisms investigated. “Excision repair” is the general mechanism of removal of DNA damage in which a segment of a DNA strand, including the damage, is enzymatically removed. The integrity of the DNA is subsequently restored by de novo synthesis in the gapped duplex (37). In the case of 06-alkylguanine, an unusual repair mechanism exists in all organisms. First discovered in E. coli in 1980, the repair process involves a direct stoichiometric transfer of the alkyl group from the guanine adduct to the repair protein, resulting in its inactivation (38, 39). The amino-acid residue accepting the alkyl group is a unique cysteine (40). The S-alkylcysteine produced is stable, and the repair protein does not regenerate. Thus this is the only repair protein that, while not a true enzyme, restores the integrity of DNA in a single-step reaction. While the protein has been variously named by different workers, its commonly accepted name is 06-methylguanine-DNA methyltransferase (EC 2.1.1.63),abbreviated by us as MGMT (21,41).MGMT has been accepted by the Human Gene Mapping Nomenclature Committee as the formal name of the human gene and the protein (41).The bacterial and yeast methyltransferase genes and polypeptides have been given distinct names (21).In this review, we use the term MGMT as a generic name for all 06-methylguanine-DNA methyltransferases. MGMT activity has been detected in all organisms tested so far (35, 4244). Even though the overall sequences of various MGMTs are rather different, the sequence surrounding the alkyl acceptor cysteine residue is highly conserved (21,41,45,46)(Fig. 1).The reason for the evolutionary conservation of this unique repair protein is not clear. It is possible that among all mutagenic lesions in DNA, 06-alkylguanine holds a unique position in that even though it does impede DNA polymerase (12), it does not act as an absolute replication block. Additionally, its invariable tendency to base-pair with thymine makes it imperative to repair this lesion in the fastest possible way prior to replication (47). The other well-characterized adduct induced by alkylating mutagen and known to be directly mutagenic is 04-alkylthymine (48, 49). However, it is induced to a much smaller extent than 06-alkylguanine (48). Furthermore, the 04-alkylthymines are also substrates, although poor ones, for the E. coli MGMTs (Ada and Ogt proteins) (21,45, 50-52). The situation is not as clear for the mammalian system. The human MGMT appears to act on 04methylthymine in DNA, but at an extremely slow rate (51, 53),and it does

Mouse MGMT

I S Y Q Q L A A L A G N P K A A R A V G G A M R S N P V P I L I P C H R V V R S D G A I G I H

n

R a t MGMT

S Y Q Q L A A L A G N P K A A R A V C G A M R S N P V P I L I P C H R V I R S D G A I G N

Human MGMT

S Y Q Q L A A L A G N P K A A R A V G G A M R G N P V P I

E. coli Ada

E. coli

Ogt

6. subtilis D a t - 1

n

V V P C H R’V I G R F V P C H R V I G K N S A L T G

-

FIG. 1. Sequence homology among MGMTs of mammalian and bacterial origin around the alkyl acceptor site. The boxes enclose regions with identical sequences. [With permission from Shiota et al. (175) and the ACS.]

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not appear to act on 04-ethylthymine at all (54).An alternative pathway for the repair of 04-alkylthymine in DNA in mammalian cells has been detected but not characterized (55). It is not surprising that MGMT accepts higher alkyl groups from 0 6 alkylguanine, but the finding that E. coli and mammalian MGMTs show different rates of reaction with different alkyl residues was unexpected. Bulky base adducts, irrespective of their chemical nature, distort the DNA helix and are removed by the excision repair pathway. The excision repair systems remove 06-alkylguanine, especially if large alkyl groups are involved (56). Such results are consistent with the observation that the major MGMT in E. coli (Ada protein) reacts poorly with 06-ethylguanine and is nearly inactive with propyl and butyl derivatives (57, 58), with rates of 10.1% that for 06-methylguanine. In contrast, mammalian MGMT reacts with ethyl to butyl derivatives of guanine at a significant rate (57, 59). For example, rat MGMT removes 06-ethylguanine from DNA at about onethird the rate of removal of 06-methylguanine (57). While the repair mechanism of large alkyl adducts of guanine has not been studied in mammalian cells, the fact that the loss of 06-methylguanine does not occur in vivo in MGMT-negative (Mex-) cells indicates that the excision repair pathway does not contribute significantly to the repair of 06-methylguanine in mammalian cells. In any case, it is tempting to speculate that evolution of repair pathways is coordinated so that the 06-alkylguanine adducts that act as replication blocks are repaired by a more complex mechanism, in contrast to 06-methylguanine, which warrants a more immediate repair.

111. Multistep Repair of N-Alkylpurines In contrast to 06-alkylguanine, N-alkylpurines, the other major adducts induced by simple alkylating agents, are repaired in multiple steps. These involve removal of the bases by DNA glycosylases followed by excision repair of the resulting AP sites. With the exception of 06-alkylguanine (and 04-alkylthymine), the repair of abnormal bases or bases modified with small adducts occurs via specific glycosyl bond cleavage of the base, and appears to follow a common pathway in all organisms (2). Many such DNA glycosylases have been identified and characterized. N-Alkylpurine-specific glycosylases are also ubiquitous. Escherichia coli AlkA protein, the major glycosylase for N-alkylpurines, also utilizes 02-methylpyrimidines as substrates (60). Repair of these pyrimidine adducts in mammalian systems has not been investigated. In contrast to the situation with 06-alkylguanine, the N-methylpurine-DNA glycosylases have not been extensively studied. We have adopted the abbreviation MPG for this class of enzymes, which has been accepted by the

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Human Gene Mapping Nomenclature Committee. The DNA glycosylases, unlike MGMT, are true enzymes; they act catalytically. The MPGs from a number of mammalian sources have been purified to various degrees, and so far, unlike E. coli, mammals appear to contain a single MPG. Interestingly, mammalian MPG behaves more like the inducible E. coli AlkA protein (3-methyladenine-DNA glycosylase 11) rather than the constitutive Tag protein (3-methyladenine-DNA glycosylase I) (61,62). The protein has a broader substrate range (including 7-alkylguanine and 3alkylguanine in addition to 3-alkyladenine) than the E. coli Tag protein, which is specific for 3-alkyladenine in DNA (21). The enzymological parameters of the MPGs have not been studied. Thus it is not proved that the same MPG is expressed in different tissues or cultured cell lines of an organism. There are also significant discrepancies in the molecular weight values reported in the literature (62). Recently, cDNAs and coding sequences of the MPG gene from human, rat, and yeast have been isolated (63-66). Based on the deduced amino-acid sequence, as expected, the mammalian proteins share a significant homology. However, these proteins are quite different from the two E. coli MPGs and the yeast enzyme (MAG protein) (Fig. 2). The yeast MAG polypeptide on the other hand, does share a significant proportion of conserved aminoThus the situation with MPG acid sequences of the E. coli AlkA protein (64). is in sharp contrast to the highly conserved active-site region of MGMT. Similarly conserved sequences in MPG could have suggested the locations of their active sites, which have not been identified as yet. However, as we discussed in a recent paper, the glutamine and arginine residues that may be involved in the recognition of adenine and guanine, respectively, are conserved in these proteins (Fig. 2). The arginine is not conserved in the E. coli Tag protein, which does not recognize the N-alkylguanines as substrates. The AP endonucleases play a central role in DNA repair because AP sites are produced not only by the action of DNA glycosylases, but also by spontaneous loss of purines as mentioned above. The excision repair of noninstructional AP sites involves cleavage of phosphodiester bonds on either

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FIG. 2. Sequence comparison among MPGs of mammalian, E . coli, and yeast cells. The numbers represent the positions of the amino-acid residues preceding or following the sequences shown. The boxes represent sequence identity. [With permission from Chakravarti et al. (@).I

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side of the AP site by AP endonucleases (AP lyases types I and II), excision of the damaged sites by exonucleases, and resynthesis of the gapped region, using the complementary DNA strand as the template. Commensurate with its importance in maintenance of DNA integrity, multiple AP endonucleases are present in all organisms (62,67-69). The E. coli AP endonucleases-one major (Ex0111protein) and several minor species-have been characterized; some contain associated and specific DNA glycosylase activities as well (6872). Only a few mammalian AP endonucleases have been identified, and fewer human enzymes have been purified to homogeneity and studied in detail (73, 74). The excision repair of AP sites in DNA involves a number of other proteins, including DNA replication complexes; these have not been characterized in mammalian systems. Nevertheless, it is reasonable to state that MGMT, MPG, and AP endonucleases are the key proteins involved in repair of alkylation damage in DNA. However, this review is confined to MGMT and MPG. AP endonucleases have recently been reviewed elsewhere (67-69).

IV. Properties of Mammalian 06-Methylguanine-DNA Methyltransferases Until the recent cloning of cDNAs (41, 65, 66), which allows purification of significant quantities of the plasmid-encoded mammalian proteins from E . coli cells, highly purified mammalian MGMT and MPG were not available for biochemical investigation. In addition to the low concentration of these proteins in mammalian tissues, purification procedures of the human MGMT in general led to extensive loss of material apparently due to “stickiness” of the protein (75). MPG had been extensively purified from several mammalian cell lines and tissues, but the final preparations were far from homogeneous (61, 62). Some biochemical studies have been carried out with partially purified human and rodent MGMTs. One of the unresolved questions about the MGMT reaction is the fate of the alkylated protein after alkyl group transfer. It is reasonable to suggest that the alkylated and active MGMT may have significantly different conformations and thus an altered affinity for DNA. Thus the early observation (76), that the methylated MGMT has a much larger Stokes radius than the unmethylated MGMT, was consistent with this idea of an alkylation-induced change in conformation of the polypeptide. However, we could not confirm this observation in our more recent series of experiments (75, 77). On the other hand, we did find evidence for a subtle change in polypeptide conformation as indicated by a change in isoelectric point following methylation (75). The issue of how surveillance by repair proteins for the presence of

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lesions in the large mammalian genome occurs is not clear. The repair proteins, like other DNA-specific proteins (e.g., repressors), may have two components in their mode of recognition for DNA, one for a nonspecific lowaffinity binding, and the other for a high-affinity binding for specific recognition (78). As expected, MGMT was found to be chromatin-bound (77, 79). Although a nuclear location of other repair proteins has not been uniformly demonstrated, all of the characterized repair proteins have varying degrees of affinity for DNA (2, 74, 75).We observed, by affinity chromatography on DNA-cellulose, that the non specific DNA binding of human MGMT was unaffected by the methylation status of the protein (75). However, the importance of the 06-alkylguanine-specificbinding was uncertain because the rate of the MGMT reaction was inversely proportional to the amount of DNA, either in the single-stranded or the duplex form (75). RNA is not as good a competitive inhibitor as is DNA (75). These results predict that 06-alkylguanine repair in the presence of a vast excess of unaltered DNA may be far less efficient than its repair in uitro when a limited amount of DNA is present. However, we would need to reinvestigate such inhibition of MGMT when DNA is present in the form of chromatin before drawing a definite conclusion. The other unusual property of the human (and presumably all mammalian) MGMT is inhibition by salt. While similar studies have not been carried out with the bacterial MGMTs, the human MGMT reacts in physiological ionic strength (0.2 M) at about one-fifth the rate of that in the absence of added salt (10 mM ionic strength of the buffer). The inhibition is not ionspecific and may be related to the reduced affinity for DNA in higher ionic strength (75, 80, 81). Nonetheless, it appears that 06-alkylguanine repair should be significantly slower in uivo than the observed rate in uitro. Even though the primary sequence of all MGMTs characterized so far surrounding the alkyl acceptor cysteine residue is remarkably conserved (Fig. I), their reaction rates can be widely different, in addition to the differences in their substrate preferences, as already discussed. We have determined that the second-order rate constant of the E. coli Ada protein is at least 103 times that of partially purified human MGMT (75, 82). While it should eventually be possible to explain such differences by the structural differences of these polypeptides, it makes sense that the bacterial protein should have a much higher reaction rate, because the shorter interval between DNA replication cycles warrants rapid repair of mutagenic lesions. It should also be noted in this context that the number of MGMT molecules in mammalian tissues is in the range of 3 X 104 to 20 X 104 per cell (35, 83, 84) compared to 10 molecules or less per cell in uninduced E. coli (85, 86). Because the mammalian genome is more than lo00 times the size of the E. coli genome, and because the mammalian cells have about 104 times the

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MGMT of E. cob, the slower reaction rate of the former may be partially offset by its larger amount.

P

V. Clonin of Mammalian Alkylation Re air Genes %y Phenotypic Rescue of E. co i Several strategies have been routinely used for isolation of cDNAs and genomic clones of mammalian genes. The simplest approach would be to screen mammalian cDNA libraries with the analogous cloned genes of E. coli as probes. However, it appears unlikely that a sequence homology at the nucleotide level exists in the bacterial and mammalian genes over a stretch of 15-20 nucleotides to make it unique even in the most conserved regions of polypeptides. This is due to a large difference in the bias of codon usage between mammalian and bacterial genes (87). In fact, it did turn out that the nucleotide sequences encoding the absolutely conserved pentapeptide ProCys-His-Arg-Val are different in different organisms (Fig. 1). Alternative procedures for cloning genes include screening cDNA or expression libraries with oligonucleotides or antibodies as probes (88). In both cases, nearly homogeneous protein (at least as pure as that present in a polyacrylamide gel band) is necessary before the oligonucleotide sequence (inferred from the peptide sequence) can be deduced or an antibody produced. The approach utilized successfully elsewhere in cloning several human DNA repair genes (ERCC genes) was to transfect repair-deficient CHO cells with human DNA, isolate repair-proficient transfectants, and then retrieve the human DNA containing the repair gene from the transfectants (89). Early attempts to clone the human MGMT gene were based on this approach because Mex- CHO cells could readily be made Mex+ with human DNA (90-92). However, such efforts uniformly failed because of two limitations of the approach; the possibility of activation of endogenous hamster gene in Mex+ cells could not be eliminated, and the method can be successful only if the mammalian repair gene is smaller than the DNA fragments used for transfection. It turned out, as elaborated below, that the human MGMT gene (>150 kb) is much larger than the DNA fragments used for transfection. Thus the MGMT activity in all Mex+ Chinese hamster ovary cells were encoded by the activated endogenous gene as shown directly by the lack of human MGMT gene sequences in these cells (93). In our continuing efforts to clone human MGMT cDNA, we tried other, more involved procedures, such as differential screening of cDNA libraries of Mex+ and Mex- human cells. In principle, this technique should succeed if the differential expression of only a few genes, including MGMT, occurs in the selected pair of cells. The identity of MGMT cDNA among the

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putative clones could be established subsequently by more extensive screening of a variety of Mex- cells and by isolating Mex+ clones from them after transfection with an expression vector derived from the cloned cDNA. In practice, construction and screening of subtraction libraries are difficult. We and at least one other group failed to isolate MGMT cDNA clones after expending a significant amount of effort (K. Tano, R. S. Foote, and M. Mitra, unpublished experiments; A. Fornace, personal communication). Another potential approach for cloning the mammalian MGMT genes (cDNA) is based on transient complementation of Mex- cells with microinjected mRNA transcribed from a cDNA expression library (94). However, this procedure is extremely labor-intensive. We finally entertained the simple idea, based on our past results about the comparative properties of human and E. coli MGMTs, that the human protein expressed in E. coli may be active and thus that a&- E. coli may become resistant to an alkylating agent as a result of this expression. This idea was supported by the earlier complementary experiment that the E. coli Ada protein confers resistance to alkylating agents on mammalian cells (95-96). The concept of phenotypic rescue is not new and had been used in the past for cloning eukaryotic genes in bacteria (97). However, the genes cloned earlier are involved in intermediary metabolism. We and L. Samson's group were the first to exploit this strategy for cloning eukaryotic DNA repair genes (41,63). The limitation of this strategy should also be obvious. It is unlikely that mammalian repair proteins that function in oligomeric protein complexes would be functional in E. coli. Furthermore, appropriate mutants of E. coli must be available for cloning. Finally, a simple way to challenge the cells in order to eliminate the repair-defective mutant cells should be feasible. The strategy of cloning of DNA repair genes (and their cDNAs) by positive selection is extremely powerful. However, it is important to note two parameters that affect such selection. First, the discrimination in killing of repair-positive and repair-negative E. coli may vary widely for different repair genes and genotoxic agents. Second, the mammalian repair proteins expressed in E. coli may not fully confer the wild-type phenotype. We postulated that the screening strategy should be effective even with a lower ratio of discrimination between the control mutant of E. coli and the repair protein-positive cells if' a suboptimal condition of selection is used. While this will undoubtedly lead to an increase in the level of background noise, a significant enrichment of the desired clones could be achieved by cyclic exposure to an appropriate genotoxic agent and enrichment of the plasmid by cyclic transformation. We exploited this approach in cloning human MGMT and human MPG cDNAs from a HeLa cell cDNA expression library (41, 66). Phenotypic

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screening was subsequently used elsewhere for cloning cDNAs of rat MGMT and MPG (65,98). Human MGMT cDNA has been cloned independently by others using other strategies (53,99).

VI. Regulation of Mammalian MGMT and MPG In view of the fact that persistence of alkyl adducts in DNA leads to severely adverse biological consequences, it is surprising that the repair activity, at least for alkylation damage, is highly regulated. The regulation of MGMT has an important practical implication in the therapeutic effectiveness of the alkylating drugs, such as procarbazine, DTIC, and derivatives of CNU (e.g., Carmustine), which exert their cytotoxic action via formation of 06-alkylguanine derivatives (20). There is an excellent correlation between the development of resistance to these drugs and increased levels of MGMT in tumors of brain and other organs (100-103). In a few cases where such correlation was not observed, it appears likely that the critical cytotoxic lesions may be N-alkylpurines, and the drug resistance may be due to elevated levels of MPG and AP endonucleases involved in repair of these lesions (104). Regulation of enzyme activity can occur at various levels. While the regulation at the gene level, i.e., at the level of transcription without any other concomitant change, may be the common mechanism of controlling enzyme activity, other more complex ways (e.g., change in the stability of mRNA, the half-life of the active protein, and translational control) have been shown to be operative for some well-regulated proteins (105, 106).The molecular basis of regulation of DNA repair proteins in general, and of MGMT and MPG in particular, have not been extensively studied. In the limited experiments carried out so far, MGMT appears to be regulated exclusively at the level of transcription (41, 107, 108, 168). In one extensive study with a number of human brain tumor lines, a good correlation was observed among the MGMT activity, the amount of MGMT polypeptide measured immunochemically, and the amount of MGMT message (103). Even fewer data are available for mammalian MPG. However, we observed recently that cells of rat origin have a much lower level of stable MPG message than do human and mouse cells, even though all of these cells have comparable MPG activity (66). Thus it appears that the regulation of rat MPG activity occurs at a level other than transcription of the gene.

A. Tissue-Specific Level of Expression Ever since the proposal that 06-alkylguanine, in spite of its being a minor alkyl adduct, is the critical mutagenic and carcinogenic lesion (lo),extensive studies of its removal from rodent tissues have been carried out. After the

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observation that the adduct persists to a much higher extent in brain than in liver, with an intermediate level in the kidney, of rats exposed to N-alkylnitrosamines, and with the subsequent discovej of MGMT, a correlation of MGMT activity and the rate of removal of 06-alkylguanine was documented (92, 109, 110). Because it is much easier to quantitate MGMT activity than the 06-alkylguanine adduct in DNA, the levels of MGMT in different tissues of various eukaryotes have been assayed (42, 44,111,112). The liver has uniformly been shown to contain the highest, and brain and lymphocytes the lowest, activity (0.2-0.01 that in the liver). We were surprised to find that the MGMT activity in mouse ovary is as high as that in mouse liver. The methyltransferaseactivity in other organs also vaned over a range of three- to fivefold (113). The significance of tissue-specific variation in MGMT level is not clear. One possible explanation is that liver DNA may be the target for the highest level of adduct formation, because the active species for alkylation are often produced by P-450-mediated metabolic activation of alkylating mutagens and carcinogens (114).Furthermore, the need for repair may be rather low in brain because the adduct formation in this organ is low, probably due to inefficient transport of the alkylating species across the blood/brain barrier, and because the alkyl adduct may be tolerated in nondividing cells. On the basis of this hypothesis, we expected that the highest level of other alkylation repair proteins would be present in liver as well. It was therefore extremely surprising to observe that, in mouse, the MPG level is the highest in stomach, higher than that in the liver, and that the brain has also a high level of activity (113, 115). It appears that a better understanding of the tissuespecific levels of DNA repair proteins can be achieved only after a comprehensive study of the variety of DNA repair pathways is completed.

B. Age-Dependent Modulation of the Methyltransferase (MGMT) and Glycosylase (MPG) Activities Many investigators have proposed a linkage between aging in mammals and accumulation of lesions in DNA or their misrepair (28,30). As discussed, while some of these lesions could be induced by exposure to environmental agents, others, including alkyl adducts, may arise from spontaneous endogenous chemical reaction. 7-Methylguanine accumulates in older mice that have not been deliberately exposed to alkylating agents (31). The observation that excision repair, measured as “unscheduled DNA synthesis” (UDS), needed for the elimination of N-methylpurines was significantly reduced in mice exposed to MNU is indicative of inefficient repair of N-methylpurines from the DNA of older animals (29). Thus these results support the possibility of accumulation of 7-methylguanine, although its half-life in DNA due to spontaneous release is approximately 150 h.

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We measured MPG activity in several organs of two inbred stocks of mice of three different age ranges. The enzyme activity of cell-free extracts was calculated as units per microgram of total DNA in the extracts and therefore represents relative activity per diploid cell. It was surprising to observe that the MPG activity was lower both in suckling animals and in mature adults than in young adults. The same trend was observed in all four organs tested, namely, liver, lung, brain, and ovary, and in both strains of mice (113). However, we cannot conclude from these results alone that N-alkylpurines will accumulate in the DNA of older animals without exposure to alkylating agents, because even the reduced glycosylase MPG activity may be more than enough to remove the small amount of N-methylpurines (e.g., 7-methylguanine) spontaneously induced. Nevertheless, it appears reasonable that a systematic study should be undertaken to determine the level of alkyl adducts in several organs of a test animal as a function of age following a chronic low level of exposure to alkylating agents. The methyltransferase activity is lower in human and rodent fetal tissues than in adults (116-118), but no systematic measurement of MGMT in human tissues as a function of age has been carried out. Again, our results show that suckling mice have lower MGMT activity than young adults, analogous to the situation with MPG. However, in contrast to MPG, MGMT activity is not lower in mature adults than in young adults. Finally, whether the age-dependent changes in MGMT and MPG activities are a reflection of the transcription rates of their genes, or result from altered stability of their mRNAs and their translation, is not known. While the significance of these results in regard to the change in level of alkylation repair in aging is not clear, it is obvious that the MGMT and MPG activities may not be coordinately controlled in viuo in mammals. The situation in E. coli is very different, in that the inducible repair of all alkylation damages is under a single control (21,119).

C. Cell-Cycle-Specific Regulation of the Methyltransferase Many genes, e.g., those encoding chromosomal DNA replication, are regulated in a cell-cycle-dependent fashion in mammalian cells. Thus, the expression of DNA polymerases ci and 6, dNTP synthesizing enzymes, and proliferating cell nuclear antigen (PCNA) is activated just prior to or early in the S phase (120, 121). Cell-cycle dependence of the repair of DNA adducts is also critical, because the lack of repair prior to DNA replication will lead either to replication block or to mutation due to misreplication. Both the activity and the mRNA level of uracil-DNA glycosylase (the enzyme that removes uracil generated in DNA due to deamination of cytosine) is higher in proliferating cells than in resting cells, presumably beaause of the need to remove the mutagenic lesion prior to DNA replication (122). By the same token, we expected the transferase activity to be the greatest

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in cells prior to the S phase. It was therefore surprising that, in a mouse cell line parasynchronized by serum starvation, MGMT activity appeared to be minimal 6-8 h before the onset of S phase (123). These results were later verified at the level of transcription of the MGMT gene in diploid human fibroblasts. In these cells a relatively high level of MGMT mRNA [compared to the amount of 3-phosphoglyceraldehyde dehydrogenase (GAPDH) mRNA measured as a control] was observed in the Go phase after serum starvation. Following supplementation of serum, the MGMT mRNA level declined (relative to GAPDH and p-actin mRNA) with a minimum just prior to the S phase (Fig. 3) (124). This is in contrast to some other enzymes of DNA

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FIG.3. Cell-cycle-dependent modulation of expression of MGMT mRNA in a human fibroblast line. Time 0 represents the addition of serum to serum-starved GMlO cells. The upper graph shows the kinetics of thymidine incorporation in DNA. The lower graph shows the level of MGMT mRNA normalized to the level of glyceraldehyde-phosphatedehydrogenase (GAPDH) or p-actin.

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metabolism that are present at a very low level in resting (Go) cells (120, 121). The significance of the unexpected cell-cycle-dependent fluctuation of the transferase level is not clear. Because little is known about the cell cycle dependence of the glycosylase and other repair proteins in mammalian cells, it is not possible to draw any general conclusion about this phenomenon.

D. Epigenetic Regulation of the Methyltransferase in Cell Lines and Human/Rodent Cell Hybrids A remarkable feature of regulation of MGMT, so far unique among all repair proteins, is its complete extinction in some cultured cell lines. Many human cell lines of both human fibroblast and lymphoblast origin, after transformation with SV40 and Epstein Barr virus, lose their ability to counter the toxic effects of MNNG, and a concomitant loss of 06-methylguanine repair activity was observed (125, 126). The MGMT-negative cells were called Mex- or Mer-. Subsequently a large number of Mex- (Mer-) cells were identified among human and rodent cell lines (127-130). In addition to the virus-transformed lines, some 20-30% of the lines derived from human tumors are Mex- (131). Except for a few cell lines (e.g., GMll), all diploid human cell lines are Mex+. The G M l l line is unusual because, unlike a similar but unrelated line GM10, it is Mex+ at a low passage level, but loses MGMT activity as a function of age (132, 133). After 20-30 cell doublings, the MGMT activity was undetectable, although no other macroscopic and karyotypic changes in the cells could be detected. The situation in rodent cells is somewhat different. While many tumorderived human cell lines are Mex-, in the case of rodents, a number of “normal” cell lines derived from different tissues are also Mex-. In fact, none of the routinely used hamster cell lines, including Chinese hamster ovary (CHO), lung (V79) cells, and Syrian hamster kidney (BHK21), has detectable MGMT activity (128, 132). Syrian hamster fibroblasts become Mex- after two or three passages of the Mex+ primary culture (134).One of four embryonic mouse lines is Mex- (135). In an attempt to map the structural and regulatory locus for human MGMT, we investigated the MGMT activity of Mex+ human/Mex- mouse or hamster cell hybrids, which selectively lose the human chromosomes. In one such study, none of the hybrids, derived from human skin’fibroblasts W138 and mouse RAG lines, had detectable MGMT activity (132).These and similar results with humadhamster hybrids, in which some or all of the human chromosomes were present, preclude an unequivocal assignment of a human chromosome linked to MGMT gene. As a control, we did observe that the Mex+/Mex+ hybrids had the Mex+ phenotype. These results suggest that Mex- is the dominant phenotype.

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On the other hand, most hybrids of Mex+ human/Mex- human cells are Mex+ (84). This supports an earlier study (136). Thus it appears that the epigenetic control of the Mex phenotype is much more complex than if simply caused by a single trans-acting factor. Analysis of MGMT activity of a panel of Mex+ human/Mex- CHO cells suggests the possibility that the MGMT activity in some of the hybrids arose from activation of the endogenous hamster gene and were not encoded by the human gene. A recent surprising observation is that the extinction of MGMT in Mex- human lymphoblastoid cells is accompanied by the loss of expression of unrelated genes, namely, thymidine kinase and galactokinase (137) and overexpression of a ribosomal protein gene (138). Because thymidine kinase and galactokinase genes are located on chromosome 17 and the MGMT gene is on chromosome 10 (99, 139), it appears possible that a nonspecific trans-acting factor encoded by a gene on chromosome 17 contributes to the Mex+-toMex- transition. One obvious explanation of the Mex- phenotype is the loss of MGMT structural genes. This possibility could not be investigated until the recent cloning of MGMT cDNA and chromosomal mapping of the gene. Although such studies have not been carried out extensively, it is clear that loss of the structural gene is precluded by the observation of reactivation of the MGMT gene in the Mex- cells under certain circumstances. Based on the evidence discussed in Section IX, both V79 and CHO cells appear to have the complete MGMT gene. The appearance of Mex- lymphoblastoid lines following viral transformation of Mex+ cell lines suggests that the transition from Mex+ to Mexphenotype is a stochastic process (130, 134, 140). Furthermore, the Mexcells did revert to Mex+ phenotype, although at a very low (10-7-10-s per cell per generation) frequency (141).It is intriguing that these numbers are in the same range as that of the spontaneous mutation rate of mammalian genes, such as that governing HPRT (142).However, there is no evidence as yet that the Mex- phenotype is a result of mutation in the MGMT gene as discussed above. Nevertheless, questions have often been raised as to whether the Mexphenotype is an artifact of cell culture, or whether Mex- cells occur naturally. For example, were the original tumor tissues from which Mex- cell lines were established also Mex-? A recent study provides an answer to this question (143).It appears that a small number of excised liver tumors from humans has no detectable MGMT activity. What is the molecular basis of the nearly complete extinction of MGMT in many Mex- cells? Does it involve positive or negative regulatory elements, or both? Is there a unlfying mechanism that can explain the appearance of the Mex- phenotype in the diverse circumstances described

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above? The answers to these questions will be evident only after a comprehensive understanding of the promoter and enhancer elements of MGMT gene and the trans-acting factors that recognize them. We have recently cloned the 5' proximal region of the transcribed sequence of the human MGMT gene and observed promoter activity in a segment of the region by measuring transient expression of a reporter gene (chloramphenicol acetyltransferase) (144).As is common for many housekeeping genes, the promoter region has no TATA or CAAT boxes, but is extremely G-C-rich and contains a repeat of the 5'-CCGCCC motif. Gradual reduction in the promoter strength observed with sequential deletion of the promoter fragment suggests that multiple regulatory elements control MGMT expression. However, the nature of these elements and of the sequence-specific transcription factors and their interaction in regulating the MGMT level have yet to be elucidated. It was somewhat surprising that, in our preliminary experiments, no difference was observed in the MGMT promoter-driven transcription of the reporter gene in transfected Mex+ and Mex- cells (K. Tan0 and B. Kaina, unpublished experiment), (145).Thus, the lack of MGMT transcript in Mex- cells is unlikely to be caused by the loss of specific transcription factors or to the presence of a specific repressor in those cells that recognize the cloned promoter fragment. One way to investigate the role of trans-acting factors for MGMT regulation that may be encoded by genes located in distinct chromosomes is to reexamine the genetic origin of MGMT expressed in Mex+ human/Mexrodent somatic-cell hybrids (132).Because of the nearly identical size of the MGMT polypeptides and mRNAs in human and rodent cells, it is rather dimcult to establish such a genetic origin. However, a polyclonal antibody directed against a unique peptide sequence of human MGMT not conserved in the mouse coding sequence has recently been raised (146,147). It does not recognize the mouse and presumably other rodent MGMTs (147)and should therefore be useful in establishing unequivocally whether a specific human chromosome is responsible for activating the rodent MGMT gene. One trivial explanation for MGMT extinction is the presence of mutations in the regulatory sequence that would prevent synthesis of MGMT transcripts. However, this appears unlikely for most of the Mex- cells in view of the observations on Mex- -+ Mex+ transition stated earlier. Other arguments against this explanation are as follows. First, we and several others have detected no gross deletion or rearrangement of the MGMT gene It in Mex- human and rodent cells by Southern blot analysis (41,108,168). also appears unlikely that in many of the pseudodiploid Mex- cells (e.g., CHO cell line), both copies of the MGMT gene will be mutated to yield the Mex- phenotype. Only two Mex- lines that also lack the MGMT gene were

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identified, the human HeLa/S3 (MIT) clone (41) and rat line 208F (158).A restriction fragment length polymorphism (RFLP) was observed in the MGMT gene in Mex- HeLa MR line (158),but whether the polymorphism is related to the lack of MGMT transcription is not clear. Second, although no MGMT mRNA was detected in Mex- cell mRNA by Northern blot analysis (41, 99,107,168), a minute amount of the MGMT message was identified by the more sensitive PCR method in Mex- human tumor and CHO cells (108, 158).This suggests that, in most Mex- cells, the MGMT gene is not inactivated by mutation and that the lack of MGMT activity is caused by repression of the gene. Finally, Mex+ cell clones have been isolated from Mex- human lymphoblastoid cells without any selective treatment (130, 141)by simple selection with an alkylating agent in the case of V79 cells (148),and by transfection of CHO cells with human DNA (90-93).

E. Lack of Correlation between Methyltransferase Repression and Oncogene Expression Because of the Mex- phenotype of a significant number of tumor cells, and because infection with tumor viruses such as EBV and SV40 often leads to a Mex- phenotype in the resulting transformed cells, it was of interest to investigate whether the extinction of the MGMT gene is related to oncogene

FIG. 4. Northern blot analysis of induction of MGMT and MPG in mRNA H4IIE cells following treatment with X-rays, MMS, and MNNG. Lane 1, control; 2, 24 h after 200-rad Xrays; 3, 48 h after 200 rad; 4, 24 h after 300 rad; 5, 48 h after 300 rad; lanes 6 and 7, 24 and 48 h after exposure to 0.2 mM MMS; lanes 8 and 9,24 and 48 h after 10 pM MNNG treatment; lanes 10 and 11,24 and 48 h after 20 p M MNNG treatment; lanes 12 and 13,24 and 48 h after 30 p.M MNNG treatment. GAPDH mRNA levels were determined as an internal control.

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activation. No available information supports the possibility of a direct connection. In has been observed that SV40 transformation of human diploid cells does not immediately give rise to the Mex- phenotype (140).It appears that the Mex+-to-Mex- transition usually occurs at the time of crisis during the establishment of cell lines. Immortalizing cells with SV40 large T antigen led to both Mex+ and Mex- strains. These results suggest that MGMT extinction is not a direct consequence of expression of viral oncogenes. This conclusion is further supported by the observation that activity after transient overexpression of c-Ha-ras, c-fos, and v-mos oncogenes in Mex+ NIH 3T3 cells is not associated with a change in the level of MGMT mRNA (149; B. Kaina, unpublished).

VII. Role of DNA Meth lation in Methyltransferase Gene f;egulation A. Level of Methylation of MGMT Gene Sequences in Mex+ and Mex- Cells In view of the extensive substitution of 5-methylcytosine for cytosine in CpG sequences in mammalian genomes, and a large body of evidence in support of the dogma that transcriptional activity of a gene is inversely correlated with the level of its methylation (primarily at the 5’ ends of genes), it was attractive to investigate whether the Mex- phenotype is related to hypermethylation of the MGMT gene. It is now believed that methylation of cytosine causes a change in the local configuration of the gene that negatively affects its promoter activity, and that binding of some transcription factors (e.g., CREB) to specific recognition elements in the promoter is sensitive to methylation (150, 151).Even though the promoters are enriched in CpG sequences “CpG islands” (150), the dinucleotides are also present in the coding sequences of genes. Because the promoter elements and their sequences of the MGMT gene have not been fully identified, we made a preliminary investigation of the methylation sequence of the exon and surrounding sequences of the M GMT DNA isolated from Mex+ and Mex- cells. A nearly identical pattern of methylation was observed in three independent studies with DNA isolated from SV4O-transformed and tumor lines with Mex- phenotype (152). In all cases, the presence of m5C in CpG sequences was determined by comparative probing of genomic DNA digested with restriction endonuclease isoschizomers HpaII and MspI in Southern blots. Both enzymes recognize the 5’-CCGG sequence in DNA but HpaII cannot digest DNA when the internal C is methylated. The DNA of all Mex+ cells expressing MGMT at various levels was methylated to a much higher level than the DNA of Mex-

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cells (152). While this unexpected result is the opposite of the pattern previously observed in general, it is by no means unique. For example, a direct correlation between methylation and gene expression has been observed in the major histocompatibility complex gene H-2K of the mouse (153).Similarly, there was no correlation between DNA methylation and transcriptional inactivation in the chicken lysozyme gene (154). However, we should keep in mind that the genes in the two last examples are developmentally regulated, unlike MGMT and other DNA repair genes that should be typical “housekeeping” genes. Is it possible that there is no causal relationship between methylation of exon sequences and gene activity for MGMT? Despite attempts to explain the phenomenon, it is not clear how and why an increase in the methylation level of cytosine can lead to opposite effects for different genes. We hope that answers to these questions will be forthcoming, once we have dissected the promoter sequence and functions of the human and rodent MGMT genes.

B. MGMT Activation with 5-Azacytidine 5-Azacytidine, a potent agent for inhibiting methylation of cytosine, has been routinely used to demonstrate the role of 5-methylcytosine in gene activation (155), even though the drug has other activities and affects genes that do not contain 5-methylcytosine (156).Treatment of murine sarcoma virus-transformed Mex- NIH 3T3 cells with 5-azacytidine led to stable expression of MGMT in these at about the same level as that of the nontransforming parent cells (157). Attempts by us and others to reproduce these results with Mex- Chinese hamster lines (CHO and V79) and with human HeLa MR cells have been unsuccessful (158; W. C. Dunn and S. Mitra, unpublished; R. S. Day, personal communication). With V79 (Chinese hamster) cells, 5-azacytidine treatment gave rise to cell clones that acquired resistance to HeCNU. However, these cells still lacked MGMT activity and were not cross-resistant to other alkylating agents, e.g., MNNG (158). A strong correlation of MGMT activity and CNU resistance has been observed for the hamster and other mammalian cells. Thus it appears that the induction of MGMT by 5-azacytidine cannot be achieved in all Mexcells and that activation of other, as yet undefined, genes may also be responsible for alkylation resistance.

VIII. Inducibility of Alkylation Repair Genes The seminal discovery in the understanding of alkylation damage repair in E. coli was that of “adaptive response” during which E. coli cells treated with a subtoxic dose of a simple alkylating agent became resistant to the toxic and mutagenic effects of the same and other alkylating agents (159). Subse-

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quent studies showed that several genes in the ada regulon involved in alkylation repair are coordinately induced during the adaptation treatment (21,119). The 39-kDa Ada protein with its distinct amino- and carboxylterminal domains has methyltransferase activities for alkyl phosphotriesters and 06-alkylguanine in DNA, respectively. The alkyl acceptor residues in the protein are Cys-69 and Cys-321 (21).The Ada protein methylated at Cys-69 acts as the inducer of its own as well as one of the glycosylase (aZkA) genes in addition to other genes of unknown function (21,160). Adaptive response has been observed in some other bacteria as well (46, 86,161).Experiments to identify an adaptive response in mammalian cells gave intriguing results. Early in uiuo studies showed a two- to threefold increase in MGMT activity in livers of rats chronically fed with dimethy1-Nnitrosamine (162).It was later observed that a similar increase in MGMT level can also be induced by a variety of hepatotoxic agents (35).Partial hepatectomy and ionizing radiation, and even interferon inducers and hormones, are effective inducers of MGMT (35,163). Many of the inducing agents are not unrelated to DNA repair because these apparently do not cause DNA damage. Thus it is possible that the apparent induction of MGMT arises from the stimulation of cell proliferation. It is surprising that MGMT induction was not observed in livers of other rodents (e.g., mice and hamsters). In in uitro experiments with cultured cell lines, we and others did not observe an induction of MGMT activity following exposure to various alkylating and other DNA-damaging agents (128,164).However, such an induction was indeed seen only in several rat and a human hepatoma, and a rat rhabdomyosarcoma line treated with alkylating agents and UV light (148, 164a, 165).More recently, a 5- to 10-fold increase in MGMT activity concomitant with an increased protection against alkylating agents for neoplastic transformation was observed in mouse C3H 10T1/2 cells after exposure to Xrays (166,167). These results discount the possibility that the adaptive response is generally present in mammals. In a few exceptional cases in rodent tissues and cell lines where there is some evidence of MGMT induction, it is clear that this was due to a response to nonspecific DNA damage. Thus MGMT activity increased in H4 and C3H 10T1/2 cells after exposure to X-rays and bleomycin and restriction endonucleases, all of which cause DNA strand breaks (165,166,168).It is now evident that mammalian cells are capable of a DNA damage-inducible global response in which induction of specific genes involved in repair of DNA damage as well as other seemingly unrelated genes (e.g., oncogenes) occurs (169,170).It is interesting that MGMT and MPG genes have not been identified so far among the damage-inducible repair genes except in a few rodent cells. However, DNA polymerse @, the enzyme involved in “very-short-patch excision repair of DNA lesions, including N-

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alkyl adducts following the removal of MPG, is strongly inducible by alkylating agents (171). It was not possible, prior to the cloning of alkylation repair genes, to establish that the increase in MGMT activity in mammalian cells pretreated with DNA-damaging agents reflects true induction at the level of transcription. Alternate explanations, e. g., stabilization and increased half-life of MGMT by the damaging agent, could not be eliminated. Our recent results, as well as the experiments of others, have now established that the increase in MGMT activity in rat cells exposed to alkylating agents is indeed due to an increase in transcription of the gene (168, 172) (Fig. 4). In H41IE and FTO-2B, both of which are rat hepatoma cells that express liver specific enzymes (e.g., tyrosine aminotransferase), a two- to fivefold increase in MGMT mRNA was observed after exposure to MNNG, MNU, MMS, HeCNU, X-rays, and UV light. Furthermore, MGMT transcription could also be induced by PuuII, a restriction endonucleae producing blunt ends in DNA (168).That the accumulation of mRNA was prevented by actinomycin D, an RNA-synthesis inhibitor, indicates that this increase in RNA level was due to de nouo synthesis. The induction response appears to be late and long-lasting. Thus, MGMT mRNA accumulation continued up to 72 h after exposure of the rat cells to MNNG; addition of actinomycin D 6 h after MNNG prevented the increase (168).Abolition of this induction at both protein and mRNA levels by the protein-synthesis inhibitor cycloheximide suggests that de nouo protein synthesis is essential for this inducible response (168). Because MGMT activity appears to be correlated with the state of digerentiation in rat hepatoma cells (173),it would be worthwhile to test whether the inducibility of the MGMT gene is linked to a specific liver function. A 10-fold increase in MGMT mRNA level was also observed in uiuo in the liver of rats exposed to 2-acetylaminofluorene (173). However, no studies on the possible induction of MGMT transcription in other rat tissues and cultured cells have come to our attention. No MGMT induction was observed in human hepatoma and fibroblast lines under the conditions that induce the MGMT gene in rat hepatoma lines (168). In view of the coordinated regulation of inducible MGMT (Ada) and MPG ( A M ) proteins in E. coh, it was obvious to ask whether MPG is simultaneously induced with MGMT in the rat cells and liver exposed to genotoxic agents. Figure 4 shows our results. The MPG gene is indeed induced in H4 cells at the same time MGMT is induced (172).However, the level of induction of the gene is at best about twofold under the condition in which more than a fivefold increase was observed in the MGMT mRNA level (172, 172a, 172b, 172c). It should also be noted that, unlike for MGMT, MPG induction has not been shown as yet to be due to de nouo RNA synthesis. While the inducibility of MGMT (and MPG genes) in rat liver and

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hepatoma cells has been unambiguously established, why is it that such a phenomenon cannot be clearly demonstrated in human fibroblasts or even other rodent cells and tissues? Furthermore, why is the 2- to 10-fold induction of MGMT observed in rat cells much lower than the several hundredfold induction observed in E. coli? One possible answer to the latter question is that even though, on the basis of genome size, the number (2 x 104 to 10 X 104) of MGMT molecules per cell in mammalian cells is not much different from that of 20 MGMT molecules per cell in E. coli (85, 86), each mammalian cell has 103 more MGMT molecules than do the bacteria. A large increase in MGMT activity may not be necessary when a high basal level is present. The answer to the first question is more uncertain. Is it possible that the MGMT gene in other rodents and even humans is also damage inducible, just as in rats, but that the optimum conditions for such induction have not been established? Alternatively, because the induction phenomenon both at the level of activity and transcription is limited to rat liver cells with a few exceptions (165, 166, 168), is it possible that the regulatory region of the rat MGMT (and MPG) gene is uniquely different from those of other mammals? The cloning and a comprehensive characterization of the MGMT gene from different mammals will be needed for an answer to this question. We may make a point in this context that the primary sequences of the rat and mouse MGMTs are nearly identical and are significantly different from that of the human protein (174, 175).

IX. Alkylating Dru Resistance and Regulation of %NA Repair Alkylating drugs constitute a major class of antitumor agents that exert their cytotoxic action by damaging DNA in various ways. It thus follows that repair competence of cells for the potentially lethal lesions should have a direct impact on the ultimate therapeutic potency of these drugs. It has long been known that a large variation exists in the tissue-effectiveness of drugs in general. In particular, development of resistance to drugs is a common phenomenon among tumors. While the precise nature of critical DNA adducts that primarily contribute to cytotoxicity has not been elucidated for many alkylating drugs, extensive studies have been carried out for the class of CNU derivatives (20, 176). These drugs cross-link DNA strands by forming the intermediate 06-chloroethylguanine (20, 100, 176). Several other drugs (e.g., procarbazine and DTIC) may also exert their cytotoxic effect by inducing 06-alkylguanine (176). Furthermore, all of the alkylating agents, as expected, also induce in DNA N-alkyl adducts that are removable by MPG. MGMT activity has been measured in a variety of tumor cell lines and

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xenografts, and an excellent inverse correlation has been observed between the MGMT level and the cytotoxic response of cells to CNU and its derivatives. A clear correlation between MGMT level and CNU (HeCNU) resistance was also shown in CHO cells expressing human MGMT (177).While these results suggest that alkylation of guanine at the 0-6 position is the major toxic event induced by CNU, certain exceptions were observed where the BCNU (or HeCNU)-resistant cells did not have a high level of MGMT (158,178;S. C. Schold, personal communication). It has been proposed that, in certain BCNU-resistant human glioma lines, repair of N-alkyl adducts is enhanced (101). With the availability of a human MPG cDNA clone, it should now be possible to confirm whether alkylation resistance in some cells is due to an elevated expression of the MPG gene, and whether coordinate regulation of MGMT and MPG is a common phenomenon in resistance to alkylating drugs. These studies have also underscored another important issue, namely, the relative toxicity of different alkyl adducts in DNA. Our recent results reconcile a recent controversy about the toxic nature of 06-alkylguanine (179, 180). We found that MGMT expression protects cells from MNNG, MNU, and, to a certain extent, EMS and MMS-induced cell killing, but not at all from ENU toxicity (177). The differential resistance toward various alkylating agents has led us to propose the hypothesis that, in addition to 06alkylguanine, a second group of cytotoxic alkyl adducts are induced by alkylating drugs (92). These could be one or several N-alkylpurine adducts. The relative contribution to the overall toxicity of a drug will depend on the relative proportion of the class of adducts induced by it (92, 177).

A. Activation of the Endogenous Methyltransferase Gene and Drug Resistance As stated in Section VI, MGMT-positive clones were isolated from MexCHO cells after transfection with human DNA. In all cases, the selective agent was CNU or its derivatives and the MGMT activity was due to derepression of the endogenous gene. The obvious question is whether the human DNA activates transcription of the hamster MGMT by providing a trans-acting factor. This situation will be analogous to the MGMT regulation in humadrodent cell hybrids described in Section VI,D. Isolation of Mex+ clones from Mex- V79 cells by simple selection in the presence of CNU (148) suggests that Mex+ cell populations arose without involvement of foreign DNA. However, as we have discussed (92), we did not observe reversion of Mex- to Mex in the nontransfected CHO cells used as a control, in the presence of CNU. Second, the MGMT activity in the Mex+ V79 clone appears to be much lower than that present in the CHO transfectant lines isolated by others (92, 148). +

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In any event, regardless of the mechanism, development of CNU resistance is in most cases directly correlated with MGMT activity. A similar correlation was not observed for MPG. We showed that, at least with our MGMT-positive transfectants, in uiuo removal of O6-methy1guaninebut not of N-methylpurines was affected in the CNU-resistant cells (91).

6. Expression of the Glycosylase Gene and Alkylating Drug Resistance Unlike MGMT-negative cells, no MPG-negative cell line has yet been isolated. It is therefore not possible to establish a correlation of MPG repair activity and sensitivity to alkylating drugs. However, we investigated the possibility of increased drug resistance as a result of overexpression of MPG. MMS-resistant CHO cell lines have been isolated either as spontaneous variants or after transfection with human DNA (19,181). These MGMTnegative cell lines do not have a higher level of MPG activity, nor do they express a higher level of MPG mRNA than the control cells. Furthermore, neither the removal of 3-methyladenine in DNA nor the MPG mRNA level was altered in an MMS-hypersensitive mutant derived from CHO cells (182).We have tentatively concluded from these results that N-alkylpurines in DNA in Mex- cells may be toxic, but during the multistep repair of these adducts, MPG activity may not be rate limiting. It appears that this repair protein is present in excess in normal cells. Thus a specific increase in MPG activity will not affect the overall repair of N-alkyl addwcts in DNA. The increased alkylation resistance of various MGMT-negative cells would then be due to increased expression of an activity involved in a subsequent step, e.g., AP endonuclease. We have obtained additional support for this hypothesis by carrying out complementary studies in which CHO cells were transfected with a human MPG cDNA expression vector. Several clones expressing up to seven times the MPG mRNA and MPG activity of the control cells were isolated. None of these overexpressing cell lines is more resistant to MMS, DMS and ENU than the parent line (183).

C. The Tolerance Phenomenon and Alkylation Resistance An alternative hypothesis for a lack of correlation of drug resistance with increased DNA repair has recently been proposed by several groups who have cloned Mex- human and rodent cell lines that are resistant to MNNG and yet are unable to remove 06-methylguanine. These cell lines were isolated either by simple selection (180,184-186) or cloned following their transfection with genomic human DNA (181,188).Because these lines were uniformly MGMT-negative and showed no difference in the removal of Nalkylpurines (181,184,187),it appears that the cells developed tolerance to 06-alkylguanine and/or other toxic lesion(s). Interestingly, the cells were

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cross-resistant only to other methylating agents but not to UV light, CNU derivatives, and mitomycin C. It is surprising that they were also resistant to 6-thioguanine (181, 185, 188, 189; B. Kaina and G. Fritz, unpublished result). This resistance appears to be due to tolerance of the analog rather than inhibition of its incorporation into DNA (190). It was unexpected that resistance to cell killing was not associated with a reduction in mutation and sister-chromatid exchanges induced by alkylating agents (187, 191). The molecular mechanism of tolerance to alkylating agents is not clear, but it appears that cellular toxicity to alkylating drugs may involve a number of seemingly unrelated processes. For example, rodent cells that did not express the metallothionein gene but that were made metallothioneinpositive by transfection became resistant not only to heavy metals but also to simple alkylating agents, e.g., MNNG and MNU, as well as to other antitumor drugs such as cisplatin and chlorambucil(192-194). It should be noted that the metallothionein gene is inducible by various DNA-damaging agents (195, 196). It is thus possible that metallothionein may indirectly regulate DNA repair or tolerance functions, perhaps by control of the intracellular level of zinc (192).

X. Amplification of the Methyltransferase Gene and Drug Resistance The link between drug resistance and amplification of genes whose products provide the resistance to the specific antimetabolites is well documented (197,198). In the earlier studies, the drugs used in such experiments had specific targets. For example, methotrexate inhibits dihydrofolate reductase in a competitive fashion. An increased resistance to methotrexate may arise from the increased activity of DHFR expressed from multiple copies of its gene. In the case of “multiple drug resistance,” the MDR protein reduces the intracellular concentration of a variety of drugs (e.g., adriamycin and vincristine) by acting as an effluxing pump (199). Thus the increased resistance of cells to these drugs is due to an increased level of the MDR protein as a result of gene amplification. In contrast to the above examples, amplification of mammalian DNA repair genes has not previously been observed. We reasoned that a higher level of expression of MGMT that results in an increased cellular resistance to CNU may sometimes result fiom an amplification of the MGMT gene. Thus, in a recent study with NIH 3T3 cells, we showed that cells chronically exposed to increasing concentrations of CNU give rise to clones with 3-10 times higher levels of MGMT activity, which in turn resulted from three- to fivefold increase in the number of copies of the MGMT gene (200). A detailed analysis showed that the MGMT gene amplification was not accom-

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panied by a similar amplification of MPG, nor was there an amplification of DHFR and MDR genes. Because the mouse MGMT gene is located at the distal end of chromosome 7 (201),we looked for but did not observe amplification of any other gene on chromosome 7, including IGF-2, which is located nearest the MGMT gene. There was also no evidence of DM chromosomes or HSR in chromosome 7, the classical indicators of high-level gene amplification (198). This was the first demonstration of amplification of a DNA repair gene in mammals. There are some unusual features of this amplification. We never observed more than 10 copies of the gene in spite of attempts for stepwise selection for variants carried out in the usual way. These results are in sharp contrast to the hundreds of copies of DHFR genes observed in methotrexate-resistant cells. However, the low copy number of the MGMT gene is consistent with the lack of DM chromosomes and HSR sequences associated with its amplification (198). We should point out here that our inability to isolate cells with a high copy-number of the repair gene may be due to the fact that we could not use a selecting agent that specifically targets MGMT or acts in an indirect fashion, e.g., an alkylating agent that exclusively produces 06-alkylguanine. In contrast to target-specific drugs such as rnethotrexate, C N U produces a number of toxic lesions. Even though 06-alkylation of guanine may be the predominant event for toxicity, the cells could not handle a higher concentration of C N U because the other minor toxic adducts would reach significant levels and become major contributors to cell killing. This has indeed been shown with cells transfected with an MGMT expression vector (177). We would thus predict that resistance to high concentrations of alkylating drugs would require simultaneous amplification of mope than one DNA repair gene. This prediction can be tested experimentally. What is the biological significance of the amplification of DNA repair genes? In contrast to the common occurrence of gene amplification in cultured (particularly rodent) cells, amplification of genes (mostly those for multiple drug resistance and oncogenes) is rather rarely observed in uiuo and also exclusively in tumors (202). Increased activity of proteins responsible for drug-resistance results more commonly from an up-regulation of its gene than as a result of increased gene dosage (199).While the same situation may be true for DNA repair genes as well, it is important to investigate how often resistance to alkylating drugs does result from amplification of MGMT and other DNA repair genes, both in uiuo and in cultured cells in uitro.

XI. Outlook Research on the molecular biology of mammalian DNA repair in general and alkylation damage repair in particular has entered an exciting phase with

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the recent success in the cloning of a number of DNA repair genes and cDNAs from human and rodent sources. The availability of nucleic-acid and antibody probes for many of the alkylation repair genes and proteins, and elucidation of the structure and identification of the regulatory elements of these genes, provide an opportunity for a comprehensive understanding of regulation of alkylation damage repair. The prospect of large-scale production of the human alkylation repair proteins in E. coli for the subsequent determination of their structure by X-ray crystallography and NMR looks quite good. Rational design of drugs €or specific inhibition of repair proteins may be possible when the three-dimensional structures of these proteins are known. Inhibitors could be very important as adjuvant therapeutic agents for alkylating drugs. Antisense oligonucleotides could also be used for inhibiting transcription and/or translation of repair genes both in uiuo and in uitro. In contrast to inhibition of repair genes at the level of their expression, these genes could also be inactivated in cultured cells by homologous recombination. Starting with mutations in repair genes of pluripotent embryonic stem cells, repair-deficient or repair-negative mice could be generated. Such animals may make excellent models for mutagen, carcinogen, and aging studies. Finally, an understanding of the molecular basis of repair regulation may lead to targeted mutagenesis for up-regulation of the alkylation repair genes in the mouse, particularly, MGMT. Such a change may also profoundly affect the mutagenic, carcinogenic, and toxic responses of the animals to environmental agents. ACKNOWLEDGMENTS The work described in this article was supported at Oak Ridge National Laboratory by the Office of Health and Environmental Research, U.S. Department of Energy, under contract DEACO5-84OR21400 with the Martin Marietta Energy Systems, Inc. and by U.S.P.H.S. Grants CA 31721 and CA 53791, and at Karlsruhe Kernforschungszentrum by Deutsche Forschungemeinschaft KA 724-22. We would like to thank Dr. Rufus Day for his critical reading of the manuscript and for suggestions that significantly improved its quality. Finally, we gratefully acknowledge our colleagues’ contributions, which made this review possible. REFERENCES 1 . T. Lindahl, This Series 22, 135 (1979). 2. T. Lindahl, ARB 51, 61 (1982). 3. S. K. Randall, R. Eritja and B. E. Kaplan, JBC 262, 6864 (1987). 4. B. Straws, Adv. Cancer Res. 45, 45 (1985). 5. E. C. Miller and J. A. Miller, in “Chemical Carcinogens” (C. E. Searle, ed.), pp. 737-762. American Chemical Society, Washington, D.C., 1976. 6. P. D. Lawley, in “Chemical Carcinogens and DNA” (P. L. Glover, ed.), pp. 1-36. CRC Press, Boca Raton, Florida, 1979. 7. J. K. Selkirk and M. C. MacLeod, Biosci. 32, 601 (1982). 8. D. T. Beranek, Mutat. Res. 231, 11 (1990). 9. B. Singer and D. Grunberger, in “Molecular Biology of Mutagens and Carcinogens,” Plenum, New York, 1983.

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Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides JEANPAULLEONETTI, GENEVIBVE DEGOLS,JEAN NADIR PIERRECLARENC, MECHTI, AND BERNARD LEBLEU~ UA CNRS 1191 G d d t q u e Mol&ulaire Unioersitd de MontpeUier I1 Sciences et Techniques du Languedoc 34095 Montpellier Cdder 5, France Historical Background . . ............................ AIGE Concept . . . . . . . From the Antisense Approac Limitations of the SNAIGE Approach ...... Internalization and Targeting of Oligonucleoti Intracellular Distribution of Oligonucleotides Mechanisms of Action of Antisense Oligonucl in the VSV Model ....................... VII. Conclusion and Perspectives .............. References .............................

I. 11. 111. IV. V. VI.

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1. Historical Background Down-regulating or specifically turning off the expression of individual genes represents a powerful tool for studies of their biological role in both in uitro cell cultures and in uivo in animal or plant models. This should also have great potential for therapeutic applications, as in the search for more selective antiviral or antineoplasic drugs. The antisense concept, in which mRNA translation is controlled by chemically synthesized complementary oligonucleotides (or oligomers), originated as early as 1967 (1).At about the same time, the DNA-mRNA hybridarrested cell-free translation assay was reported (2), while Zamecnik and Stephenson described the control of Rous sarcoma virus expression by synthetic oligodeoxynucleotides (3). It has since been realized that gene expression may be finely tuned in prokaryotes by complementary RNAs. This particular strategy has been used frequently for the control of bacterial plasmid expression. Although it is not our purpose to review this broadly documented field (see 4 for a recent 1

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review), two aspects appear important to mention. First, various strategies have been exploited; these include translation arrest by complementary RNA, RNase-III-mediated degradation of the target RNA, and transcription interference. Second, extensive genetic analysis has led to the unexpected “kissing” model, which postulates an interaction between complementary sequences initiated at the tip of a stable loop structure in the antisense moiety (5). To our knowledge, this has not been exploited for the design of synthetic RNA or DNA antisense oligomers. A broader approach to specific gene regulation has been demonstrated by Inouye (prokaryotes) and by Weintraub (eukaryotes) in which the control of various genes is achieved in cells transfected (or microinjected in the case of Xenopus laevis oocytes) by complete genes or gene fragments inserted in inverse orientation downstream from various promoters (reviewed in 6-8). Several transgenic species with an altered gene expression pattern have been obtained as well. As an example, altered phenocopies have been obtained by the injection of antisense RNAs in Drosophila embryos (9). Similar studies with plants appear to be one step closer to potential applications, i.e., the successful down-regulation of the polygalacturonase gene in transgenic tomato plants results in a better control of fruit ripening (10). Paralleling these advances in genetic manipulations, rapid progress in oligomer chemistry has rendered the automated synthesis of oligodeoxyribonucleotides accessible to nonspecialized laboratories, using either phosphoramidite (11)or, more recently, hydrogen-phosphonate chemistry (12). This has stimulated a growing number of studies, demonstrating the potential of the antisense oligomer approach (reviewed in 13-15). Reliable protocols for the synthesis of oligoribonucleotides have only recently emerged, due to the difficulty of devising appropriate protecting groups for the 2’-OH group of the ribose moieties (reviewed in 16). Initial successes have also allowed the synthesis of oligomer analogs with modifications within the internucleotidic linkages, the sugar moiety, or more recently, the base. Derivatization of oligomers with reporter groups, peptides, or lipids at various positions is also possible (reviewed in 17). Collaborative efforts of chemists and biologists both in the academic arena and in an increasing number of companies have been devoted to the potential diagnostic and therapeutic applications of nucleic acids; this will undoubtedly lead to more progress in the near future. Whether transfection of antisense genes (or of genes coding ribozymes) or administration of chemically assembled synthetic oligomers will have the brighter future cannot now be ascertained. The first approach bears the problems and the potential of gene therapy. The latter is closer to the problems classically encountered in drug use, e.g., target access and recognition, metabolism, and toxicity; these are reviewed in Section 111.

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II. From the Antisense to the SNAIGE Concept Since these early pioneering efforts, the antisense concept has been vastly expanded. Apart from being antimessengers, synthetic oligomers provide several other strategies for the artificial regulation of gene expression. These include triple-helix formation, inhibition of protein binding, and artificial nucleases, such as ribozymes. We therefore propose SNAIGE (Synthetic or Small Nucleic Acids Znterfering with Gene Expression) as a generic term to describe more appropriately these various approaches (Fig. 1).

A. The Antisense Approach Most of the early studies were performed with oligodeoxyribonucleotides complementary to mRNAs; hence, the antimessenger or antisense concept. Although extensive studies have been performed in both cell-free extracts and intact cells (i.e., X. Zaeuis oocytes), the precise mechanisms involved have seldom been unraveled. A physical interaction between sense and antisense sequences was proposed initially, either at the mRNA level or at the pre-mRNA level in eukaryotic cells. This in turn would lead to an inhibition of mRNA processing, nucleocytoplasmic transport, or mRNA translation. Alternatively, RNA.DNA hybrids are substrates for cellular or viral RNase H, which hydrolyzes the RNA strand of these hybrids and effectively converts antisense oligomers into powerful sequence-specific nucleases. The precise role played either by physical interaction or by RNase-H-mediated cleavage in intact cells is still a matter of conjecture and might vary with the biological system under consideration; these aspects have been reviewed in detail recently (18) and are discussed in Section VI. Likewise, it has generally been difficult to assess whether the primary site of action of an antisense oligomer is within the cytoplasm (e.g., mRNA translation) or the nucleus (e.g., pre-mRNA maturation and transport) (see also Section V).

B. The Triple-Helix Concept The association of a homopyrimidine DNA sequence in the major groove of duplex DNA was originally demonstrated in 1957 (19). Hydrogen bonding can be achieved between thymidine or protonated cytosine in the third strand and, respectively, the conventional A-T and G-C base-pairs in the double helix, following the so-called Hoogsteen rules. This results in the sequence-specific annealing of a pyrimidine sequence to homopurine-homopyrimidine tracts in DNA. Interestingly, appropriate sequences appear to be relatively widespread around regulatory regions in natural genes, thus opening prospects for the control of gene expression. This could, in princi-

dsoligomer

m

deDNA

Oligomer

m

mRNA

Oligomer

m

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ple, take place in various ways. First, a stable triple helix could block the progression of RNA polymerase or interfere with the recognition of DNA binding proteins essential for transcription initiation or for the integration of a viral genome, to give just a few examples. Second, synthetic oligomers forming triple helices could be used to concentrate various DNA-binding drugs, thereby greatly increasing their specificity; the latter might be as diverse as intercalating, cross-linking, or DNA-hydrolyzing groups. The feasability of these various approaches has been documented in cellfree model systems using photocrosslinking (20) or cleavage (21) agents (see also 18 and 22 for reviews). The restrictions imposed by the initial Hoogsteen pairing rules might be overcome, since additional possibilities are being found; for example, a purine-rich oligomer can be associated in antiparallel orientation to the pyrimidine strand of double-stranded DNA through G.(T.A) hydrogen bonds (23). Another potential limitation to the formation of a triple helix in intact cells resides in their high sensitivity to pH and ionic conditions. Additional rules allowing stable annealing at physiological pH have now been demonstrated (24) and allow the sequence-specific control of c-myc gene transcription in intact cells (25).

C. Synthetic

Ribozymes

Studies on the structures and autocatalytic properties of plant and animal ribozymes have defined the consensus sequences required for their nuclease activity; the best defined is the “hammerhead structure (reviewed in 26). Other ribozymic activities have also been described as an essential step in delta hepatitis virus multiplication in human hepatocytes (27). Although the alignment of the catalytic moiety and of the cleavage site is provided by intramolecular hydrogen-bonding in the natural ribozymes, both entities may belong to separate molecules. Synthetic hammerhead ribozymes can thus be engineered for the sequence-specific cleavage of any complementary RNA sequence; the only target requirement is a G-U-H sequence (H = “not G”) on the 5’ side of the cleavage site (28). The feasability of the approach has been demonstrated in cell-free experiments on various targets. Ribozyme-mediated degradation of various RNAs has also been demonstrated in cell cultures, although problems dealing with cleavFIG. 1. The SNAIGE concept: different strategies can potentially be used to inhibit gene expression by synthetic oligomers. (1) Competitionof transcription factors with double-stranded oligomers. (2) Competition with transcription factors, or transcription blockage with triplehelix-forming oligomers. (3) Inhibition of the mRNA functions with antisense oligomers. (4) Cleavage of the mRNA by a ribozyme. (5) Competition of RNA binding proteins (i.e., transactivators) with synthetic ribonucleotides.

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age yield and stability of the catalytic RNA still must be solved (for example, 29, 30). Progress in oligomer chemistry now allows for the design of composite synthetic ribozymes combining ribo- and deoxyribonucleotides. A better knowledge of ribozyme fine structure and mode of action will obviously be helpful in defining simple and more efficient structures for possible therapeutic use.

D. Sense Oligonucleotides Gene expression is conditioned by sequence-specific interactions between nucleic acids and regulatory proteins at both genomic and mRNA levels. Synthetic double-stranded or single-stranded oligomers can thus be used as competitors for these proteins. This approach obviously requires appropriate knowledge about the sequence and the structure of these protein binding sites. As an example, double-stranded oligomers or analogs thereof have been introduced into cells by direct microinjection (31)or incubated with intact cells (32), and have been successful in regulation of gene expression, most probably through competition with transcription regulatory proteins. Trans-activating proteins represent another class of possible targets, e.g., Tat and Rev, two gene-regulating proteins expressed in HIV-infected cells. Increasing knowledge of the structural elements essential for the interaction between these proteins and their RNA binding sites, e.g., TAR (33)and RRE (34),will allow the design of competitor oligomers. A last example illustrates both the potential and some of the pitfalls of synthetic oligomers. Phosphorothioate oligomer derivatives designed as antisense oligomers aiming at interfering with HIV expression turned out to be efficient but non-sequence-specific inhibitors of the viral reverse transcriptase through competition with genomic RNA for the enzyme template binding site (35).

111. limitations of the SNAIGE Approach As summarized above, the SNAIGE concept is straightforward and, in principle, should give rise to specific gene-expression modifiers. However, difficulties have often been underevaluated, giving rise to a flurry of illcontrolled data and of failures, as well as undisputed successes. The main problems encountered in in uitro utilization of synthetic oligomers deal with metabolic stability, cell penetration, intracellular distribution, availability of the nucleic acid or protein target, and processing of target-oligomer complexes; these points are dealt with in this section. Further, in uiuo applications must cope with large-scale production and manufac-

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turing costs, toxicology, mutagenicity, immunogenicity, and body distribution; this has been discussed in a few recent publications (36,37).Finally, we have contributed to the development of antibody liposomes (vide infru) as a first step toward site-specific delivery of the encapsidated antisense oligomers.

A. Target Choice The choice of a target sequence is not always easy, due to (1)a lack of knowledge about the three-dimensional structure of most RNA targets within their natural environment; (2) the near impossibility of predicting accessible nucleic acid sequences within ribonucleoprotein complexes or chromatin structures; and (3) our as yet poor understanding of the rules governing sense-antisense or nucleic acid-protein interactions. Splice sites on premRNAs (38),as well as 5’ untranslated regions on mRNAs, generally appear as the most efficient targets in the antisense approach, but exceptions have been documented (reviewed in 18) (Fig. 2).

B. Metabolic Stability The metabolic stability of oligonucleotides is low due to the action of nucleases (mainly 3’-exonucieases) in extracellular fluids, endocytic compartments, and the intracellular environment. Various analogs modified at either the internucleotidic phosphate backbone [e.g., methylphosphonates (39) or phosphorothioates (40)],the sugar configuration [e.g., a-oligomers (41)or e’-O-methyl oligomers (42)],or the 3’ end have been synthesized and are adequate solutions to this particular problem. The abundant literature along these lines has been extensively reviewed recently (13, 17). Reconciling these modifications with the structural features required for sequencespecific nucleic acid recognition at a useful T,, and eventual processing of the hybrids by RNase H, have, however, turned out to be more difficult than initially foreseen. Moreover, little is still known about the toxicity and the mutagenicity of the metabolites arising from oligomer analogs. Let us illustrate these points with a few examples. The a-anomeric oligomer analogs can be assembled with good coupling yields using standard automated methods (43).They hybridize, with good T , values, to their complementary targets, although in parallel orientation (45), and are not recognized by most nucleases (44). However, 15-mer oligomers specific for vesicular stomatitis virus (VSV) or interleukin-6 are devoid of biological activity either in a cell-free translation assay or in intact cells (45);absence of processing of these DNA-mRNA hybrids by RNase H might be an explanation. On the other hand, an oligomer complementary to the cap site of P-globin mRNA inhibits its translation in reticulocyte lysates (46).Likewise an a-oligomer complementary to the primer binding site in pglobin mRNA, taken here as a model system, inhibits its transcription by

Nuclear events

0

'2,

Nuclear or cytoplasmic events

Cytoplasmic events

Ribosome

mRNA

FIG.2. Possible mechanisms of action of antisense oligomers. The mechanism of action is p r l y understood, but is generally supposed to inhibit translation. However, this inhibition can be direct or quite indirect due to interferences in the nuclei or the cytqplasm of the cells. The hybridization of the oligomer can change the mRNA structure and inhibit splicing (1)or nucleocytoplasmic transport (2). The mRNA oligonucleotide duplex can be recognized by RNase H and subsequently the mRNA can be degraded (3). When located at the 5' end of mRNA, the oligomer can inhibit the binding of translation initiation factors (4). The oligomers can also directly inhibit the translation of the mRNA by ribosomes (5).

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MoMuLV reverse transcriptase (47). Taken together, these experiments seem to restrict the use of a-oligomers as sequence-specific inhibitors to non-RNase-H-dependent effects. Methylphosphonate derivatives combine the two advantageous properties of being uncharged and resistant to nucleases. Yet their biological activity in various models is somewhat disappointing; they must be added to cell culture media in a 50-200 pM range (48). This might be due to lack of recognition by RNase H, cell penetration through diffusion rather than receptor-mediated endocytosis, or chirality of the modified phosphate backbone. Interestingly, psoralen methylphosphonate derivatives photoactivatable by UV become active in the same models around 5-10 pM (38). An alternative approach consists of the association of unprotected oligomers with drug delivery systems such as liposomes, lipoprotein particles, nanoparticles, or protein conjugates, as developed initially by our group (see Section IV).

C. Cell Uptake and lntracellular Compartmentalization Another problem encountered with the use of synthetic oligomers deals with cell uptake and intracellular compartmentalization (Fig. 3). Oligomers

I Antibody targeted liposome FIG. 3. Endocytosis of free oligomers as compared to their targeted counterparts. Oligomers are taken up by the cells by receptor-mediated or fluid-phase endocytosis. Poly(L1ysine)-conjugatedoligomers interact with the negative charges of the cellular membranes, and are taken up by nonspecific receptor-mediated endocytosis. On the contrary, oligomers encapsulated in antibody-targeted liposomes are taken up by specific receptor-mediated endocytosis. The oligomers accumulate in the endocytic compartments, and must escape from these compartments to reach their target in the cytoplasm or in the nuclei.

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are now believed to be taken up by pinocytosis and/or by receptor-mediated endocytosis after binding to cell surface proteins; several candidate receptors have recently been described and partially characterized (49, 50). Conjugation to synthetic polypeptides such as poly(L-lysine)(51)and to lipids such as cholesterol (52), or encapsidation in antibody-targeted liposomes (XI), increases cell uptake and biological efficacy, strongly suggesting cell uptake of unmodified oligomers as a limiting step; this point is detailed in Section IV. Whether oligomers are used in their free form or in association with the delivery systems mentioned briefly above, their internalization involves receptor-mediated (or fluid-phase) endocytosis. Escaping the endocytic compartments to reach intracellular targets in the cytoplasm or in the nucleus is another problem. Trapping of the oligomers in endocytic compartments and/or degradation by lysosomal nucleases might be a strong limitation to this approach. Tools allowing the cytoplasmic delivery of oligomers should overcome these potential problems. However, neutral methylphosphonate analogs that bypass endocytosis suffer other limitations, as outlined above. An unexpected feature of the intracellular behavior of oligomers has arisen from microinjection studies that indicate a rapid diffusion to the nucleus (%, 55). Whether this favors the interaction of synthetic oligomers with nuclear targets or segregates oligomers in the nuclei is not known and is discussed in Section V.

D. Fate of Oligonucleotide-Target Hybrids The fate of oligomer-target hybrids represents an additional ill-understood event with relevance to biological efficacy. As discussed above, physical association through hybridization could lead to the activation of endogenous RNase H with an expected increment in inhibitory activity (45, 56, 57). A new generation of oligomers engineered to destroy their target RNA (or DNA) or to covalently bind to them is now being studied in many laboratories. It includes oligomers linked to alkylating, free-radical generating, or photoactivable moieties (Fig. 4). Oligomers conjugated to intercalating drugs, with the aim of increasing the binding constant to their DNA or RNA target should also be mentioned (reviewed in 17 and 58). In addition, cells are equipped with a collection of RNA unwinding activities, particularly evident for the ribosome machinery. This would explain why translation elongation cannot be blocked by antisense oligomers unless RNase H is able to destroy mRNAs (Fig. 2). Unwinding and, more recently, unwinding-modifying activities have been documented in both X. Zueois embryos (59) and in mammalian cells (60). Such activities impair the biological efficacy of antisense RNAs in developing X. Zueuis embryos (59).It deserves additional study, since a better understanding of these unwinding

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-cross-linkingof the oligomer and the mRNA (alkylatingagents, psoralen) -alteration of the mRNA by the functionalized oligomer (porphyrin) -stabilizationof the interactionsbetween the oligomer and the mRNA (acridine) FIG.4. Strategies to increase the interaction properties of antisense oligomers.

activities might ultimately allow the design of oligomer analogs or of protecting groups preventing their recognition.

E.

Side Effects

Finally, we have to cope with often-unforeseen side-effects of oligomers and their analogs. Striking examples have been documented in various attempts to control in uitro HIV infection by synthetic oligomers. In & nouoinfected T-lymphocyte cell-lines, little sequence specificity of antisense oligomer was found. A polycytidylate with phosphorothioate internucleotidic linkages (SdC),, turned out to be the most active compound, with an EC50 (concentration giving a 50% inhibition) -0.5 pM (61). Non-sequencespecific biological activity probably results from competitive inhibition for substrate binding on viral reverse transcriptase (35)or from interferences with virus adsorption to the CD4 membrane receptor (62). Likewise, oligomer-cholesterol derivatives probably act through sequence-independent mechanisms. We have initiated a comparative study of 12-mer oligomers complementary to the Tat splice acceptor site in collaboration with the group of J. L. Imbach (Lab. Chimie Bio-Organique, Universitk Montpellier 11);our results also exhibit little or no sequence specificity, and the following order of efficiency: a or PS % a or Met P > p.

IV. Internalization and Targeting of Oligonucleotides As previously mentioned, one of the main problems in using synthetic oligomers is to get a sufficient number of molecules into the cells and prevent hydrolysis before they reach their target. Despite the presence of putative receptors at the cell surface (49,50), it seems that oligomers are not internalized very efficiently. Much interest has been devoted in our group to develop tools allowing protection of antisense oligomers against serum nu-

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clease degradation, efficient transfer across the plasma membrane, and the possible targeting to cells expressing specific determinants at their surface (Fig. 5). Conjugation to poly(L-lysine) (51) or encapsulation in antibodytargeted liposomes (53) have provided efficient ways to deliver oligomers into cells. The efficiency of cellular uptake of antisense oligomers was significantly improved. However, both of these tools present some limitations, as we discuss below.

Liposome -encapsulated oligomers Small unilamellar liposome

0 I

0-P-0 I

Oligomer linked b poly(L-Lysine) wc

NH,-CH-COO

I

w ? ) 4

I Ntl,

I (y%lr (NH -CH-COO\nNH-CH-COO(NH

I I

-CH-COO),NH

I

-CH-COOH

I

(CH3,

( 7 1 4

(7YI4

NY

NY

NHZ

FIG.5. Specific and nonspecific targeting of antisense oligomers. To increase their uptake in the cells, oligomers have first been synthesized with an adenosine at their 3' end. After oxidation of the ribose, the dialdehyde formed reacts with the a-amino groups of poly(r,-lysine), leading after reduction to an N-morpholine ring. Oligomers have also been encapsulated in small unilamellar liposomes linked to protein A. These liposomes can be efficiently targeted to cell surface determinants by monoclonal antibodies.

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A. Poly(L4ysine) Conjugation of Oligonucleotides Poly(L-lysine)is a well-known polypeptidic carrier; it has been used to potentiate the uptake of various drugs and macromolecules such as methotrexate and horseradish peroxidase (63, 64).We have conjugated oligomers to this carrier in order to potentiate their antisense properties. Oligomers were chemically linked to the €-amino groups of polylysine through an Nmorpholine ring following periodic acid oxidation and borocyanohydride reduction of their 3' end ribose (65). The best results were obtained by coupling oligomers ranging in size from 10 to 15 nucleotides to M , 14,000 polylysine. Statistically, the 14-mer sequence occurs once in the RNA of a higher eukaryote. 3'-Modified natural oligomers have been used throughout in order to minimize problems and possible adverse effects associated with alternative chemistries.

B. Biological Activity of Oligonucleotide-Poly(L-lysine) Conjugates The efficiency of polylysine conjugates was illustrated in several biological models (51, 66-69). Initially, conjugated oligomers of the appropriate sequence were demonstrated to inhibit VSV multiplication (51, 65). More recently, similar approaches were successful in developing an antiproliferative activity with anti-c-myc oligomers (67') or to decrease the cytopathic effects of HIV-1 in de nouo-infected MT4 T-lymphocytes (66). Polylysine-conjugatedoligomers complementary to the 5' end of the VSV N protein mRNA are 10 to 50 times more active than unconjugated oligomers on L929-infected cells. Sequence-specific antiviral activities of such conjugates are observed at concentrations lower than 1pM (as summarized in Table I; see also 51). As a point of comparison, methylphosphonate derivatives inhibit VSV expression in a 50 pM concentration range (39). In the c-myc oncogene model, it is important to mention that nonconjugated oligomers only exhibit biological efficacy when incubated in a culture medium devoid of serum nucleases (as obtained by heat decomplementation of the serum or by omitting the serum at the time of experimentation). In contrast, polylysine-conjugated oligomers are active without any manipulation of the culture medium (67). The mechanisms through which polylysine increases the antisense effect of oligomers are not clearly understood. We have demonstrated that the uptake of fluorescently tagged oligomers conjugated to polylysine is accelerated and increased as compared to unconjugated material (70). The conjugate is taken up by a nonspecific receptor-mediated endocytic pathway. It seems to accumulate in acidic compartments, where proteolysis of the carrier would release some oligonucleotide material. Data involving pOly(Dlysine) and inhibitors of the endocytic pathway are in line with this scheme. Other effects of poly(L-lysine) cannot be excluded in the potentiation of the

TABLE I EFFECTSOF ANTISENSE NUCLEOTIDESON VSV MULTIPLICATION^

Oligomer target

5’ end of N mRNA 5’ end mismatch of N mRNA Internal site of N mRNA Intergenic region (-) Control oligomer Viral polymerase binding site

VSV reduction

++ + ++ -

In uitro translation

In uitro transcription

Primary viral transcription

Viral transcription

+

-

-

++

-

nd nd

nd nd -

nd nd

++ nd

-

nd

-

nd

++ nd

015-Mer oligonucleotides complementary to various sites on VSV mRNAs or genomic RNA have been compared for inhibition of antiviral activity, translation in reticulocyte lysates complemented with RNase H, cell-free transcription from isolated virions, primary transcription in actinomycin D-treated cells, and total virion transcription in cells. + +, Drastic (10- to 100-fold reduction) inhibitory activity; +, moderate (
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biological activity of conjugates. Steric hindrance of the polylysine moiety and modifications of the oligomer at its 3' end should protect it against nucleases. It should be recalled here that phosphodiesterases that degrade nucleic acids in a processive manner from their 3' end play a major role in oligomer catabolism. Whatever the fate of the polypeptide moiety after cell uptake, we are left with a 3'-modified oligomer. A contribution of the positively charged lysine residues to the hybridization of the oligomer with its target could also be considered (711, provided that some lysyl residues remain associated to the oligomer after passage through the endocytic compartments. Poly(L-lysine)conjugation thus constitutes an interesting alternative to increase the penetration of oligomers; nevertheless, polylysine is not devoid of cytotoxicity above certain doses, with large variations among cell lines. Moreover, since oligomer-polylysine conjugates are not hlly protected against nucleases, antisense effects of such conjugates are temporary; conjugation of oligomer derivatives with a nuclease-resistant backbone has not yet been evaluated. Poly(L-lysine) has also been used recently in gene transfection studies since protein conjugation of the polycationic moiety allows its targeting and endocytosis in defined cell lines (72, 73). Plasmid DNA has been condensed to polylysine modified with transferrin or asialo-glycoprotein residues and efficiently internalized in the targeted cell. This offers interesting prospects for the selective introduction of plasmids expressing antisense RNA or ribozymes. However, the efficiency of these conjugates needs to be improved. Using the same experimental conditions, we have obtained efficient targeting specificity of the SV40 genome; nevertheless, preliminary results suggest that only a few percent of the cells express large-T antigen. It remains to be ascertained if these results are due to the competence of the K562 cells for large-T antigen expression, or are a consequence of a low uptake of the SV40 genome.

C. The Biological Activity of Poly(L-lysine) Conjugates Is Potentiated by Polyanions Polyanions, such as heparin, dramatically reduce the cytotoxicity of polylysine, even at high concentrations (74);this we confirmed by monitoring cell growth or release of radioactivity from cells loaded with 51Cr (Fig. 6). We thus evaluated the biological activity of polyanions complexed with oligomer-polylysine conjugates (67). Polyanions appear to be beneficial to the antisense approach in various ways. They potentiate the sequence-specific activity of these conjugated oligomers directed against c-myc: the active oligomer concentration is significantly reduced and the antiproliferative activity is maintained for longer periods of time.

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-!! 6 Q)

c

6

u)

1

2

time

3

4

5

(hours)

FIG. 6. Cell permeation due to poly(L-lysine). L929 cells were loaded (200,000 cellslml) with 5 W r for 14 hr and then treated with polylysine (2 pM)or polylysine + heparin (100 pg/ml). Release of 51Cr was measured at different times atter treatment. U, Untreated cells; A, PLL treated cells; 0 , PLL + heparin-treated cells.

It is worth mentioning that sulfated polyanions, such as heparin, have been extensively explored as potential antiviral agents against herpes simplex virus, VSV, or HIV (75, 76). Taken together, the intrinsic antiviral effects of sulfated polyanions and the enhanced antisense effects of ternary complexes might lead to interesting new prospects in antiviral chemotherapy. In a preliminary study, we have evaluated the efficiency of such ternary complexes upon HIV-1 infection. In the presence of heparin, polylysineconjugated oligomers directed to the 5' end of Tat mRNA promote strong antiviral effects in HIV-1 de mo-infected MT4 cells: no reverse transcriptase activity could be detected in cell supernatants for more then 2 months (G. Degols, J. P. Leonetti, and B. Lebleu, unpublished observations). This inhibition is strictly sequence-specific,and preliminary results suggest cooperative effects between heparin and the conjugates.

D. Cell Targeting of Antisense Oligonucleotides through Antibody-bearing Liposome Encapsulation Despite the increased biological activity of antisense oligomer-polylysine conjugates, this approach lacks specificity in cell recognition and the

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conjugated oligomers still remain sensitive to nucleases. We have examined, in collaboration with the group of L. Leserman (Centre dImmunologie INSERM-CNRS de Marseille-Luminy) an alternative strategy for increasing cell association and enhancing the resistance to nuclease degradation of oligomers, e.g., their encapsulation in liposomes (53). These lipid vesicles, by virtue of their limited permeability, restrict access by the milieu and protect their content against enzymatic degradation. Liposomes may also be coupled to various ligands that permit their targeting to defined cell populations. We have used small unilamellar liposomes whose phosphatidylethanolamine moiety has been covalently coupled to Staphylococcus aureus protein A (Fig. 5) (77). This provides a versatile drug carrier, since the cell surface determinant to which they bind will be defined by adding the cognate antibody. Moreover, chemical coupling of antibodies sometimes impairs their antigenic potential (unpublished observation). These small liposomes are taken up by receptor-mediated endocytosis and release the encapsulated product intracellularly(78).These liposomes can be used for the intracellular delivery of various polynucleotide material such as (2’-5’)(A)n (79), doublestranded RNAs (80), or antisense oligomers (53). Liposomes containing oligomers complementary to the 5’ end of VSV N protein mRNA have been targeted to L929 cells with an H-2K-specific antibody, and inhibit VSV infectivity: a dose-dependent reduction in VSV titer was observed at an oligomer concentration well below 1pM.Antiviral effects are thus observed in the same concentration range as for oligomer-polylysine conjugates. Liposome encapsulation fully protects the oligomer from degradation by nucleases, as expected. Incubation of the liposomes with DNase-I at a concentration of 50 pglml did not inhibit their antiviral potential, while in the same conditions the antiviral activity of oligomer-polylysine conjugates was largely diminished. Similar to that observed for polylysine conjugates, the antiviral effect from liposome-encapsulated antisense oligomers remains sequence-specific. Moreover, the antiviral effect was strictly dependent on the use of a monoclonal antibody specific for the H-2K molecules expressed by the target cells; no inhibition was observed if incubation occurred in the presence of antibodies of the same class that did not bind to the cell surface. Thus antibodybearing liposomes containing antisense oligomers allow a double specificity, since a particular cell is selected by the targeting antibody and a particular mRNA in the cell is selected by sequence complementarity with the oligomer. Owing to the versatility of this system, liposome delivery of oligomers can in principle be adapted to other biological models. Preliminary results show that anti-HIV antisense oligomers encapsulated in liposomes targeted to the CD7 determinant are efficiently internalized in a human T-lympho-

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cyte cell line (G. Degols, J. P. Leonetti, and L. Leseman, unpublished observations). Moreover, other types of nucleic acid material, such as ribozymes, could be delivered with liposomes. It is also worth mentioning that liposomes targeted to CD3 have been used with success to internalize anti-HIV antisense RNAs (81). Unfortunately, a major drawback to the use of these small unilamellar liposomes for the delivery of nucleic acid material is their poor encapsulation efficiency (around 3%); work aiming at increasing encapsulation is in progress.

V. lntracellular Distribution of Oligonucleotides The antisense concept was initially based on the assumption that oligomers might interact with mRNA to block their translation. However the actual site of action of antisense oligomers has not been firmly established in intact cells. Since the targeted sequence is present in the mRNA but also in the primary transcript, interference with pre-mRNA maturation or nucleocytoplasmic transport could also be envisaged. Free, polylysine-conjugated or liposome-encapsulated oligomers are internalized through an endocytic pathway (Fig. 3). Indeed, punctuated cytoplasmic labeling, which is characteristic of accumulation in the endocytic vesicles, is observed when fluorescently tagged oligomers are incubated with cells. How oligomers escape these vesicles to reach their cellular target remains a matter of conjecture. Moreover, conflicting results about the cellular distribution of oligomers have been reported (50, 82, 83). We decided to avoid the problem of their internalization pathway and ascertain their intracellular location by microinjection of oligomer into the cytoplasm of somatic cells (84).A 1 8 m e r oligomer conjugated at its 5’ end to tetramethylrhodamine isothiocyanate accumulates in the nuclei of microinjected REF-52rat embryo fibroblasts. This location is apparently not influenced by the technique of cell fixation, by the nature of the fluorochrome, or by the release of this molecule. Experiments performed under inverted fluorescent microscopy on living cells reveal the swiftness of this phenomenon: translocation to the nuclei is nearly complete 1 min after microinjection. This process remains unaffected in its rate and magnitude upon ATP depletion or low-temperature incubation. These arguments favor a diffusion process through the nuclear pores, compatible with the small size of the oligomers. However, the accumulation of nuclear material implies the existence of nuclear binding sites. First steps in the identification of oligomer binding sites have been made. Photosensitive oligomers incubated with isolated nuclei bind a small number of nu-

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clear proteins ranging from 36 to 48 kDa (84).Although the negative charge of the oligomer seems to be involved in these interactions, highly basic proteins such as histones do not participate in the accumulation process. These results are in agreement with other studies (54)showing that oligomers colocalize with snRNP when these proteins are immunologically detected. We have tried to repeat this observation. Hypertonic treatment of cells during interphase and mitosis inhibits the intracellular transport of snRNP. However, when fluorescently tagged oligomers were microinjected in REF-52 cells cultured for 2-4 h in an hypertonic medium, no change in their cellular distribution was observed, while the location of snRNP detected by immunofluorescence became more cytoplasmic and diffuse (J. P. Leonetti et al., unpublished observations). However, these data should be evaluated with care, as the nature of the binding proteins remains to be ascertained. Indeed, a partial depletion of nuclear snRNP can be insufficient to change oligomer location, in the case when cytoplasmic unassembled snRNP does not bind oligomer. These observations provide interesting prospects for a better understanding of the mechanisms of action of antisense oligomers. However, it remains to be shown that microinjection really mimics the fate of oligomers added to the culture medium. It seems that there may be several mechanisms of action of oligomers. Although various data have demonstrated the biological activity of oligomers specifically directed to nuclear targets, we have observed antisense oligomer inhibition of the multiplication of VSV, a virus with a replicative cycle restricted to the cytoplasm.

VI. Mechanisms of Action of Antisense Oligonucleotides in the VSV Model At the beginning of “antisense history,” oligomers were generally designed to interfere with mRNA translation; hence molecules were also called “antimessengers.”Their potential and mechanisms of action have been well defined in cell-free translation systems and in X.Zueuis oocytes. As an example, the control of globin mRNA translation by natural and modified antisense oligomers in cell-free extracts, or in microinjected X. Zaeuis oocytes, has been extensively studied and can be schematized as follows (see 18 for a review). Oligomers complementary to the region between the cap site and the initiator AUG codon can block the translation machinery in an RNase-Hindependent mechanism. This has been demonstrated either by using mammalian reticulocyte lysates (which are rather poor in endogenous RNase H) or by using oligomer analogs (methylphosphate derivatives or a-anomeric oligomers)whose hybrids are not processed by RNase H. Once the translation machinery has been completely assembled (e.g., downstream from the

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initiation codon), unwinding activities associated with the ribosomes will process the hybrids and render antisense oligomers inefficient unless RNase H has hydrolyzed the target RNA. On intact cells, however, little is known about the actual mechanisms of action of oligomers, although a large number of genes have been inhibited using this concept. The presence of oligomers in the cytoplasm, their hybridization with the targeted mRNA, and the mechanism of translation inhibition have generally not been formally proved. A direct answer will probably have to await tools to analyze the intracellular distribution of RNase H activity, as well as genetic manipulation of its content and/or activity in intact cells other than X. Zueois oocytes. Furthermore, if an oligomer can potentially act at the translation level, it can also interfere with other stages of mRNA maturation such as splicing, nucleo-cytoplasmic transport, metabolic stability, etc. (Fig. 2). Assays with control oligomers that cannot hybridize to the targeted mRNA are most cwmmonly used to demonstrate the specificity of action of antisense oligomers. However, such controls remain imperfect, since these oligomers can potentially hybridize to other cellular sequences or inhibit the action of various enzymes. The specificity of action of an antisense oligomer is more adequately proved using sequence variability at the targeted mRNA. For example, the common sequence at the 3' end of all viral mRNA is different in type B and type A influenza viruses. A heptanucleotide linked to an acridine derivative complementary to the type A 3' end of viral RNAs inhibits the type A virus cytopathic effect but has no activity on the type B influenza virus (85). We have recently obtained similar results with HIV-1 isolates diverging by only two nucleotides at the targeted sequence in Tat mRNA. Each of these isolates can be inhibited with a polylysine-conjugated oligomer targeted against the fully complementary Tat sequence, while the same oligomer is inefficient on the divergent isolate (G. Degols, J. P. Leonetti, and B. Ixbleu, unpublished observations). However, these controls do not lead to a better understanding of the antisense oligomers mechanism of action. We have tried to define more precisely the actual target in two models. An anti c-myc oligomer-polylysine-heparin complex specifically decreased c-myc mRNA levels, as compared to glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA (66). Similar observations have been made on the c-myb model (86),and can be explained by the degradation of the mRNA by RNAse H. However other mechanisms, which do not involve the action of RNAse H, can be considered, such as a decrease of mRNA stability due to conformational changes induced by oligomer hybridization. We have also tried to explain the action of synthetic oligomers linked to polylysine on VSV multiplication. In this model, several potential targets on

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genomic RNA and mRNA sequences were tested in cell-free assays and in infected cells (see Table I for an outline of the VSV multiplication cycle and a summary of these data). As already observed in other systems, oligomers directed to the 5' end of the nucleocapsid (N) protein mRNA were the most effective on infected cells. In reticulocyte lysates complemented with RNase H, however, an oligomer complementary to the internal coding site of N protein mRNA was more efficient in inhibiting translation than an oligonucleodite spanning the 5' end. This points out that cell-free translation experiments cannot predict and do not necessarily reflect the inhibition process on intact cells. Moreover, complementation of reticulocyte lysates with RNase H does not necessarily mimic the endogenous RNase H activity, as discussed above. An oligomer complementary to the intergenic regions on viral genomic RNA also promotes strong antiviral effects (63). These intergenic sequences are implicated in the transcription attenuation process; however, the complementary oligomer is unable to interfere with virus primary transcription in intact cells or in cell-free transcription assays directed by the virionassociated RNA polymerase (65). An action at the replicative level could account for these antiviral effects. On the other hand, oligomers complementary to the VSV polymerase binding site are totally inactive. This could be explained by the inaccessibility of this genomic RNA region, which is indeed largely protected by nucleocapsid proteins. This study reflects one of the main problems encountered in the use of the antisense approach: the choice of the targeted sequence. The knowledge of the mode of action of oligomers is still rather fragmentary and may be different from one targeted gene to another. Moreover, mRNA secondary structures and their interaction with proteins are important points to consider.

VII. Conclusion and Perspectives As briefly reviewed here, synthetic nucleic acids interfering with gene expression (SNAIGE) are being developed as specific regulators of gene expression both for fundamental studies and/or for potential (medical) applications. Although straightforward in principle, the approach still faces many unknowns. Cell-free studies with model systems have helped us to learn much about the various modes through which oligonucleotides bind to their nucleic acids or protein targets. Increasing numbers of modifications allowing improved resistance toward nucleases or more efficient interaction with various targets have been devised and more will be forthcoming. A most exciting prospect has been enabled by synthetic material: the normal

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backbone of DNA has been replaced by a polyamide linkage, giving rise to “polyamide nucleic acid” (87), which retains specificity in base recognition. It is our feeling that much effort should be concentrated on the mechanisms through which these small nucleic acids, in their various configurations, operate in intact cells. Although a large number of genes have been successfully down-regulated in various biological systems, the underlying mechanisms have indeed seldom been pinpointed. The tools of cell biology will help us in solving pending questions, such as intracellular routing of SNAIGE, involvement of RNase H in their action, and accessibility of various sites in mRNAs, pre-mRNAs, or DNAs in their cellular environment. This will, we hope, help us to devise new tools on a more rational basis and to choose the best strategy in each case. Moving to in uiuo experiments as a prelude to possible applications in humans will be the next step; experiments along these lines have already begun. Again, more knowledge about SNAIGE behavior in various cellular contexts will have to be gained. Topical applications, as in the treatment of papilloma or herpes viral infections, might be a realistic goal for the coming years. Most other targets will, at least according to our feeling, require the use of appropriate delivery systems.

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47. C. Boiziau, N. T. Thung and J. J. ToulmB, PNAS 89, 768 (1992). 48. C. H. Agris, K. R. Blake, P. S. Miller, M. P. Reddy and P. 0. P. Ts’o, Bchern 25, 6268 (1%). 49. L. A. Yakubov, E. A. Deeva, V. F. Zarytova, E. M. Ivanova, A. S. Ryte, L. Yurchinki and V. V. Vlassov. PNAS 86, 6454 (1989).

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57. J. Minshull and T. Hunt, NARes 14, 6433 (19%). 58. D. G. Knorre, V. V. Vlassov and V. F. Zarytova, in “Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression” 0. S. Cohen, ed.), p. 173. MacMiUan, London, 1989. 59. B. L. Bass and H. Weintraub, Cell 55, 1089 (1988). 60. R. W. Wagner, J. E. Smith, B. S. Cooperman and K. Nishikura, PNAS 86, 2647 (1989). 61. M. Matsukura, K. Shinozuka, G. Zon, H. Mitzuya, M. Reitz, J. S. Cohen and S. Broder, PNAS 84,7706 (1987). 62. C. A. Stein, L. H. Neckers, B. C. Nair, S. Mumbauer, G. Hoke and R. Pal,J . Acq. Zmm. Defic. Syndr. 4, 696 (1991). 63. H. J. P. Ryser and W. C. Shen, PNAS 75, 3867 (1978). 64. W. C. Shen and H. J. P. Ryser, PNAS 75, 1872 (1978). 65. J. P. Leonetti, B. Rayner. M. Lemaitre, C. Gagnor, P. G. Milhaud, J. L. Imbach and B. Lebleu, Gene 72, 323 (1989). 66. G. Degols, J. P. Leonetti, P. Milhaud, N. Mechti and 8. Lebleu, Antioir. Res. 17, 279 (1992). 67. G . Degols, J. P. Leonetti, N . Mechti and B. Lebleu, NARes 19, 945 (1991). 68. M. Stevenson and P. L. Iversen, J . Gen. Virol. 70, 2673 (1989). 69. P. Westermann, B. Gross and G. Hoinkis, BBA 48, 85 (1989). 70. J. P. Leonetti. G. Degols and B. Lebleu, Bioconj. Chem. 1, 149 (1990). 71. H. B. Levy, G. Baer. S. Baron, C. E. Bruckler, C. Gibbs, M. J. Ladamla, W. T. London and J. Rice, J . Infect. Dis. 132, 434 (1975). 72. M. Zenkz, P. Steinlein, E. Wagner, M. Cotten, H. Beug and M.L. Birstiel, PNAS 87,3655 (1990). 73. C . Y. Wu and C. H. Wu, JBC 162, 4426 (1987). 74. D. M. L. Morgan, J. Clover and J. D. Pearson, J . Cell. S c i . 91, 231 (1988). 75. M. Ito, M. Baba, A. Sato, R. Pauwels, E. De Clerq and S. Shigeta, Antioir. Res. 7, 361 (1987). 76. M. E. Gonzales, B. Alarcon and L. Carrasm, Antimicrob. Agents Chemother. 31, 1388 (1987). 77. L. D. Leserman, J. Barbet, F. M. Koulrilsky and J. N. Weinstein, Nature 266, 602 (1980). 78. P. Machy and L. D. Leserman, BBA 730, 313 (1983). 79. B. Bayard, L. D. Leserman, C. Bisbal and B. Lebleu, EJB 151, 319 (1985). 80. P. G . Milhaud. P. Machy, B. Lebleu and L. Leserman, BBA 987, 15 (1989). 81. K. Renneisen, L. D. Leserman, E. Matthes, H. C. S ch r aer and W. E.G. Miiller. JBC 265, 16337 (1990). 82. J. Goodchild, R. L. Letsinger, P. S. Sarin, M. Zarnecnik and P. C. Zarnecnik, in “Human Retrovirus, Cancer and AIDS: Approaches to Prevention and Therapy,” p. 423. Alan Liss, New York, 1988. 83. A. S. Boutorin, L. V. Gus’lova. E. M. Ivanova, N. D. Kobetz, V. F. Zaritova, A. S. Ryte, L. V. Yurchenko and V. V. Vlassov. FEBS Lett. 254, 129 (1989). 84. J. P. Leonetti, N. Mechti, G. Degols, C. Gagnor and B. Lebleu, PNAS 88, 2702 (1991). 85. A. Zerial, N. T.Thuong and C. HblBne, NARes 15, 9909 (1987). 86. A. M. Gewirtz and B. Callabretta, Science 242, 1303 (1988). 87. P. E. Nielsen, M. Egholm, R. H. Bergand and 0. Buchardt, Science 253, 1 (1991).

Enzyme Organization in DNA Precursor Biosynthesis CHRISTOPHER

K. MATHEWS

Department of Biochemistry and Biophysics Oregon State Unioersity Cornallis, Oregon 97331

I. Enzyme Organization and Contemporary Enzymology . . . . . . . . . . . . . . . 11. Early Evidence for dNTP Compartmentation ....................... 111. T4 dNTP Synthetase: A Multienzyrne Complex for Deoxyribonucleotide

Synthesis ......................................... IV. Is the T4 dNTP Synthetase Complex Linked to DNA Replication Machinery? ....................................... V. Enzyme Organization in Bacterial Cells ............................ VI. Organization of dNTP Synthesis in Eukaryotic Cells ................. VII. General Discussion .............................................. References .....................................................

167 171 177 181 186 187 200 20 1

1. Enzyme Organization and Contemporary Enzymology From its beginnings early in this century until about the mid-197Os, the principal goals of enzymology were the isolation of each enzyme in pure form, identification of the reaction catalyzed, characterization of the reaction mechanism, and identification of the metabolic role of the enzyme. The latter goal was accomplished largely through reconstitution of a metabolic process in uitro from purified components. Of course, it has always been apparent that the environment within which an enzyme functions in a living cell differs dramatically from the conditions of most in uitro experiments, where enzyme assays must often be conducted in very dilute aqueous solutions (1, 2, 3). Only in recent years, however, have approaches been developed for analyzing enzyme function and regulation within the cell. Some of these approaches include analysis of reaction flux rates by in uioo NMR or radioisotope-labeling procedures, metabolic experiments with mutants, immunocytochemical methods for intracellular enzyme localization, and a variety of techniques for analyzing protein-protein interactions. Thus, while enzymology maintains its traditional focus upon mechanisms of individual reactions, an equally important contemporary focus is upon learning where and how an enzyme functions in its natural environment. 167 Rogress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright 6 1993 by Academic Press, Inc.

AU rights of reproduction in any form reserved.

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A developing theme is the structural organization of functionally related enzymes. It has long been known, for example, through studies on membrane-bound enzymes such as mitochondrial electron-transport complexes, that many enzymes engaged in common processes are colocalized within cells. Similar patterns of enzyme organization have now been revealed in most metabolic pathways, including those catalyzed by readily soluble enzymes. DNA replication, the topic of this article, has been shown in both prokaryotic and eukaryotic systems to involve the cooperative function of numerous proteins associated at replication forks (4).In this review, I discuss the process by which DNA precursors are synthesized and delivered to replication sites and consider the possibility of physical or functional linkage between enzymes of deoxyribonucleoside 5‘-triphosphate (dNTP)’ synthesis and those directly involved in replication.

A. Patterns of Enzyme Organization Many metabolic pathways involve multifunctional enzymes or tightly associated multienzyme complexes (2). Examples of the former are the fattyacyl-CoA synthetase enzyme in vertebrate cells and the eukaryotic “CAD protein” and UMP synthetase, which catalyze the first three and last two reactions, respectively, of the six steps in de novo pyrimidine-nucleotide synthesis. Examples of tightly but noncovalently bound aggregates include the well-known pyruvate and a-ketoglutarate dehydrogenase complexes of mitochondria. Closer to the topic of this article, the two tetrahydrofolatedependent transformylase enzymes in purine-nucleotide synthesis form a complex with serine transhydroxymethylase and the trifunctional enzyme formyl-methenyl-methylene-tetrahydrofolatesynthetase (5). Srere (2) proposed that most metabolic pathways are catalyzed by organized multienzyme aggregates, which may be multifunctional enzymes or tightly bound complexes. Alternatively, these aggregates may be loosely bound complexes, which Srere calls metabolons. Attempts to isolate metabolons often fail, because forces that stabilize them do not survive the dilution or the ionic changes that occur when cell-free extracts are prepared by traditional means. The fact that intracellular protein concentrations are often as high as 350 mg/ml suggests the existence of many concentration-dependent enzyme associations that may not survive cell extraction. Such interactions need not involve only noncovalent associations among

1 Abbreviations: dNMP, dNDP, and dNTP, deoxyribonucleoside 5’-mOnO-, di-, and triphosphate, respectively; rNMP, rNDP, and rNTP, ribonucleoside 5’-mono-, di-, and triphosphate, respectively; hm-dCMP, hm-dCDP, and hrn-dCTP, 5-hydroxymethyldeoxycytidine 5’mono-, di-, and triphosphate, respectively; PAGE, polyacrylamide gel electrophoresis.

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soluble enzymes. Wilson, for example (6), has discussed the concept of “ambiquity”-the idea that key enzymes might exist either dissolved in the bulk aqueous phase or associated with a particulate structure. For example, brain hexokinase binds reversibly to the outer mitochondrial membrane, with the proportion of enzyme bound being related to control of the glycolytic flux rate. Consistent with a picture of enzyme organization that involves many noncovalent protein associations were the results of an important experiment by McConkey (0,showing that at least half of the proteins displayed in twodimensional gel electrophoretic resolution of total proteins from hamster and human cells are indistinguishable, one from the other, in terms of molecular weight and isoelectric point. This finding suggests that protein evolution involved constraints upon size and surface distribution of charged aminoacid residues that might have been imposed by the need to retain such interactions. The potential biological advantages of enzyme association are fairly obvious and might include: (1)maintenance of locally high metabolite concentrations without the need for physical compartmentation within a membrane; (2) protection of the solvation capacity of cell water, because average concentrations of most metabolites are kept low, even though local concentrations may be much higher; (3)efficiency of regulation of both enzyme synthesis and metabolic fluxes; and (4) relative rapidity with which a metabolic process can respond to changes in the intracellular environment. All of these characteristics are related to the presumed ability of metabolons to channel metabolic pathways, i. e., to facilitate the transfer of intermediates from one enzyme to the next, by restricting diffusion into the surrounding milieu. Because they are stabilized by weak interactions, metabolons-or loosely bound complexes-are difficult to isolate and purify in the native state. Hence, their existence is often difficult to demonstrate unequivocally, and the metabolon concept is thus somewhat controversial. If one is to convince skeptics, a range of complementary lines of evidence must be brought to bear. Suitable approaches include (1)demonstration of metabolic channeling in uitro, as shown by efficient passage of substrates through a multistep process or failure of a nonradioactive intermediate to dilute radioactivity from a radiolabeled precursor; (2) kinetic experiments supporting direct transfer of intermediates from enzyme to enzyme; (3) genetic evidence for the specificity of interactions observed; (4) colocalization of enzymes within cells; (5) studies on metabolite compartmentation in uiuo; (6) metabolic experiments with permeabilized whole cells; (7) protein crosslinking studies; (8)demonstration of interactions among purified individual enzymes thought to constitute a metabolon; and (9) immunological evidence, particularly the

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analysis of anti-idiotypic antibodies. All of these lines of evidence have been brought to bear upon the metabolic process that concerns us here, namely, the biosynthesis of DNA precursors.

B. Importance of Enzyme Organization in DNA Metabolism DNA replication in prokaryotic organisms is a particularly appropriate metabolic process in which to expect some pattern of enzyme organization (4). First, the deoxyribonucleoside triphosphates (dNTPs) play extremely limited roles outside their function as DNA precursors. Thus, unlike the ribonucleotides, which play multiple roles, the dNTPs need to be present in significant amounts only where they are used for DNA replication. Second, DNA chains grow rapidly, but at few intracellular sites. For example, an Escherichiu coli cell at moderate growth rates replicates its 4OOO-kbp genome in 40 min at two replication forks, each of which contains two growing DNA chains. One can readily calculate the growth rate per chain at about 850 nucleotides per second. A rapidly growing E . coli cell has six forks, with a correspondingly greater total rate of DNA synthesis, while a T4 phage-infected E . coli cell, which synthesizes DNA at up to 10 times the rate of an uninfected cell, has about 60 forks (8). One can argue qualitatively that dNTPs for DNA replication cannot be provided to these sites at sufficient rates by simple diffusion from remote sites of synthesis. We have estimated that, in a T4 phage-infected E . coli cell, the dNTP pools in the immediate vicinity of replication sites must turn over completely about 10 times per second (9). Third, prokaryotic replicative polymerases have rather high K , values, necessitating the maintenance of high dNTP levels at replication sites in order for DNA polymerases to be saturated. In T4-infected E . coli, DNA polymerase is almost certainly saturated with dNTPs in uiuo (10).However, in permeabilized T4-infected cells or in a replication system reconstituted from purified proteins, the concentrations of dNTPs needed to give maximal rates of incorporation in uitro are three- to fourfold higher than average dNTP concentrations estimated from pool measurements (11).These factors suggest the existence of dNTP concentration gradients at replication sites in uiuo. How might such concentration gradients be maintained? Some years ago we proposed that enzymes of dNTP synthesis could be organized into multienzyme complexes that were somehowjuxtaposed with replication sites (12). Such a complex could facilitate both the synthesis of deoxyribonucleotides and their flow into DNA. A similar proposal had already been made on the basis of quite different, but complementary, lines of evidence (13). In the remainder of this article, I summarize experiments that led to this proposal, describe this “dNTP synthetase complex” as studied in T4 phage infection, and discuss the extent to which the T4 model is appropriate for understand-

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ing relationships between dNTP and DNA synthesis in cells of higher organisms.

II. Early Evidence for dNTP Compartmentation A. Kinetically Separated dNTP Pools in E. coli Two important early observations were made in 1971, both indicating some form of compartmentation of DNA precursors. Antimetabolites can inhibit DNA replication in HeLa cells under conditions where the intracellular dNTP pools are barely affected, suggesting that much of the dNTP content of these cells is not available to the replication apparatus (14). In bacterial cells, where physical compartmentation in organelles would not be expected, when E. coli cells are treated with radiolabeled thymidine, the incorporation of radioactivity into DNA reaches its maximal rate long before the dTTP pool is fully labeled (15). This means either that dTTP is not a proximal DNA precursor or that dNTP pools are compartmentalized, with DNA drawing from a small pool that becomes labeled rapidly but whose labeling is obscured by a larger and less active pool. Because of the absence of organelles, it was concluded (15)that compartmentation is not involved, and that dNTPs are not proximal DNA precursors. Although that interpretation is incorrect, the observation was valid, as was shown a few years later (16). One may rationalize these results in terms of a functional or kinetic form of compartmentation; thymidine molecules entering a cell near a replication fork would generate dTTP molecules with a much higher probability of being incorporated into DNA than those molecules, in the vast majority, entering at a distance from replication forks. Thus, the cell would have two kinetically distinct pools: a small, rapidly replenished pool, located near replication sites, and a much larger, slowly replenished pool distributed throughout the rest of the cell. Presumably this latter pool could be used for DNA repair, possibly accounting for another early observation, namely, that DNA replication and repair are diEerentially labeled by exogenous thymine, thymidine, or bromodeoxyuridine (17). Consistent with the above picture, when a mutant E. cob culture carrying a thermolabile rNDP reductase is subjected to a temperature up-shift, DNA synthesis is immediately inhibited, even though the dNTP pool sizes are substantially unchanged (18). This suggests that most of the dNTP content of a bacterial cell is not immediately accessible to replication sites.

B. dNTP Compartmentation in T4 Phage-Infected E. coli If the above model can explain kinetics of DNA labeling by exogenous precursors, what about relationships between endogenous dNTP synthesis

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and DNA replication? Here most of the crucial early insights came from observations on T4 bacteriophage-infected E. coli. T4 is advantageous for studies on dNTP metabolism, first, because the virus encodes virtually all of its own enzymes of dNTP and DNA biosynthesis, and second, because useful mutants are available in all of the genes controlling these processes. Figure 1shows phage- and host-coded reactions in dNTP biosynthesis, while Fig. 2 identifies structural genes encoding the relevant phage-coded enzymes. Particularly important to our discussion will be dCMP hydroxymethylase, the product (gp42)of gene 42, which catalyzes the synthesis of the modified nucleotide, 5-hydroxymethyl-dCMP, from dCMP: dCMP t 5,lO-methylenetetrahydrofolate+ hmdCMP

+ tetrahydrofolate

1. GENETICINTERACTIONS INVOLVING DCMP HYDROXYMETHYLASE In the early 1970s, it was reported (19, 20) that certain temperaturesensitive gene-42 alleles had a mutator phenotype, suggesting a relationship between an enzyme of dNTP biosynthesis (dCMP hydroxymethylase) and the DNA replication apparatus. Later, a gene-42 mutation was described

dTiP

hm-dCTP

dGT P

~i A FIG. 1. Reactions of dNTP biosynthesis in T4 phage-infected E . coli. Reactions catalyzed by virus-coded enzymes are identified with heavy arrows, and those catalyzed by host cell enzymes are identified with light arrows. Except for reactions leading to biosynthesis of the unique phage nucleotide, hm-dCTP, most of the enzymes duplicate preexisting activities.

173

ENZYMES IN DNA PRECURSOR BIOSYNTHESIS ,,,--thymidino thiaradorin

hinaae E C 2.7.221

th

(,:!

DNA polymaraao EC 2.7.7.7

dCMP hydrorymathylarr-42 E C 2.1.2.b dCTPaaa- du7Paaa-s6 E C 3.6.1.12 rxonucloaaa E C 3.1.4.5 AY d . 1 1

endanuelaaaa B E C 3.1.4.5 1

,160 d

.

n

B /

EC 1.5.1.3

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-rNDP

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EC 1.17.4.1

FIG.2. Map positions of T4 phage genes encoding enzymes of dNTP metabolism. The numbers in the interior represent distances in kilobase pairs from the rZZAlrlZB cistron divide.

(21)that was suppressed by a mutation mapping in gene 43, which encodes DNA polymerase. This observation suggested intracellular interactions, either physical or functional, between these two enzymes. Subsequent genetic analysis suggested comparable interactions between phage-coded dihydrofolate reductase and gp41, the primosome-associated helicase (22), and between the small subunit of ribonucleotide reductase and gp39, a subunit of the phage-coded topoisomerase (23).While none of this work even hinted at the nature of these interactions, it did suggest an intracellular enzyme organization in which dNTP synthesis is closely coordinated with DNA replication.

2. THYMIDYLATE SYNTHASEREACTION FLUXin Viuo Tomich et ul. devised an ingenious method for estimating the intracellular activity of thymidylate synthase (13).T4-infected bacteria are administered [5-3H]deoxyuridine. After deoxyuridine uptake and conversion to dUMP, the action of thymidylate synthase displaces tritium from the pyrimidine ring. Radioactivity appearing in water gives a measure of intracellular thymidylate synthase activity. By this criterion, the enzyme does not become active in uiuo until several minutes after the enzymatically active protein is present, as shown by assays of extracts. The investigators (13)

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suggested that the enzyme becomes activated only as the result of incorporation into a multiprotein complex, and that assembly of this complex occurs concomitantly with initiation of DNA replication. Flanegan and Greenberg (24) modified this approach to estimate flux rates in uiuo for both dTMP synthase and dCMP hydroxymethylase. The ratio of these flux rates was 2:1, very close to the relative abundance of dTMP and hm-dCMP in T4 DNA. Greenberg and co-workers (24-26) proposed that individual activities within the putative complex are controlled so as to ensure that all four dNTPs are synthesized at rates corresponding to their rates of utilization for phage DNA synthesis, and that these relative rates are maintained over a range of environmental conditions.

3. SPECIFIC INTERACTIONSOF E . coli NUCLEOSIDE DIPHOSPHOKINASE WITH T4 ENZYMES Nucleoside diphosphokinase (NDP kinase) is the onjy E. coli protein known to play an indispensable role in synthesis of dNTPs for T4 DNA replication (27). This highly active and nonspecific enzyme is used for synthesis of both ribo- and deoxyribonucleoside triphosphates. Two of our early observations suggested specific interactions between this host cell protein and enzymes encoded by the infecting virus. First, we analyzed thymidine nucleotide pools after shutoff of DNA synthesis in cells infected with a temperature-sensitive, replication-defective T4 mutant (Fig. 3). Although the dTTP pool is normally larger than the dTDP pool, the arrest of DNA synthesis caused both nucleotides to accumulate at equal rates (10). Because NDP kinase activity in extracts is so high, this observation suggested that not all of the enzyme is available for synthesizing dNTPs, as if a small fraction of the total NDP kinase were sequestered by specific association with a complex of phage-coded enzymes. The other observation involved an NDP kinase inhibitor, desdanine. It had been reported (28) that E . coli cells grown anaerobically are resistant to desdanine, because anaerobic bacteria produce high levels of a nonspecific pyruvate kinase that, like NDP kinase, can synthesize all ribo- and deoxyribo-NTPs from NDPs and phosphoenolpyruvate: phosphoenolpyruvate

+ (d)NDP + pyruvate + (d)NTP

We asked whether T4 DNA replication is similarly resistant to desdanine inhibition after anaerobic growth and infection (29). In fact, phage DNA synthesis is acutely sensitive to desdanine under these conditions, implying that pyruvate kinase cannot supply precursors for phage DNA synthesis and suggesting an indispensable role for NDP kinase-as .expected if NDP kinase is part of a specific multienzyme complex.

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3

FIG.3. Effect of a reversible inhibition of T4 phage DNA replication upon thymine nucleotide pools and rate of DNA synthesis. DNA synthesis was inhibited by a temperature upshift of a culture infected with a t s phage mutant bearing a temperature-labile DNA polymeraseaccessory protein. dTTP and dTDP accumulated at nearly equal rates after the up-shift (identified by an upward arrow). Note also that the DNA synthesis rate after down-shift (identified by downward arrow) is nearly identical to that before up-shift, suggesting that the replication rate before up-shift was not limited by dNTP availability. [From Mathews and Sinha (II).]

4. PROPERTIES OF PERMEABILIZED T4-INFECTED BACTERIA With T4-infected E. coli cells permeabilized to allow passage of exogenous nucleotides, at least three observations are consistent with the idea that dNTPs used for replication are synthesized near replication sites by an organized enzyme complex (see Fig. 4).First, permeabilized cells incorporate distal DNA precursors-dNMPs or rNDPs-two- to threefold more efficiently than they use dNTPs (29, 30), suggesting more efficient utilization of nucleotides that enter the pathway through the “front door,” by interaction with ribonucleotide reductases (rNDPs)or nucleotide kinases (dNMPs). In a comparable study, thymidine is incorporated (by a thymidine kinasedependent process) more efficiently than dTTP or dTMP (31).Second, during synthesis supplied by a dNMP mixture, dNTPs do not accumulate within permeabilized cells to detectable levels, consistent with the idea that the replication machinery draws its dNTPs from extremely small pools (29). Third, genetic defects in hm-dCTP synthesis cannot be bypassed in uitro.

host cell DNA

1

\\

dCMP dAMIP-dGMP dTMP

ribonucleotiL synthesis

regulatory dNTPs

repair dNTPs

FIG.4. A model ofdNTP biosynthesis in T4 phage infection in which a multienzyme complex for dNTP synthesis is juxtaposed with the replication machinery. In T4 infection, ribonucleotide reductase represents the major source of dNTPs, but about 10% of the deoxyribonucleotides in phage DNA are synthesized from dNMPs derived from breakdown of host cell DNA. The numbers in the circle refer to T4 replication proteins.

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Cells infected with T4 mutants in either gene 42 (dCMP hydroxymethylase) or gene 1 (a dNMP kinase that acts upon hm-dCMP) show little or no DNA synthesis after permeabilization, even when they are supplied with all four dNTPs, including h m - d m P (32-35). This is consistent with the hypothesis that only dNTPs generated at replication sites can be used efficiently for DNA replication. In addition, permeabilized T4-infected E. co2i incorporate thymine into DNA, apparently by incorporating the exogenous base into localized pools of replication precursors (36).

111. T4 dNTP Synthetase: A Multienzyme Complex for Deoxyribonucleotide Synthesis The above observations are all consistent with the model outlined in Fig. 4. In this model, deoxyribonucleotides are synthesized by a multienzyme complex that both facilitates this process and channels dNTPs to DNA replication sites, by virtue of juxtaposition of the complex with the replication machinery. Proximity of enzyme active sites to one another limits diffusion away from these sites and helps to maintain high local dNTP concentrations at replication forks, even though the pools created at these sites are extremely small. To test the above model directly, we examined gently lysed cells and obtained the first direct evidence that such lysates contain an aggregated form of dNTP-synthesizing enzymes (12). As displayed in sucrose gradients, the aggregate behaves as though its molecular weight lies in the range of 106 to 2 x 106. A small but kinetically adequate amount of bacterial NDP kinase cosedimented with this aggregate of phage enzymes, consistent with our earlier observations of thymine nucleotide pool dynamics.

A. Kinetic Coupling in the T4 dNTP Synthetase Complex Before attempting to purify and further characterize the enzyme aggregate, which we now call the T4 dNTP synthetase complex, we needed to establish that it represents a specific complex and is not created by artifactual aggregation of enzymes during preparation and fractionation of cell extracts. Part of the needed evidence came from a mixing experiment (30);a mixture of 14C-labeled low-molecular-weight T4 proteins was isolated by gradient centrifugation of a radiolabeled extract. When this mixture was added to phage-infected bacteria just before lysis and sucrose gradient analysis, none of the radioactive proteins sedimented rapidly, indicating that artifactual protein aggregation did not occur under our conditions of extraction and analysis. However, most of our analysis of the specificity of interactions in this complex has used a combination of kinetics and genetics. The kinetic analysis

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CHRISTOPHER K. MATHEWS

depends upon the expectation that juxtaposition of sequentially acting enzymes will reduce average concentrations of intermediates in a multistep pathway because of limited diffusion away from the enzymes, even though local concentrations near catalytic sites are relatively high. We also expect an enzyme complex to show reductions in transient time, a parameter related to the time needed to fill all intermediate pools (37). The first sequence that we analyzed for this form of kinetic coupling (12) was the three-step pathway involving dTMP synthase, dNMP kinase, and NDP kinase: dUMP + dTMP + dTDP + dTTP. When we analyzed the rapidly sedimenting enzyme fraction for its ability to carry out this pathway, we found the steady-state concentration of dTDP to be reduced to about onetenth and the transient time to about one-twentieth relative to values calculated for nonassociated enzyme mixtures. A simulation of the pathway suggested that these kinetic improvements were gained by maintaining local reactant concentration gradients about 50 times higher. The above results were obtained with a relatively crude preparation of the dNTP synthase complex-simply a sucrose-gradient fraction obtained from centrifugation of an extract. Kinetic coupling has now been demonstrated in several other multistep pathways, both in crude and in highly purified preparations. These pathways are listed in Table I (12, 30, 38-40).

B. Genetic Specificity of Enzyme-Enzyme Interactions In principle, nonspecific enzyme aggregation could generate data suggestive of kinetic coupling simply because aggregation of enzymes brings them into proximity. We have asked whether the kinetic advantages seen depend upon assembly of a particular structure, by exploring the effects of removing or altering individual enzymes. For example, we isolated a crude preparation of the complex after infection of E. coli by a t s gene-42 mutant, which specifies a thermolabile form of dCMP hydroxymethylase. When this preparation was analyzed for coupling in the three-step sequence dUMP +

TABLE I REACIION SEQUENCES SHOWING KINETIC COUPLING IN PREPARATIONS OF THE T4 dNTP SYNTHETASE COMPLEX Reaction sequence

dUMP + dTMP + dTDP + d n P dCTP + dCMP + dUMP + dTMP dCTP + dCMP + dUMP + dTMP + dTDP + dTTP UDP + dUDP + dUTP + dUMP + dTMP + dTDP + dTTP CDP + dCDP + dCMP + hm-dCMP

Ref. 12 38 39 30 40

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(29) that coupling in uitro was abolished at 42°C with this preparation, even though the dCMP hydroxymethylase reaction is not directly involved in this sequence. More recently (41), we analyzed a series of gene42 amber mutants with respect to their ability to couple the sequence dCTP + dCMP + dUMP + dTMP. Again, gp42 played no direct role in this sequence. However, mutants that make highly truncated gene-42 proteins show no coupling in the above sequence, while mutants that make nearly full-length proteins show normal kinetic behavior. These results suggest that a nearly normal-sized protein can play an essential structural role in organizing the complex, even though that enzyme is not involved in catalyzing the pathway under study. Using mutants, we have identified several other T4 genes whose protein products are essential to assembly of the complex (39). In addition to gene 42, as indicated above, several other genes must function for the complex to form properly, including cd (dCMP deaminase), nrdA and nrdB (ribonucleotide reductase large and small subunits), and regA (a translational repressor of T4 early genes). By contrast, other mutations, in gene 44 (DNA polymerase accessory protein) or in gene 55 (late gene transcription factor), have no discernible effect upon the integrity of the complex. By demonstrating specific requirements for assembly of the complex, these results indicate that the complex as isolated represents a biologically significant pattern of intracellular enzyme organization.

+ + dTTP, we noted

C. Properties of the Purified dNTP Synthetase Complex To date we have purified the complex from T4-infected bacteria by ammonium sulfate fractionation and column chromatography using DEAEcellulose, QAE-Sepharose, and Sephacryl S300 gel filtration medium (39). Individual enzymes in the complex are enriched by as much as 500-fold, although yields are low. The purified material retains its kinetic coupling ability. Also, the gene products identified in Section III,B are essential for physical integrity of the complex. Figure 5 (41) shows gel filtration profiles for preparations from cells infected with wild-type phage (top panel) or with two gene-42 amber mutants that make extremely short truncated hydroxymethylase proteins. The complex as purified contains seven phage-coded enzyme activities, those shown in Fig. 4, and two encoded by the host genome-NDP kinase and adenylate kinase, which also phosphorylates dAMP. DNA polymerase activity is undetectable. The molecular mass estimated from gel filtration is 1300 kDa. The enzymes we have detected, at a stoichiometry of one molecule each, give an aggregate molecular mass of 900kDa. The remainder may represent either proteins that we have not yet identified in the complex (several additional bands are seen by SDS-PAGE) or the presence of multi-

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CHRISTOPHER K. MATHEWS

3

2

1

%

c .-

w .c

3

T4 a m C 8 7

0

a W

€ 2 %

N

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W

.-$

1

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Fraction Number FIG. 5. Gel filtration profiles from normal and abnormal dNTP synthetase complexes. Escherichio coli was infected either with wild-type T4 phage (top panel) or two gene42 amber mutants, and each of the three preparations was carried through our multistep fractionation scheme. This experiment shows that a full-length (or nearly full-length) form of dCMP hydroxymethylase is essential to the physical integrity of the complex. [From 'Ihyl6n and Mathews (401

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ple copies of some proteins. DNA is evidently not present to a significant extent, because DNase-I treatment of pooled gel filtration column fractions has no effect upon the elution profile upon subsequent passage through the same column (39). Chiu et al. (40) have also isolated a multienzyme aggregate from T4infected E. coli cells. Their preparation is more complex than ours, as reflected by SDS-PAGE analysis of total proteins, and DNA polymerase activity is present in nondenatured preparations. However, their preparation is less highly enriched than ours. Whether replication proteins are specifically associated with the complex in vim or whether they are contaminants that have not yet been removed in their preparation is an important unresolved question, which we discuss further in Section IV. In its highly purified form, the complex is sensitive to its ionic environment (39). NaCl concentrations higher than 50 mM cause dissociation. MgCI, concentrations above 10 mM also disrupt the complex.

IV. Is the T4 dNTP Synthetase Complex linked to DNA Replication Machinery? Investigations of the T4 dNTP synthetase complex, described above, began because we and others suspected a physical connection between the enzymes of dNTP synthesis and those of DNA replication. Somewhat disappointingly, unequivocal evidence on this point is not yet available. While the existence of a multienzyme complex for dNTP synthesis in T4 infection seems well established, its connection with replication proteins is not. On one hand, the preparation from the Greenberg laboratory contains replication proteins, but it may not be sufficiently highly purified to provide assurance that all proteins present are associated with each other. On the other hand, the preparation from our laboratory contains no DNA polymerase, but in light of low recoveries in our fractionation scheme, specifically associated proteins may have been stripped away during purification. One other important observation was the identification of dCMP hydroxymethylase among those proteins in a deoxyribonucleoprotein complex isolated from T4-infected bacteria (42). However, the functional nature of that physical association has not yet been established. In support of channeling of dNTPs to DNA replication sites are our observations with permeabilized T4-infected bacteria (29, 35). Presumably, these cells retain intracellular macromolecular associations. Therefore, the more efficient use of rNDPs compared to dNTPs for DNA synthesis in this system is hard to explain by any mechanism other than channeling, particularly since our permeabilized cell preparations seem to take up all common nucleotides equally efficiently.

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On the other hand, V, values for individual dNTP synthetic enzymes in citro are considerably lower than the calculated DNA ahain growth rate in uiuo, which represents V,, for DNA replication (43,44):This means that if there is a physical link between dNTP and DNA synthesis, several dNTP synthetase complexes must be associated with each replication fork. Clearly, new approaches are needed, and in this section I describe the results of more recent lines of investigation.

A. Genetic Evidence for Replication-active dNTP Pools Although Fig. 4 is highly schematic, it creates an impression that dNTP channeling creates pools of DNA precursors that are distinguished by physical location, small size, and rapid turnover. Obviously, these pools cannot be analyzed by direct biochemical measurements. However, the results of two recent genetic investigations support the concept that replicative DNA polymerases draw from pools distinct from those that one measures biochemically in extracts. The first study (9, 45) analyzed the mutator phenotype of t s gene42 (dCMP hydroxymethylase) mutations; when phages bearing such mutations infect cells at 34"C, a semipermissive temperature, spontaneous mutation frequencies are increased. The most straightforward way to explain this is that partial impairment of dCMP hydroxymethylase activity decreases hm-dCTP pools, creating increased likelihood of replicative mismatches opposite template G and causing GC + AT transitions. Sure enough, when we sequenced several dozen mutant sites, both in the rZZ and tk genes, we found that most mutations were GC + AT transitions. The remaining few were GC + TA transversions, which could have arisen through competition between dATP and diminished hm-dCTP for incorporation opposite template G. However, although all mutations that we sequenced could have been caused by a deficiency of hm-dCTP at replication sites, we found that infection at 34°C had no detectable effect upon dNTP pools, in infection either by wild-type phage or by the t s gene-42 mutant (Fig. 6). This implies that DNA replication draws from pools so small that changes in them are obscured by the much larger bulk pools, which do not change under conditions of our experiments, because of their much lower turnover rates. A similar conclusion was drawn from our studies of 5-bromodeoxyuridine mutagenesis (46,47). Br-dUrd can mutagenize either by an AT + GC transition pathway, in which Br-dUTP substitutes for dTTP during replication, or by GC -P AT transitions, in which Br-dUTP is misincorporated opposite template G. The latter pathway is stimulated at high Br-dUrd concentrations, because Br-dUTP mimics dTTP as a feedback inhibitor of CDP reduction by ribonucleotide reductase, decreasing the pool of dCTP with which Br-dUTP must compete for incorporation (hm-dCTP in T4 infection). We

183

ENZYMES IN DNA PRECURSOR BIOSYNTHESIS

100 rll SM03

10

1

.1

Plating efficiency .01 25

35

45

Temperature (“c) FIG.6. Temperature sensitivity of various biological parameters in infection by a temperature-sensitive gene42 mutant. Note that spontaneous mutagenesis (r22 SN103 reversion)is greatly stimulated at temperatures well below values where any changes in dNTP levels can be detected. [From Ji and Mathews (9).]

found that in T4 infection, Br-dUrd preferentially stimulated GC + AT transitions at high concentrations, although there were no significant effects upon dNTP pool sizes. Again, we concluded that any decrease in hm-dCTP levels at replication sites was obscured by the much larger size of the bulk dNTP pools, in which slower turnover led to less change during the time course of our experiments.

B. Affinity Chromatographic Analysis of Protein Associations Two informative approaches to identlfying enzyme associations start with purified enzymes. Most of the genes encoding enzymes depicted in Fig. 4 have been cloned into expression vectors, and the purified proteins are available in quantity. One approach is enzyme-affinitychromatography, patterned after an analysis by Formosa et al. (48)of protein-protein interactions involving T4 gp32, the single-stranded DNA binding protein. These investigators immobilized purified gp32 on AfEGel (BioRad), and used two-dimen-

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CHRISTOPHER K. MATHEWS

sional gel electrophoresis and radioautography to identify those [sS]methionine-labeled T4 proteins that bound specifically to the column. Several proteins of DNA replication, repair, and recombination were identified among the bound proteins. We have applied the same approach to gp42 (dCMP hydroxymethylase). A surprisingly large number of proteins was adsorbed rather tightly to this column-specifically bound at 0.2 M NaCl and eluted at 0.6 M (49). These included several enzymes in the dNTP synthetase complex: rNDP reductase large and small subunits; dCTPase-dUTPase; dTMP synthase; dihydrofolate reductase; and dCMP hydroxymethylase. The latter finding was not unexpected, because dCMP hydroxymethylase is a homodimer, and subunit exchange could be expected on the column. Also pmsent in the hydroxymethylase-bound proteins were several replication proteins, including gp32 (single-stranded DNA binding protein), gp45 (polymerase-accessory protein), and gp6l (DNA primase). In addition, two DNA repair proteins, UvsX and UvsY, were prominent among the bound proteins. Results of this study are summarized in Table 11.

RETENTION OF

TABLE I1 T4 PROTEINS BY dCMP IiYDROXYMETHYLASE COLUMN4 Phage strain

Gene 42

td frd 56

nrdA nrdB 32 436 44 45

61 62 46b

uosx UVSY

pseT &t/RNaseH ~~

Gene product dCMP IIMase dTMP synthase DHF reductase dCTPase/dUTPase rNDP reductase R1 rNDP reductase R2 ssDNA binding DNA polymerase Polymerase accessory Polymerase accessory Primase Polymerase accessory Nuclease RecA andog Repair protein Phosphatase/kinase Glc-transferase/HNase H

Function

T4D

T432am

dNTP synthesis dNTP synthesis dNTP synthesis dNTP synthesis dNTP synthesis dNTP synthesis DNA replication DNA replication DNA replication DNA replication DNA replication DNA replication Recombination DNA repair DNA repair ? DNA modification

+ + + + + + +

+

2 -

+ +

-

+ + + + -

T4tdam

-+ -r-

+

2

+ t

+ + + -

-

+ +

+ + ~

4Bound specifically at 0.2 M NaCI; eluted at 0.6 M NaCI. bnetected in 0.2 M NaC1 eluate but not present at appreciable levels in 0.6 M NaCl eluate. The amher mutants listed are mutant in dTMP synthase (td) or single-strand DNA binding protein (gene 32). [From Wheeler et al. (49).]

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Table I1 shows another interesting finding. When we realized how many T4 proteins bound specifically to the hydroxymethylase column, it was evident that they could not all be bound directly to the immobilized enzyme. Rather, it seemed likely that indirect interactions, or “piggybacking,”were probably involved. Identification of such interactions might help to establish neighbor relationships among proteins of dNTP and DNA synthesis in uiuo. We were intrigued to note that among the unidentified proteins bound to gp32 in the study of Formosa et al. (48)was one that we tentatively identified as thymidylate synthase. This suggested that gp32 might be bound to our dCMP hydroxymethylase column by virtue of a direct interaction with dTMP synthase, which was in turn bound directly to immobilized dCMP hydroxymethylase. That idea received support when we labeled proteins after infection with umber mutants defective in either gene 32 or gene td, the structural gene for dTMP synthase. When these extracts were analyzed on the dCMP hydroxymethylase column, several proteins failed to bind to the column, while several new bound proteins appeared. Most significant, no gp32 was bound to the column in analysis of the td- mutant extract. This finding, plus the large number of other changes observed (summarized in Table II), indicates that thymidylate synthase and gp32 do interact directly, and that this interaction is important in directing and stabilizing a number of other interactions in what may represent a biologically significant multiprotein structure. It is of interest that the amino-acid sequences of E. coli and T4 thymidylate synthases are closely related, except for a few distinct areas. One might predict these areas of nonhomology to represent domains involved in species-specific protein interactions. We are now applying affinity chromatographic analysis to several other enzymes in the dNTP synthetase complex. Results with nucleoside diphosphokinase are particularly interesting, because this is a bacterial enzyme that evidently is sequestered by forming specific interactions with phage proteins (So). Among those T4 proteins moderately tightly bound to an NDP kinase column (bound at 0.2 M NaCl, eluted at 0.6 M) are those identified as gp32, gp45 (polymerase accessory protein), gp61 (DNA primase), and gpuvsY (DNA repair protein). DNA polymerase (gp43)also binds to the NDP kinase column, but with lower affinity.

C. Anti-idiotypic Antibodies as Tools to Identify Protein-Protein Associations Associations identified by affinity chromatography are stable at salt concentrations comparable to, or somewhat exceeding, those encountered in uiuo. However, in other respects the conditions under which associations are seen are quite unphysiological, and one cannot conclude that all of the interactions observed by this approach are biologically significant.

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An alternative approach, the generation and analysis of anti-idiotypic antibodies, is designed to identify interactions that are significant in uiuo (51). If two enzymes, E l and E2, interact in uiuo, and if one prepares a polyclonal antiserum to E l , some of the antibodies may recognize epitopes in the E l domain that interacts with E2. Therefore, if one purifies antibodies in the E l antiserum and from these prepares a polyclonal antiserum to antiE l , then that secondary antiserum may have antibodies that mirror the binding geometry of the El-E2 binding domain. Thus, these anti-idiotypic antibodies should react with E2, identifying the El-E2 interaction. This approach need not be confined to enzyme proteins, and indeed the approach is identical in concept to the use of anti-idiotypic antibodies to ligands, for identification of receptors. Using this approach, we have developed anti-idiotypic antisera against T4 dCMP hydroxymethylase (52). One such serum specifically immunoprecipitates two phage proteins, which we have identified as dCMP hydroxymethylase and dTMP synthase, respectively. Anti-dTMP synthase antibodies in the serum can be separated from anti-dCMP hydroxymethylase antibodies by chromatography on immobilized dCMP hydroxymethylase. Thus, distinct antibodies are reacting with each enzyme. Presumably the anti-dCMP hydroxymethylase antibodies recognize epitopes in the enzyme dimerization region, and experiments are under way to test this idea. The antiserum does not react with E . coli thymidylate synthase, which reinforces the concept of species specificity in those structural features involved in protein-protein interactions. So far this approach has not identified an interaction between aT4 enzyme of dNTP synthesis and a DNA replication protein. However, the interaction between dTMP synthase and dCMP hydroxymethylase is of considerable interest, partly because the affinity chromatography discussed above suggests a direct interaction between these two enzymes. Of equal interest is the fact that this interaction was predictable from the observation (24)that in uiuo flux rates for these enzymes are strictly maintained at a ratio of 2:l synthase: hydroxymethylase. It was suggested that some form of interaction between these enzymes might be involved in maintaining this ratio. As discussed in Section VI, D, the generation of anti-idiotypic antibodies has identified a physical link between dNTP and DNA synthesis in another system that we are studying, namely, vaccinia virus.

V. Enzyme Organization in Bacterial Cells Considering the amount of attention focused upon the T4 phage system, on one hand, and mammalian cells, as discussed below, it is perhaps surprising that relatively little work has been done on uninfected E. coli beyond the

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early findings, mentioned in Section II,A, indicating that some form of dNTP compartmentation exists in these cells. Other early findings related to the intracellular milieu for ribonucleotide reductase. In attempts to explain why activities of the isolated enzyme were so much lower than calculated activities needed to sustain DNA replication in uiuo, three laboratories reported that activities were far higher when assayed, not in extracts, but in in situ preparations, where intracellular macromolecular associations were believed to be retained. These systems included bacterial cells permeabilized by ether treatment (53),cells gently lysed on cellophane discs [where dilu],gently lysed spheroplasts (55). tion of cell contents was prevented (a)and In the latter study, E. coli rNDP reductase was readily isolated as a large, membranous aggregate. However, other enzymes that one might expect to find in a dNTP synthetase complex were not present in this aggregate. Two reports suggest the existence of unspecified intracellular interactions involving E. coli rNDP reductase. First, analysis of proteins in an E. coli extract that bound to an immobilized monoclonal antibody against the large subunit showed two proteins bound, in addition to both reductase subunits (56). The other finding was that treatment of bacterial cells with protein crosslinking reagents generated an aggregated form of rNDP reductase, under conditions where very few proteins were crosslinked (57). So far, none of the additional proteins involved have been identified.

VI. Organization of dNTP Synthesis in Eukaryotic Cells The above-described research on prokaryotic systems, particularly phage T4, has provided a conceptual framework within which to ask how enzymes of dNTP synthesis are organized in eukaryotic cells. Much of the additional background for understanding the situation in eukaryotes has come from analysis of nucleotide compartmentation, DNA labeling kinetics, in uiuo reaction flux measurements, dNTP pool turnover, and enzyme localization in mammalian cells. The situation in eukaryoticcells is clearly more complex than that in prokaryotes, and some of the data to be discussed can be interpreted in more than one way. In this section, I discuss investigations suggesting dNTP compartmentation and their implications for understanding enzyme organization. Next, I summarize results of investigations on multienzyme aggregates of dNTP metabolism in eukaryotic cells. Following this is a review of the current status of dNTP synthetic enzyme organization in eukaryotic cells, primarily from the standpoint of data published within the past 5 years. Finally, I discuss recent work from my laboratory that establishes a strong connection between dNTP synthesis and DNA replication in one “eukaryotic” situation, namely, the replication of vaccinia virus in cultured primate cells.

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CHRISTOPHER K. MATIIEWS

A. dNTP Pool Dynamics and Compartmentation in Eukaryotic Cells Many investigators have presented data suggestive of dNTP pool compartmentation in eukaryotic (primarily mammalian) cells. Early developments paralleled those described previously in this article for prokaryotic systems, and comparable data were presented: existence of dNTP pools not used for replication; dTTP pools whose radioactivity increases after DNA labeling has reached its maximal rate; analyses of dNTP pool turnover rate; differential fates of dNTPs labeled via salvage or de nouo synthetic pathways; metabolic flux rates apparently unrelated to intracellular concentration of the enzyme under study; and so forth. Many of these observations were interpreted in terms of a direct channeling of dNTPs to replication sites. Partly stemming from these observations, at least a half-dozen laboratories have described in eukaryotic cells multienzyme aggregates of dNTP-synthesizing enzymes. I first discuss the evidence supporting dNTP compartmentation.

1. RATES OF RADIOLABELING

OF

DEOXYRIBONUCLEOTIDES AND DNA

Using CCRF-CEM human lymphoblast cells instead of bacteria, Fridland (58)carried out experiments similar in principle to those of Werner (15).Like E. coli cells, labeling of these eukaryotic cells with [3H]thymidine led to continued increase in radioactivity of the dTTP pool long after DNA labeling had reached its maximal rate. However, unlike Werner, Fridland concluded that this resulted from compartmentation of dNTP pools. This was not based simply upon the existence of organelles that could physically separate metabolite pools. Instead, Fridland compared turnover rates of labeled thymidine nucleotide pools with rates of DNA labeling during a chase from labeled to unlabeled medium. Both dTMP and dTDP lost radioactivity at rates considerably lower than the rate of DNA labeling. Thus, neither could be a proximal DNA precursor. A curious feature of Fridlands data was the fact that, while the dTTP pool was labeled equally rapidly from either [3H]thymidine or [3H]deoxyuridine, the dUrd-derived dTTP labeled DNA at twice the rate of the dThd-derived dTTP. Similar observations were made with cultured human lymphocytes (59-62) and with synchronized S-phase Chinese embryo fibroblasts (62). Deoxyuridine utilization involves the de nouo biosynthetic pathway, since dUMP is anabolized via thymidylate synthase. A reasonable interpretation of this result is that nucleotides synthesized by the de nouo pathway have preferential access to replication forks, possibly because of a connection between a dNTP synthetase complex and the replication machinery. Whether or not this interpretation is correct, the data suggest that the

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specific activity of deoxyuridine-derived dTTP at replication sites is higher than that of thymidine-derived dTTP. As a first attempt to assess this, it would be desirable to determine directly the specific radioactivity of the total dTTP pool after labeling with either dUrd or dThd. To my knowledge, this has not been done. More data on dTTP pool turnover and incorporation into DNA were presented by Taheri et al. (59-61), who studied several different human leukemoblastoid cell lines. Like Fridland (S),they found that dTMP and dTDP pools turned over too slowly during a chase to be considered proximal DNA precursors. The cell lines varied considerably in percentage of the dTTP pool that was incorporated during the chase. What was not incorporated was degraded. Taheri et al. concluded that dTTP is compartmentalized, with one pool used primarily to support replication and the other being degraded. However, it seems just as likely that variations in intracellular nucleotidase activities may account for varying amounts of dTTP degradation. Taheri et al. also proposed dNTP degradation as a mechanism for regulating deoxyribonucleotide pool sizes, a concept supported by more extensive data from Reichards laboratory (63). Another pattern of compartmentation, also somewhat undefined, comes from the finding that, in regenerating rat liver, [32P]orthophosphatelabeling of DNA is stimulated several hours before incorporation of [3H]thymidine (64).This suggests that dTTP pools are compartmentalized, with an endogenous pool, synthesized by the de novo pathway, being preferentially used. More recently, the same group (65) reported that thymidine labels nuclear matrix DNA to maximal specific activity well before intracellular dTTP reaches its maximal specific activity. This could be an indication of the same type of compartmentation. On the other hand, in two other systems-HeLa cells grown in HAT (66) medium and fertilized sea urchin eggs (67)-exogenous thymidine can completely bypass internal pools and be incorporated into DNA at full specific activity. From these observations, it is not clear if variations in the use of exogenous precursors are due to daerences in cell structure and enzyme organization, or to variations in experimental methods. As we shall see, similar uncertainties plague much of the work in this area. 2. REPLICATION-INACTIVEDNA PRECURSOR POOLS

Also consistent with a dNTP channeling model are several studies suggesting the existence of dNTP pools that are not readily utilized for DNA replication. This was suggested by the early observation (14)that antimetabolites inhibit DNA synthesis, even while significant dNTP pools remain in the treated cells. Similar findings have been reported for other cell lines (6.68, 69). In one careful study, a density-shift technique with bro-

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modeoxyuridine incorporation was used to measure rates of replication fork movement in phytohemagglutinin-stimulated human lymphocytes (70).Previous studies had shown that cells from patients with megaloblastic anemia resulting from either folate or vitamin B,, deficiency contained near-normal dNTP pools, even though the nature of the metabolic defect suggested abnormalities of deoxyribonucleotide biosynthesis. Cells from several such patients showed replication fork movement rates from 40 to 92% of normal, suggesting that a functional deficiency of DNA precursors at replication sites was limiting rates of DNA chain elongation. Similar findings were reported for normal lymphocytes treated with either methotrexate or hydroxyurea. Another possibly relevant observation is that replicative DNA synthesis is more sensitive to hydroxyurea inhibition in intact cells than is DNA repair synthesis (6.71, 72).While this may reflect in large part the simple fact that replicative synthesis demands far larger quantities of dNTPs than does repair, it is also consistent with the existence of separate precursor pools for replicative and repair synthesis, with the former pool being more readily drained after inhibition of ribonucleotide reductase. The presence in mammalian cells of substantial dNTP pools, under conditions in which DNA replication is inhibited, can be explained in terms of the existence of dNTP pools specifically destined for use in DNA replication, which presumably are localized near replication sites. However, in evaluating data on dNTP pools published before about 1980, we must consider at least three experimental factors that might alter interpretation of the data. First is the presence of nucleosides in the serum used as a cell culture ingredient. Kyburz et al. (73) showed that residual dTTP pools after methotrexate treatment are lower when cells are cultured in medium with dialyzed rather than undialyzed serum. Second is the likelihood that cells not in S phase contain appreciable dNTP pools that are not depleted by antimetabolite treatment. (That possibility is considered further in the next section.) Third are possible sources of error in the dNTP pool assay (74). Most laboratories use an enzymatic assay based upon DNA polymerase and defined polynucleotides as template and primer. For example, incubation of an extract with poly(dA.dT) and an excess of radiolabeled dTTP should result in incorporation of label to a plateau value representing equimolar incorporation of limiting unlabeled dATP in the extract and nonlimiting labeled dTTP. Many laboratories extract cells with aqueous 60% methanol. We found that such extracts from HeLa cells contain active enzymes (deoxyribonucleases and nucleotide kinases) that lead to serious overestimation of dNTP concentrations in extracts (74).The magnitude of this overestimation varies considerably among different cell lines (75). Another commonly used technique, perchloric acid treatment, gives extracts that, in our hands, fail to

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yield true plateau incorporation values. We recommend sequential extraction of cells with aqueous methanol followed by perchloric acid. 3. dNTP POOLTURNOVER

As noted earlier, several groups have studied dNTP pool turnover. The investigations of Nicander and Reichard (76) are particularly significant, because these workers were the first to determine specific activities of dNTP pools during chase periods after labeling, so that turnover rates can be directly compared with DNA synthesis rates. In 3T6 mouse fibroblasts grown in HAT medium to suppress endogenous dNTP synthesis, the dTTP pool turned over at the same rate at which thymine nucleotides were incorporated into DNA. This argued that all of the dTTP in the cell was available for DNA synthesis, and hence, that all dTTP in the cell constituted a single metabolic compartment. This conclusion was reinforced by Wawra (77), who found that radiolabeled dNTPs microinjected into the cytosol of mammalian cells is efficiently and completely incorporated into DNA in the nucleus. However, compartmentation of dCTP pools is suggested by an experiment of Nicander and Reichard (76). When the dCTP pool was labeled with cytidine, it turned over at the same rate at which replication was occurring, suggesting an absence of compartmentation. However, when deoxycytidine was used as the labeled precursor, the dCTP pool turned over at three times the rate of DNA synthesis, indicating that just one-third of the deoxycytidine-derived dCTP pool was available for DNA replication, with the remainder being in a replication-excluded pool. Because the use of HAT medium, which contains aminopterin, might affect dNTP pools in unpredictable ways, it is important to realize that comparable observations were later made with medium not containing antimetabolites (78). From my laboratory comes an explanation for at least some of the above results. In Chinese hamster ovary cells synchronized by isoleucine starvation (79), S-phase-enriched cells incorporated radiolabeled cytidine or deoxycytidine into dCTP to the same specific activity. The GI-phase-enriched cells incorporated cytidine into dCTP at about that same, relatively low, value. However, administration of deoxycytidine to the GI-enriched cells labeled dCTP to a 13-fold higher value (Fig. 7). Activities of deoxycytidine kinase, through which deoxycytidine must pass en route to dCTP, are relatively constant throughout the cell cycle (80). However, ribonucleotide reductase, a key enzyme in the anabolism of cytidine, is greatly activated during S phase. Therefore, it seems likely that the dCTP compartmentation observed by Nicander and Reichard is intercellular, not intracellular; the replication-excluded dCTP pool represents the deoxycytidine-derived pool that becomes labeled in non-S-phase cells.

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CHRISTOPHER K. MATHEWS

30

1

+

/

CMP

60 0 Minuter of labeling

>f

&

CD,P

.L CMP

+

&

,

V

dCMP+dCDP+dCTP

" % m n

FIG. 7. Data supporting intercellular compartmentation as the basis for replication-excluded dNTP pools. Top: rates of labeling dCTP pools with either cytidine or deoxycytidine in either GI-phase- or S-phase-enriched CHO cells. Bottom: cell cycle-dependent changes in enzyme activities that can account for the above data. [From Mathews (3), with permission of CRC Press.]

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Spyrou and Reichard, working with synchronized mouse 3T3 fibroblasts, confirmed these observations, but they also extended them in an interesting way (81).dCTP is used not only for DNA synthesis but also for the synthesis of “1ipodeoxynucleotides”-deoxyribo analogs of CDP-choline and CDPethanolamine. These compounds are labeled preferentially by the dCTP pool that is derived from deoxycytidine. Thus, deoxycytidine nucleotides are evidently subject to both intra- and intercellular compartmentation. Evidently, purine deoxyribonucleotides are also subject to intercellular compartmentation. Work with mouse S49 lymphoma cells gave results comparable to those presented above-preferential DNA labeling by dGTP derived via the de nouo synthetic pathway from guanine, with the salvage route from deoxyguanosine providing most of the dGTP measured in a pool assay (82,83). Since purine deoxyribonucleotides are also phosphorylated by deoxycytidine kinase, this presents a route for significant accumulation of dGTP in non-S-phase cells, just as described above for dCTP. It is clear that any future analyses of intracellular DNA precursor compartmentation should be done with synchronized cell populations. 4. METABOLIC FLUXRATE STUDIES

In Section 11, I described the [5-3H]deoxyuridine tritium release assay and its importance for estimating intracellular thymidylate synthase flux rates. Applying this technique to synchronized mammalian cells showed that reaction flux is high only during S phase; however, thymidylate synthase activities in uitro, as recorded by enzyme assays of extracts, are invariant through the cell cycle (84, 85). Rode et al. (84) ascribed their results to product inhibition of thymidylate synthase, significant at all phases of the cell cycle except when dNTPs are being turned over by incorporation into DNA. However, Reddy (85) interpreted his result as support for the “replitase” model, in which thymidylate synthase flux is regulated by allosteric interactions among dNTP synthetic enzymes complex at or near to replication sites. Consistent with this was the observation (86) that dTMP synthase flux is greatly reduced by inhibition of DNA replication with aphidicolin, as expected if dNTP synthetic enzymes can sense the demand for their products through allosteric interactions with replicative enzymes. Similarly, aphidicolin treatment of 3T6 cells caused a threefold drop in dTMP synthase flux rate (78). Similar observations have been made in mouse leukemia and human tumor cells (87, 88). However, all three of the latter groups argued that their results did not support the replitase (85) model. Distinguishing between the replitase and product-inhibition models cannot now be done on the basis of these experiments, for two reasons. First, evaluating dTMP synthase inhibition by its product, dTMP, would require dTMP pool measurements through the cell cycle. Even if such data were

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available, however, one could not assess the possibility of dTMP accumulation specifically within the microenvironment near dTMP synthase molecules. More important is the fact that if tritium release rates are to represent absolute intracellular reaction rates, they must be corrected for possible changes in dUMP specific activities. Nicander and Reichard (78) related their tritium release data to dTTP pool sizes, while offering the opinion that dTTP should have the same specific activity as dUMP. More recently, Plucinski et al. (89) reported that aphidicolin treatment of S-phase Chinese hamster embryo fibroblast cells caused a 2.5-fold drop in dUMP pool size. They did not measure dUMP specific activity, but if it is increased proportionately to the decrease in pool size, then published dTMP synthase flux rates may have been significantly overestimated. Regardless of the explanation for variations in dTMP synthase flux rates, the data mentioned above all suggest a high degree of coordination between dNTP synthesis and DNA replication. 5. INABILITY TO CIRCUMVENT HYDROXYUREA INHIBITION OF DNA SYNTHESIS

Although hydroxyurea inhibits reduction of all four common rNDPs by ribonucleotide reductase, hydroxyurea treatment of mammalian cells causes preferential depletion of purine dNTP pools. Using an isotope-dilution method as an indirect assay system, Scott and Forsdyke (90)asked whether hydroxyurea inhibition of DNA replication in whole cells can be bypassed by provision of deoxyadenosine and deoxyguanosine in the medium. Their data suggested that the added nucleosides fill pools of deoxyribonucleotides that can interact with control sites on ribonucleotide reductase, but that the replication block was not bypassed. This suggests the existence of “replication-active” dNTP pools at replication sites, which are not readily accessible to purine dNTPs derived via salvage pathways. Snyder (71) and Eriksson et al. (91) confirmed that purine deoxyribonucleosides cannot bypass a hydroxyurea block to DNA replication, although Lagergren and Reichard (92) described conditions under which partial bypass was seen. More recently, in studies on vaccinia virus growth, we found that hydroxyurea inhibition can be almost completely bypassed by deoxyribonucleosides, but only when an adenosine deaminase inhibitor is added to prevent deoxyadenosine catabolism (93). The surprising finding from this study was that deoxyadenosine alone (plus the deaminase inhibitor) is as effective as all four nucleosides in reversing the inhibition to virus growth. Other data suggest that ADP reduction is a specific target for hydroxyurea inhibition of viral DNA synthesis. While we do not yet know whether that is also true for cellular DNA synthesis, our data show that the

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inability to bypass a hydroxyurea block cannot be ascribed to a special pool of DNA precursors.

6. DISTRIBUTION OF DEOXYRIBONUCLEOTIDE POOLS BETWEEN NUCLEUSAND CYTOPLASM Any consideration of deoxyribonucleotidecompartmentation has to take into account the possibility of physical compartmentation within organelles. Data from our laboratory (94) indicate the existence of physically compartmentalized dNTP pools within HeLa cell mitochondria; as one might expect from the small size of the mitochondrial genome, these pools are small relative to the total intracellular dNTP contents. Considerations of dNTP compartmentation for nuclear DNA replication must take account of the porous nature of the nuclear membrane. This makes it difficult to isolate nuclei with their internal small-moleculecontents intact for subsequent analysis. Two general methods have been described in connection with dNTP measurements-isolation in nonaqueous solvents (95, 96) and rapid isolation in aqueous media (97). The chief drawbacks to nonaqueous fractionation are the length of time required and low recoveries of material in the final extracts, while rapid fractionation techniques suffer from the possibility that nucleotide leakage from nuclei might be significant, even within just a few seconds. Nonaqueous fractionation of intranuclear dNTP pools in synchronized CHO cells showed asymmetric distribution of dNTPs across the nuclear membrane and preferential nuclear dNTP accumulation in S phase (95). More recently, glycerol gradients were used to fractionate extracts of lyophilized regenerating rat liver (96). Although this approach precluded reporting pools on a per-cell or per-nucleus basis, the data suggested that those fractions with the lowest RNA/DNA ratio, presumably representing nuclei, had disproportionately large proportions of the total dTTP pool. This supports the conclusion (95) that dNTPs are distributed asymmetrically across the nuclear membrane. The rapid nuclear isolation method involves brief treatment of cultured cell monolayers with buffer containing 1%NP40 detergent. Rupture of the plasma membrane, with loss of cytosolic contents, occurs almost immediately. After a few seconds the detergent solution is aspirated away, and the remaining material (nuclei draped in plasma membrane) is quickly washed with detergent-free buffer and extracted. The entire process takes about 20 sec. By this method, we do see some asymmetries in dNTP distribution (95, 98), but they are not as pronounced as in the previously cited studies. For example, in asynchronous CHO cells, we found that about 15-20% of the total dATP and dGTP content, but only 5-10% of total dCTP and dTTP, to be in the nuclear extracts, with neither value representing a large tendency

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to concentrate dNTPs in the nucleus (98).Also, in synchronized CHO cells, we found that dCTP accumulated in whole extracts to a much greater extent than in nuclei (97), indicating that the primary dCTP accumulation was in the cytosol. Most CHO cell lines have high dCTP pools because they lack dCMP deaminase, so these may be unrepresentative results. How reliable are intranuclear measurements based upon a nuclear isolation procedure that leaves pores open before extraction? To answer that we observe, first, that the intranuclear levels we measure remain stable over at least 2 min of detergent treatment (98). Note that our normal isolation pro; cedure involves 10 sec of detergent treatment and is complete within 20 sec. This stability is perhaps greater than one might expect given that opened nuclear pores are large enough to permit passage of small proteins. However, from the dimensions and number of nuclear pores, one can estimate that the fraction of the area of the nuclear membrane ocuupied by pores is only 0.1%(99).Thus, very few of the collisions between intranuclear molecules and the nuclear membrane will lead to passage through the membrane, and the existence of apparently stable intranuclear pools is not as surprising as it might seem at first glance. Using rapidly isolated nuclei from S-phase-enriched CHO cells, we have asked also whether dNTP biosynthesis occurs in the nucleus, as it must if DNA replication is directly linked to dNTP synthesis. Using either radiolabeled cytidine or deoxycytidine, we compared the rates of labeling of the nuclear and whole-cell dCTP pools, with the expectation that the nuclear pools would reach maximal specific activity earlier if the nucleus were the major site for dNTP synthesis (79). For each precursor, we found that the nuclear and whole-cell dCTP pools were labeled at equal rates, as expected if dNTPs are synthesized in the cytosol, followed by rapid migration into the nucleus. This is consistent with the fact that most (not all) reports of the subcellular distribution of dNTP biosynthetic enzymes place these enzymes in the cytosol.

B. Multienzyme Aggregates for dNTP Synthesis in Eukaryotic Cells To my knowledge, the first data supporting a structural link between eukaryotic DNA replication and the enzymes of dNTP synthesis were presented in two 1974 reports (100,101)showing that ribonudeotide reductase activity, in both cultured hepatoma cells and regenerating rat liver, is in a membranous postmicrosomal supernatant fraction that also contains DNA polymerase activity. Since 1974, at least four other groups have described high-molecular-weight aggregated forms of dNTP-synthesizing enzymes in mammalian cells. Table I11 lists properties of these systems in summary form. The table also identifies an interesting five-protein complex purified

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TABLE I11 MULTIENZYMEAGGREGATES OF dNTP BIOSYNTHETIC ENZYMES IN EUKARYOTES ~

Source Hepatoma, regenerating rat liver CHEFI18 hamster fibroblasts Human lymphoblastoid cells Mouse FMSA cells S-phase BHK fibroblasts HSV-infected fibroblasts Cultured carrot cells

How demonstrated Postmicrosomal membrane fraction Cytochalasin enucleation, gradients Gel filtration Sucrose gradients Sucrose gradients Sucrose gradients Conventional protein fractionation

Enzymes detecteda

Ref.

RNR, DNA pol

100,

TK, DHFR, NDPK, DNA pol

101 103, 104

DNA pol, TK, NDPK, dTMPK, TS TS, TK, DNA pol a DNA pol, TK, RNR, DHFR, NDPK Viral RNR and TK TS, DHFR, 3 unidentified proteins

105

106 107

107 102

aAbbreviations: RNR, ribonucleotide reductase; TK, thymidine kinase; dTMPK, thymidylate kinase; DHFR, dibydrofolate reductase; NDPK, nucleoside diphospbokinase; DNA pol, DNA polymerase.

from cultured carrot cells, which contains thymidylate synthase and dihydrofolate reductase (102). Most attention has focused upon an aggregate isolated by Reddy and Pardee (103)from S-phase CHEF118 hamster embryo fibroblast cells, since this was the earliest preparation shown to contain several different enzyme activities (104). This preparation was isolated from extracts of nuclei, which were obtained by cytochalasin-enucleationof whole cells. As noted in Table 111, several groups (105-107)have also described high-molecular-weight aggregates in mammalian cells. One report (105) presents what may be the only genetic evidence supporting specificity of the associations. In a thymidylate synthase-negative mouse cell line transfected with DNA encoding human thymidylate synthase, a rapidly sedimenting aggregate was seen, but the heterologous thymidylate synthase failed to cosediment with this aggregate. In addition to this one piece of genetic evidence, some who isolated these complexes have attempted to demonstrate kinetic coupling, by methods comparable to those earlier reported for the T4 dNTP synthetase complex. Although some data (105)suggest a possibility of kinetic coupling in the preparations, the aggregate from BHK cells (107) showed little or no evidence for coupling or channeling. Reddy and Pardee reported that their preparation efficiently incorporated ribonucleoside diphosphates into DNA with little or no dilu-

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tion by added dCTP. While this type of experiment provides powerful evidence for channeling, the experiments have been challenged on technical grounds (108). Perhaps the most important part of the reports of Reddy and Pardee (103) and of Noguchi et al. (104)was the claim that their preparation was localized to the nucleus and that it assembled as cells entered S phase, by migration of enzymes inward from the cytosol. Obviously, if dNTPs are to be channeled directly to replication sites, dNTP synthesis must occur in the nucleus. Although data on ribonucleotide reductase activity per se were not presented in the reports from the Pardee laboratory, the investigators clearly felt that this enzyme was part of their complex, because of their data on rNDP incorporation. On the other hand, Larsson (109) had previously reported rNDP reductase to be localized to the cytosol. To be sure, Larsson’s experiments were not done with synchronized cells. However, three laboratories in the late 1980s reported ribonucleotide reductase to be cytosolic throughout the cell cycle (97, 110-112). These reports described several d&rent methods of subcellular fractionation, and they also included immunocytochemical evidence, which does not depend upon subcellular fractionation. One of those reports (110)also identified thymidylate synthase as a cytosolic enzyme, and another (113)localized dUTPase to the cytosol. These data, plus our report that dNTPs are synthesized in cytosol (79), suggest that direct coupling of dNTP synthesis to DNA replication in the nucleus does not occur. In attempting to reconcile the Reddy-Pardee results with those of other investigations, it should be recalled that in their first report (103),nuclei were isolated after cytochalasin treatment, which generates a nucleus-enriched “karyoplast”fraction. The possibility of contamination of their preparations with cytosolic components or membranes from the perinuclear area was not specifically ruled out. This raises the intriguing possibility that a dNTP-synthesizing enzyme complex is localized just outside the nucleus. Indeed, Sikorska et al. (114)have recently presented immunocytochemical evidence for a perinuclear location for ribonucleotide reductase in a number of mammalian cell types.

C. Are dNTPs Channeled to Replication Sites in Eukaryotic Cells? When work on the T4 dNTP synthetase complex began, it seemed an appealing model for understanding the organization of dNTP synthetic enzyme in eukaryotic cells. Indeed, our growing awareness of the structural architecture of DNA replicative complexes (115)makes such a model even more attractive. However, in considering the data on eukaryoticcells and the appropriateness of the T4 model, one should keep in mind several important quantitative distinctions between prokaryotic and eukaryotic DNA metabo-

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lism (4, 116). First, eukaryotic DNA replicates as a set of several thousand replicons, so replication is not confined to just a few sites. Second, individual DNA chains grow in eukaryotes at rates that are only a tenth of those in prokaryotes. Third, eukaryotic replicative DNA polymerases have K, values only a tenth or so of their prokaryotic counterparts (116).Thus, eukaryotic DNA replicates slowly, at many intracellular sites, and replication is catalyzed by enzymes with high affinities for their substrates. Thus, one can envision dNTPs moving to replication sites at adequate rates by simple diffusion, which obviates a kinetic argument in support of channeling. Finally, there are important unresolved questions about nuclear structure and organization of DNA metabolic proteins near the nucleus. The idea that replication occurs via the action of enzyme complexes immobilized on the nuclear matrix seems to be gaining acceptance (6.115). Although much of the data remain controversial, there is an increasing body of support for the idea that most metabolic pathways involve organized enzyme complexes (cf. 1,2). Even though dNTP pools turn over in eukaryotes more slowly than in prokaryotes, the pool sizes are small, sufficient to support DNA replication for just a few minutes in most mammalian cells (63). Since the metabolic consequences of interrupted DNA precursor supply to replication sites are often lethal, it seems that some pattern of organization should exist, and we have not yet described it. My own view is that dNTPs are not directly channeled to replicating DNA, as is probably the case in prokaryotic systems, and that the key to further comprehension will be a more thorough understanding of the nuclear envelope.

D. A Connection between dNTP Synthesis and DNA Replication in Vaccinia Virus

d

I close by describing an emerging biological system wherein vidence for a physical connection between dNTP synthesis and DNA re ication is beginning to accumulate. Some years ago we began to study vaccinia virus, a large DNA virus that replicates in the cytoplasm of infected cells (117). Viral replication and transcription occur in a cytoplasmic particle called the virosome, or virus factory. The existence of this “organelle”presented an opportunity to ask whether dNTP synthesis also occurs in the virosome. Our first experiments were inconclusive; when we isolated virosomes by standard procedures and analyzed them for virus-coded ribonucleotide reductase activity, we found no evidence for association of this enzyme with virosomes (118).However, once antibodies to the viral reductase became available, we could do immunocytochemical experiments, and these confirmed that the viral enzyme is localized to virosomes within the infected cell (119). More intriguing, we have applied the anti-idiotypic antibody approach described in Section IV,C to vaccinia ribonucleotide reductase. A polyclonal antiserum

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prepared against purified antibody to the small subunit reacts specifically with a 34-kDa viral protein, which is also localized to virosomes, as shown by immunocytochemistry (119).That protein has been identified as an abundant DNA-binding phosphoprotein, with a specificity for single-stranded DNA. While we do not yet know the biological basis for the association or the role of the protein in DNA replication, this association represents perhaps the clearest indication of a functional link in any biological system between the synthesis and utilization of DNA precursors. Moreover, the association of an enzyme of small-molecule metabolism with a particulate structure is of interest in relation to the concept of “ambiquitous” enzymes mentioned earlier (6).

VII. General Discussion At the outset, I stated that a rigorous test of the metabolon concept in any biological process would require a range of different experimental approaches. Although the truth of that statement may still be evident, some readers may feel that in some of the areas discussed here, too much information is available. For example, there is equally persuasive evidence for and against DNA precursor compartmentation in different mammalian cell systems. Even when compartmentation is clearly documented, its existence does not provide unequivocal support for the concept that DNA precursors are synthesized at replication sites. Our current state of uncertainty about some of these issues merely underscores the necessity of combining further biochemical experimentation with simultaneous unraveling of the complexities of cell structure and function. It does appear evident that, at least in one prokaryotic system, the enzymes of dNTP biosynthesis are organized into a multienzyme complex. Evidence for interaction between components of this complex and replication proteins is emerging, but it is still to early to describe the interactions in hnctional terms. For eukaryotic cells, evidence is accumulating that dNTPsynthesizing enzymes are somehow associated, even though the kinetic and genetic evidence to support the specificity of the associations seen still needs to be developed more thoroughly. If such complexes exist, as seems likely to me, they are probably extranuclear in most cells. Whether there is some association with the nuclear membrane, which might act somehow to facilitate the transport of deoxyribonucleotides into the nucleus, is a fascinating, and still open, question. ACKNOWLEDGMENTS I thank numerous students and co-workers, whose efforts are cited throughout this article, for their contributions to the ideas developed here. Special thanks go to Patrick Young and

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Ralph Davis for thoughtfully and critically reviewing a draft of this article. Research in my laboratory is supported by research Grants NSF DMB-8916366and NIH R01-GM37508.

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C.-S. Chiu, K. S. Cook and G. R. Greenberg, JBC 257, 15087 (1982). C. ThylBn and C. K. Mathews, JBC 264, 15169 (1989). C. Manoil, N. Sinha and B. Alberts, JBC 252, 2734 (1977). R. G . Sargent, Ph.D. Thesis, Oregon State University, 1987. C. K. Mathews, L. K. Moen, Y. Wang and R. G . Sargent, Trends Biochem. Sci. 13,394 (1988). 45. C. K. Mathews and J. Ji, BioEssays 14, 295 (1992). 46. R. G . Sargent, J. Ji, B. Mun and C. K. Mathews, MCG 217, 13 (1989). 47. J. Ji, R. G. Sargent and C. K. Mathews, JBC 266, 16289 (1991). 48. T. Formosa, R. L. Burke and B. M. Alberts, PNAS 80, 2442 (1983). 49. L. J. Wheeler, Y. Wang and C. K. Mathews, JBC 267, 7664 (1992). 50. N. B. Ray, Ph.D. Thesis, Oregon State University (1992). 51. R. L. Somerville, J. H. Zeilstra-Ryalls and T.-L. Shieh, in “Structuraland Organizational Aspects of Metabolic Regulation” (P. A. Srere, M. E. Jones and C. K. Mathews, eds.), p. 181. Alan R. Liss, Inc., New York, 1990. 52. J. P. Young and C. K. Mathews, JBC 267, 10786 (1992). 53. H. R. Warner, J . Bact. 115, 18 (1973). 54. S. Eriksson, EJB 56, 289 (1975). 55. C. A. Lunn and V. Pigiet, JBC 254, 5008 (1979). 56. Asa Anderson, L. Hoglund, E. Pontis and P. Reichard, Bchem. 25, 868 (1986). 57. C. K. Mathews, B.-M. Sjoberg and P. Reichard, EJB 166, 279 (1987). 58. A. Fridland, Nature 243, 105 (1973). 59. M. R. Taheri, R. G . Wickremasinghe and A. V. Hofmrand, BJ 194,451 (1981). 60. M. R. Taheri, R. G. Wickremasinghe and A. V. Hofmrand, B] 196, 225 (1981). 61. M. R. Taheri, R. G. Wickremasinghe and A. V. Hofmrand, Brit. J. Haematol. 52, 401 (1982). 62. G. P. V. Reddy, J . Mol. Recogn. 2, 75 (1989). 63. P. Reichard, ARB 57, 349 (1988). 64. D. E. Kizer and B. A. Howell, BBA 561, 276 (1979). 65. P. L. Panzeter, J. L. Etheredge, D. E. Kizer and D. P. Ringer, BBRC 149, 27 (1987). 66. D. Kuebbing and R. Werner, PNAS 72, 3333 (1975). 67. C. K. Mathews, Erp. Cell Res. 92, 47 (1975). 68. D. Roberts and C. Peck, Cancer Res. 41, 505 (1981). 69. M. H. N. Tattersall and K. R. Harrap, Cancer Res. 33, 3086 (1973). 70. R. G . Wickremasinghe and A. V. Hoffbrand, J. Clin. Znoest. 65, 26 (1980). 71. R. D. Snyder, Mutation Res. 131, 163 (1984). 72. A. Collins and D. J. Oates, EJB 169, 299 (1987). 73. S. Kyburz, J. C. Schaer and R. Schindler, Biochen. Phann. 28, 1885 (1979). 74. T. W. North, R. K. Bestwick and C. K. Mathews, JBC 255, 6640 (1980). 75. S. A. Fuller, J, J. Hutton, J. Meier and M. S. Coleman, B] 206, 131 (1982). 76. B. Nicander and P. Reichard, PNAS 80, 1347 (1983). 77. E. Wawra, JBC 263, 9908 (1988). 78. B. Nicander and P. Reichard, JBC 260, 5376 (1985). 79. J. M. Leeds and C. K. Mathews, MCBiol7, 532 (1987). 80. E. S. J. ArnBr, M. Flygar, C. Bohman, B. Wallstrom and S. Eriksson, Exp. Cell Res. 178, 335 (1988). 81. G. Spyrou and P. Reichard, JBC 264, 960 (1989). 82. B. T. Nguyen and W, Sad6e, BJ 234, 263 (1986). 83. D.-S. Duan and W. Sadke, B] 255, 1045 (1988). 84. W. Rode, K. J. Scanlon, B. A. Moroson and J. R. Bertino, JBC 255, 1305 (1980). 40. 41. 42. 43. 44.

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85. G. P. V. Reddy, BBRC 109, 908 (1982). 86. G. P. V. Reddy and A. B. Pardee, Nature 303, 86 (1983). 87. P. Chiba, P. E. Bacon and J. G. Cory, BBRC 123,656 (1984). 88. W. Rode, M. M. Jastreboff and J. R. Bertino, BBRC 128, 345 (1985). 89. T. M. Plucinski, R. S. Fager and G. P. V. Reddy, Mol. P h m c o l . 38, 114 (1990). 90. F. W. Scott and D. R. Forsdyke, BJ 190, 721 (1980). 91. S. Eriksson, S. Skog, B. Tribukait and B. Wallstrom, E r p . Cell Res. 168, 79 (1987). 92. J. Lagergren and P. Reichard, B i o c h a . P h o m c o l . 36, 2985 (1987). 93. M. B. Slabaugh, M. L. Howell, Y. Wang and C. K. Mathews, J . Virol. 65, 2290 (1991). 94. R. K. Bestwick, G. L. Moffett and C. K. Mathews, JBC 257, 9300 (1982). 95. L. Skoog and G. Bjursell, JBC 249, 6434 (1973). 96. M. Andersson and L. Lewan, Int. J. Biochem. 21, 593 (1989). 97. J. M. Leeds, M. B. Slabaugh and C. K. Mathews, MCBiol 5, 3443 (1985). 98. B. Mun and C. K. Mathews, MCBiol 11, 20 (1991). 99. P. L. Paine and S. B. Horowitz, in “Cell Biology, A Comprehensive Treatise” (D. M. Prescott and L. Goldstein, eds.), Vol. 4, p. 301. Academic Press, New York, 1980. 100. E. Baril, B. Baril, H. Elford and R. B. Luftig, in “Mechanism and Regulation of DNA Replication” (A. R. Kolber and M. Kohiyama, eds.), p. 275. Plenum, New York, 1974. 101. H. L. Elford, ABB 163, 537 (1974). 102. I. Toth, G. Lazar and H. M. Goodman, E M B O J . 6, 1853 (1987). 103. G. P. V. Reddy and A. B. Pardee, PNAS 77, 3312 (1980). 104. H. Noguchi, G . P. V. Reddy and A. B. Pardee, Cell 32,443 (1983). 105. R. G. Wickremasinghe, J. C. Yaxley and A. V. Hofmrand, BBA 740, 243 (1983). 106. D. Ayusawa, K. Shimizu, H. Koyama, K. Takeishi and T. Seno, JBC 258, 48 (1983). 107. G. Harvey and C. K. Pearson, J. Cell. Physwl. 134, 25 (1988). 108. G. Spyrou and P. Reichard, BBRC 115, 1022 (1983). 109. A. Larsson, EJB 11, 113 (1969). 110. R. Kucera and H. Paulus, E r p . Cell Res. 167, 417 (1986). 111. Y. Engstrom, B. Rozell, H.-A. Hansson, S. Stemme and L. Thelander, EMBOJ. 3, 863 (1984). 112. Y. Engstrom and B. Rozell, E M B O J . 7, 1615 (1988). 113. J. A. Vilpo and H. Autio-Harmainen, Scand. J. Clin. Lab. Inuest. 43, 583 (1983). 114. M. Sikorska, L. M. Brewer, T. Youdale, R. Richards, J. F. Whitfield, R. A. Houghten and P. R. Walker, Biochem. Cell B i d . 68, 880 (1990). 115. P. R. Cook, Cell 66, 627 (1991). 116. S. L. Dresler, M. G. Frattini and R. M. Robinson-Hill, Bchem. 27, 7247 (1988). 117. B. Moss, Science 252, 1662 (1991). 118. M. B. Slabaugh, T. L. Johnson and C. K. Mathews, J. Virol. 52, 507 (1984). 119. R. E. Davis, Ph.D. Thesis, Oregon State University, 1992.

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Identification and Characterization of Novel Substrates for Protein Tyrosine Kinases MICHAEL D. SCHALLER,* AMY H. BOUTON,* DANIELC. FLYNN? AND J. THOMAS PARSONS*J *Department of Microbiology and Cancer Center School of Medicine University of Virginia Charlottewille, Virginia 22908 fMBR Cancer Center and Department Microbiology University of West Virginia School of Medicine Morgantown, West Virginia 26506

I. Detection of Phosphotyrosine-containingProteins ................... 11. Receptor Protein Tyrosine Kinases ................................ 111. Oncogenic Protein Tyrosine Kinases ............................... IV. Novel Strategies for the Identification of Substrates .................. V. Tyrosine Phosphorylation: Molecular Consequences . . . . . . . . . . . . . . . . . . References .....................................................

of

207 208 211 215

222 224

Protein tyrosine kinases ( P T K S ) ~play an important role in the transmission of signals for a broad spectrum of cellular activities, including the activation of secretory events, T-cell and B-cell activation, responses to cell-extracellular matrix interactions, differentiation, mitogenesis, and oncogenesis. Because of the desire to understand the molecular basis of carcinogenesis, the roles played by PTKs in mitogenesis and oncogenesis have been most extensively studied. Tyrosine phosphorylation of cellular subTo whom correspondence may be addressed. Terms and abbreviations:ABL, product of the abl gene; cdc, cell cycle control; CSF-1-R, colony-stimulating factor-1 receptor; CRK, product of the crk oncogene; DAG, diacylglyerol; EGF-R, epidermal growth factor receptor; FAK, fwal adhesion kinase; FGF-R, fibroblast growth factor receptor; FITC, fluorescein isothiocyanate;FMS, product of the CSF-1 receptor gene; FPS, product of the fis oncogene; GAP, GTPase-activating protein; IGF, insulin-like growth factor; Ins-R, insulin receptor; IPS, inositol1,4,5-trisphosphate; IRS-1, insulin receptor substrate; KIT, steel factor receptor; MAPK, mitogen-activated protein kinase; Mab, monoclonal antibody; mt-c-SRC, polyoma virus middle-t antigen-pp60c-6" complex; PDGF-R, 1

2

205 Progress in Nucleic Acid Research and Molecub Biology, Vol. 44

Copyright 0 I993 by Academic Press, Inc. All rights of reproduction in any form reserved.

206

MICHAEL D. SCHALLER ET AL.

strates in uioo constitutes an important step in these processes, presumably by eliciting an alteration in the function and/or intracellular organization of these proteins. Thus the identification of substrates for protein tyrosine kinases is of central importance for the understanding of both normal and abnormal growth control. Protein tyrosine kinases that function as growth factor receptors have recently been the subject of several excellent reviews (1-4). Therefore, they are only discussed selectively, to provide illustrations of the functional consequences of tyrosine phosphorylation. This review focuses upon (1)the strategies used to identify PTK substrates and (2) the recent progress made in identlfying new and interesting cytoskeletal protein components that serve as substrates for oncogenic PTKs. The receptors for a number of polypeptide growth factors are transmembrane proteins containing an extracellular ligand-binding domain and a cytoplasmic domain responsible for protein tyrosine kinase activity (1 -4). FTKs have also been identified as the translation products of a number of oncogenes (5, 6). Expression of these tyrosine kinase oncoproteins in cultured cells results in pronounced alterations in cell shape and growth control, and, in many cases, renders the transformed cells tumorigenic in syngeneic animals or nude mice. In addition, inoculation of retroviruses containing a tyrosine kinase oncogene into animals often leads to the rapid induction of leukemias or solid tumors. Thus, oncogenic PTKs subvert the regulation of normal cell growth, presumably by chronically transmitting a mitogenic signal into the cell. Whereas oncogenic PTKs are enzymatically very active, the enzymatic activities of normal, cellular receptor and nonreceptor PTKs are tightly regulated and generally are quite low. The activity of the receptor PTKs increases dramatically upon binding its ligand, presumably due to the ligand-induced conformational alteration of the kinase domain. The elevated activity of the oncogenic FTKs is mediated by the acquisition of mutations that abrogate the normal regulation of kinase activity. For example, v-erbB encodes a constitutively active derivative of the epidermal growth factor (EGF) receptor and is activated by an amino-terminal truncation deleting the ligandplatelet-derived growth factor receptor; PISK, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositoI4,5-bisphosphate:PIE,, phospholipase C,; pp, phosphoprotein;pp5Wn, product of the c-fyn gene; ppGoc.s'c, product of the c-src gene; pp62C-yes,product of the c-yes gene; PtdIns 4K, enzyme that catalyzes phosphorylation of the D 4 position of the inositol ring of phosphatidylinositol; PtdIns(4)P 5K, enzyme that catalyzes the phosphorylation of the D-5 position of the inositol ring of phosphatidylinositol (4)P; PTK, protein tyrosine kinase; RAF, product of the rafgenr; ROS, product of the ros oncogene; RSV, Rous sarcoma virus; SH2, src homology 2; SH3, src homology 3; TrpE, product of the Escherichiu coli tryptophan E gene; YES, product of the c-yes gene.

PROTEIN TYROSINE KINASE SUBSTRATES

207

binding site (7). In the case of the oncogenic PTK, v-src, enzymatic activation arises from the substitution of 12 unrelated amino acids for the carboxyla region that terminal 19 residues of the proto-oncogene product, PPGOC-~~C, contains a critical site for negative regulation of the kinase activity (3, 8, 9). The mutations that deregulate the activity of these PTKs are critical for the oncogenicity of these proteins, since their regulated cellular counterparts are not oncogenic, even when overexpressed (8, 9). The exception to this observation is that cells overexpressing receptor PTKs become transformed when chronically activated by ligand (10-12). Several lines of evidence indicate that tyrosine phosphorylation of cellular proteins by PTKs is essential for propagating intracellular signals that are either induced by ligand activation of transmembrane receptor PTKs or by expression of constitutively activated oncogenic PTKs (1-4, 8, 9). First, an increase of phosphotyrosine on cellular proteins follows the enzymatic activation of receptor and nonreceptor PTKs. Second, abrogation of PTK activity by site-directed mutations of the kinase domains of receptor and nonreceptor PTKs blocks ligand-induced signaling and oncogenic transformation. In addition, structural alterations leading to improper subcellular localization of the oncogenic PTKs also inhibit transformation, presumably reflecting a requirement for the colocalization of the PTK and its substrates (13, 14). It is clear from these studies that PTKs transmit signals by phosphorylating substrates on tyrosine, thereby altering some property of the substrate. Consequently, one of the major thrusts of PTK research has been directed toward the identification and characterization of the in uiuo substrates of these enzymes.

1. Detection of Phosphotyrosine-containing Proteins In early studies, substrates for PTKs were identified by labeling cells with [32P]phosphateand looking for either the appearance of new phosphoproteins or an increase in the extent of phosphorylation of a phosphoprotein, using two-dimensional gel electrophoresis (15-1 7). Phosphotyrosine-containing proteins were distinguished from phosphoserine- or phosphothreonine-containing proteins by the differential sensitivity of serinelthreonine and tyrosine phosphoester bonds to alkali, and, more directly, by phosphoaminoacid analysis. A major breakthrough in the analysis of tyrosine phosphorylation was the development of antibodies that specifically recognize phosphotyrosine. Both antisera and monoclonal antibodies recognizing phosphotyrosine have been generated successfully using a variety of immunogens, including phosphotyrosine (18), synthetic haptens that structurally resemble phosphotyrosine [such as p-azobenzyl phosphonate (19,20)and 0phosphotyramine (21)], or purified proteins that contain phosphotyrosine

MICHAEL D. SCHALLER ET At,,

208

(22). These reagents have proved to be specific and sensitive tools for the detection of phosphotyrosine, and have been invaluable in the identification of PTK substrates.

II. Receptor Protein Tyrosine Kinases A. Substrates for Receptor PTKs The initial identification of candidate substrates for the receptor PTKs relied heavily upon the wealth of data describing the biochemical responses of cells to growth factor stimulation. Thus cellular enzymes activated in response to ligand stimulation of individual receptors were examined to test whether they were direct targets for the activated receptor. For example, stimulation of cells with polypeptide growth factors [epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)] leads to increases in levels of inositol 1,4,5-trisphosphate (IP,) and l,%diacylglyerol (DAG) ( I d ) , which are secondary messengers that stimulate Ca2+ release and protein kinase C activity, respectively (23).This observation suggested that the phospholipaw that cleaves phosphatidylinositol4,5-bisphosphate (PIP,) into IP, and DAG, i. e., phosphatidylinositol-specific phospholipase C,-1 (PLC,), may be a substrate for receptor FTKs. Subsequent experiments showed that PLC, becomes phosphorylated on tyrosine in response to stimulation of cells with either EGF or PDGF (see Table 1). Similar approaches have led to the identification of other components of signaling pathways that become phosphorylated on tyrosine following growth factor stimulation. These candidate substrates include: phosphatidylinositol 3-kinase (PI&), another enzyme involved in lipid metabolism; GTF'ase-activating protein (GAP), a protein involved in regulating the activity of ~21""; and mitogen-activated protein kinase (MAPK), a serine/threonine protein kinase (see Table I).

B. Tyrosine Phosphorylation: Alterations in the Regulation of Enzymatic Activity Recent experiments have extended the original identification of some receptor PTK substrates by addressing the role of tyrosine phosphorylation in altering the function of the substrate. PLC, is an example of a substrate that exhibits a functional change following tyrosine phosphorylation. In response to EGF, PLC, becomes phosphorylated on tyrosine and exhibits an elevation in enzymatic activity in uiuo (3).However, enzymatic activation of PLC, by tyrosine phosphorylation in uitro requires the presence of physiological concentrations of profilin (24), a small protein that can bind to PIP, and actin in a mutually exclusive fashion (25). In the presence of either

209

PROTEIN TYROSINE KINASE SUBSTRATES

SUBSTRATES OF

TABLE I PROTEIN TYROSINE KINASES~

kCEPrOR

Kinase

Physical association

Ref.

GAP

PDGF-R EGF-R CSF-1-RL

Yes -

95,101,102 56, 57 103

PEY

PDGF-R EGF-R FGF-R KIT

Yes Yes Yes Yes

104-107 105, 108-110 111, 112 113, 114

RAF

PDGF-RL EGF-RL FGF-RL KITb

Yes -

115, 116 115 115 113

PI3K

PDGF-R EGF-RC Ins-RC CSF-1-RC KITC

Yes Yes Yes Yes Yes

53, 117 I18 98, 119 97, 120 113, 114

PtdIns 4K

EGF-R

Yes

121

PtdIns(4)P 5 K

EGF-R

Yes

121

pp~c-src

PDGF-R

Yes

122-124

pp59fvn

PDGF-R

Yes

124

pp62C-yes

PDGF-R

Yes

124

pp190 (Gap associated)

EGR-R CSF-1-Rb

-

56, 57 I03

pp62-64 (GAP associated)

PDGF-R EGF-R CSF-1-RL

-

95 56, 57 103

PP120

PDGF-R EGF-R CSF-1-R

-

93, 125 93, 125 125

IRS-1

Ins-R

-

126, 127

pp36 (calpactin or lipocortin)

EGF-R

__

128

Substrate

p42 (MAPK)d

"See footnote 1 in text for abbreviations. LLow levels of phosphorylation are observed following activation in uioo. CTyrosine pbosphorylation may not accompany association with receptor. dTyrosine phosphorylation of p42 (MAPK) is observed in response to a wide variety of mitogens.

MICHAEL D. SCHALLER ET AL.

210

profilin, or a detergent (24, 26), the in uitro activity of tyrosine-phosphorylated PLC, is severalfold higher than PLC, isolated from unstimulated cells. Therefore tyrosine phosphorylation of PLC, may be important in enhancing PLC, activity upon profilin-PIP, complexes, which may be the physiologically relevant substrate for the enzyme. Additional evidence for the importance of tyrosine phosphorylation comes from genetic analysis of PLC,. Tyrosine 783 has been identified as a major site of PLC, tyrosine phosphorylation by the PDGF receptor. Mutation of this tyrosine abrogates the activation of PLC, normally observed upon stimulation with PDGF, implying that phosphorylation of this residue contributes to the activation of enzymatic activity (27). Tyrosint. phosphorylation also plays a crucial role in regulating two serinclthreonine protein kinases, MAP kinase and px3tCdc2. MAP kinase is activated hy treatment of cells with a diverse array of stimuli and concomitantly becomes phosphorylated on both tyrosine and threonine (28). Removal of the phosphate from either the tyrosine or threonine results in the inactivation of the enzyme (28, 29), indicating that the phosphorylation of a specific tyrosine residue appears to positively regulate MAP kinase activity. The mitotic regulator pWA2 is the catalytic subunit of a serine/threonine protein kinase, which physically associates with a regulatory subunit, cyclin B (30, 3 1 ) . p.34iCdr2is phosphorylated on a tyrosine and thrconine within the ATP binding site of the enzyme by the wee-eneoded kinase (32).Phosphorylated pWd'2 is enzymatically inactive and the enzyme is activated only upon dephospborylation by the protein tyrosine phosphatase ~ 8 0 "(33, ' ~34). ~ ~

C. Tyrosine Phosphorylation: Regulation of Protein-Protein interactions One of the first proteins to become phosphorylated on tyrosine following activation of receptor PTKs is the receptor (1-4). Subsequent analysis shows that several other tyrosine-phosphorylated proteins coimmunoprecipitate with the activated receptor. Further characterization of these associated proteins revealed that they include a number of the candidate substrates described above, such a5 PLC,, PI&, and GAP, and, in addition, several other nonreceptor PTKs, e.g., p ~ 6 0 " - (see " ~ ~Table I). Reconstitution of receptor-substrate complexes in uitro, coupled with mutational analysis of receptor PTKs, clearly demonstrated that these protein-protein interactions are catalyzed by autophosphorylation of the receptor on tyrosine (3). Each of the sequences of PLC, (35),GAP (36, 37), and the 85-kDa noncatalytic subunit of PI,K (38-40) contains an SH2 (src homology 2) domain, a cxmserved motif previously identified in virtually all nonreceptor protein tyrosine kinases (41, 42). SH2 domains mediate protein-protein interactions, as initially shown for pp60-"", where it was demonstrated that

PROTEIN TYROSINE KINASE SUBSTRATES

211

the integrity of the ppGosrc SH2 region is required for physical association with a tyrosine-phosphorylatedprotein, pp130 (43; see Section IV,B,3). This observation was corroborated by the analysis of the v-crk oncogene, which encodes a small protein, devoid of enzymatic activity, consisting of an src homology 3 (SH3) and two SH2 domains (44).This protein is associated with several phosphotyrosine-containing proteins, supporting the hypothesis that S H2 domains are binding sites for protein-protein interactions (45, 46). Given the conserved nature of SH2 domains among several receptor-binding proteins and the documented role of SH2 domains in protein-protein interactions, it was proposed that the SH2 domains are binding sites for the autophosphorylated receptors. It is now clear from analysis of several receptor-substrate interactions in uitro as well as mutational studies in uiuo that the SH2 domains of these proteins bind to a specific peptide sequence in the receptor PTK only when that sequence is phosphorylated on tyrosine (42). The reason for the association of these proteins with the receptor PTKs is not clear. This may be a mechanism for recruitment of substrates to the kinase, or for the colocalization of enzymes with their substrates (e.g., PLC, with PIP,). Alternatively, physical association of the receptor with these proteins may directly modulate their activity, as has recently been suggested for PLC, when it complexes with the EGF receptor (47). Regardless of the reason for these specific associations, these analyses clearly demonstrate that tyrosine phosphorylation plays a role in regulating these protein-protein interactions.

111. Oncogenic Protein Tyrosine Kinases A. Substrates Common to Receptor and Oncogenic P T k Many of the substrates for receptor PTKs are also substrates for oncogenic FTKs (see Table 11). These substrates may be important for transformation, since phosphorylation of these substrates by the oncogenic PTKs may mimic the mitogenic signal(s) transmitted by the activated receptor PTKs . 1. PHOSPHATIDYLINOSITOL3-KINASE PI,K is a heterodimeric protein complex that phosphorylates phosphatidyl inositides on the D3 position of the inositol ring (48).It is composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (49). Although the physiological function of the phosphorylated phosphatidylinosit01s generated by PI,K is not known, they are presumed to play a role in mitogenesis and oncogenesis since, in several cases, there is a close correlation between mitogenic or oncogenic activity and activation of PI,K (3).

Siilxtratr

M.lalP

Kinase

Physical association

Cellular location

SRC

-

Focal adhesion

66

SRC

-

Focal adhesion

64,65

SRC

-

Focal adhesion

63

SRC

-

Focal adhesion

84, 85. BN

S RC

Yes

F t d adhesion kiriasr

85

SHC

-

Focal adhesion

84

SRC mT-cSRC ABL

Yes Yes Yes Yes Yes Yes Yes

51, 52 53, 54, 129 130 129 129 129 129

43, 83, 85, 131

FPS YES

CRK" ROS 3D1, 4F4, 5F9

SRC

Yes

4c3

SRC

Yes

hctin filament binding proteinc

SRC

-

Cortical actin binding protein'

1H1, 1H3, 3A7, 3B12, 3D9, 4Fl1, 5A7, 6E12

ticf.

43, 83, 85 85, 92

-

pp36 (calpactin)

SRC

Cytoskeleton

15, 62

GAP

SRC ABL FPS FMSe

Cytosold Cytosold Cytosold Cytosold

56-58 56 56 56

pp1W (GAP associated)

SRC ABL FPS FMS

GAP GAP GAP GAP

associated associated associated associated

56, 57 56 56 56

pp62-pp64 (GAP associated)

SRC ABL FPS FMS

GAP GAP CAP GAP

associated associated associated associated

56, 57 56 56 56

1E12, 2B12

SRC

Membrane associated

80, 81, 85, 93

2C5, 4E10,4G8, 4H6, 5H10

SRC

85

EGF-R

-

SRC

Plasma membrane

IGF-I-R

-

SRC

Plasma membrane

72

p42 (MAPK)

-

SRC

-

16

Connexin-43

-

SRC

aMab-producing cell lines. bCRK elevates cellular F'TK activity and complexes with cellular proteins (46) (see text). CIn transformed cells, these proteins redistribute to rosettes (podosomes)(see text). dTyrosine-phosphorylated GAP is membrane associated (95). eOnly low levels of phosphorylation were observed following activation in uiuo.

Gap junctions

73

76, 77

214

MICHAEL D. SCHALLER ET AL.

Immune complexes of active variants of ppW" contain a phospholipid kinase activity (a), and it is now clear that this activity is due to the physical association of PI,K with ppWrc (51). PI,K enzymatic activity can also be recovered from transformed cells by immunoprecipitation with antiphosphotyrosine antibodies (52). However, it is not clear whether this is due to immunoprecipitation of PI,K directly or to coimmunoprecipitation with another phosphotyrosine-containingprotein. The 85-kDa subunit of PI,K does become phosphorylated on tyrosine in cells transformed by the middleT antigen of polyoma, a protein that transforms cells by associating with, and activating, several cellular nonreceptor PTKs, including p ~ 6 0 " - (53, " ~ ~54). 2. GTPase-ACTIVATING PROTEIN

GAP, the GTPase-activating protein for p21ras, stimulates the GTPase activity intrinsic to p2lraS, converting GTP-p21" to GDP-p21ras and thus attenuating the rus signal (55).GAP has also been proposed to be the effector molecule for p21rm, through which the signal transmitted by p2lraSis propagated (55). GAP and two GAP-associated proteins of M, 190,OOO and 62,000-64,OOO have recently been shown to become phosphorylated on tyrosine in cells transformed by several different oncogenic PTKs (56, 57). It has also been reported that GAP can associate physically with ppWrc, although the stoichiometry of association is low (58). The observed tyrosine phosphorylation of GAP is provocative, since microinjection of neutralizing antibodies directed against p21rm (59)or overexpression of GAP blocks transformation by v-src (60, 61).Thus transformation by ppWrc appears to operate through p21ras, and phosphorylation of GAP provides a possible mechanism through which these two proteins may be linked.

B. Cytoplasmic and Cytoskeletal Substrates Several strategies have been used to identify and characterize additional PTK substrates. Labelling of normal and transformed cells in parallel with 32P resulted in the identification of two cytosolic phosphoproteins, pp36 (15, 62) and pp42 (16,17),and three cytoskeletal proteins, vinculin (a), talin (64, 65), and the P,-integrin subunit (66), whose tyrosine phosphorylation was increased in ppW-srC-transformedcells (see Table 11). pp36 has since been identified as calpactin (liportin), a member of a family of calcium- and phospholipid-binding proteins that exhibit Ca2 -dependent membrane and cytoskeletal association (67).pp42 has been identified as a MAP kinase, one member of a family of serine/threonine protein kinases that are activated in response to many cellular stimuli (28, 68). It does not appear that pp36 and pp42 are intimately linked to transformation, since (1)phosphorylation of pp36 on tyrosine has been dissociated from many transformation phenotypes +

PROTEIN TYROSINE KINASE SUBSTRATES

215

using a variety of mutants of pp60v-srcand (2) phosphorylation of pp42 on tyrosine in pp60v-src-transformedcells is highly variable (8). Vinculin, talin, and the P,-integrin are localized to focal adhesions in cells in culture. The PI-integrin physically associates with one of a number of a-integrins to form heterodimers that function as cell surface receptors for extracellular matrix proteins (69). Binding studies in uitro show that talin binds to the PI-integrin and to vinculin, and this multiprotein complex is proposed to participate in physically linking the actin cytoskeleton to the extracellular matrix through further associations with two actin-binding proteins, tensin and a-actinin (70). The P,-integrin that is phosphorylated on tyrosine is impaired in its ability to bind both to fibronectin and talin in in vitro binding assays (71)-a provocative observation, since one hallmark of transformation is a gross reorganization of the cytoskeleton. However, the stoichiometry of phosphorylation of vinculin and talin is low, suggesting that phosphorylation of these proteins may not regulate their interaction with other cytoskeletal proteins (64, 65).

C. Other Substrates In cells transformed by ppWrC, two growth factor receptors-the EGF receptor and IGF-I receptor-are constitutively phosphorylated on tyrosine (72, 73). This does not appear to be a consequence of autophosphorylation due to activation of the receptor by an autocrine mechanism. In fact, the site of tyrosine phosphorylation on the EGF receptor in v-src-transformed cells is unique, and is not phosphorylated upon EGF stimulation (73). These observations argue for a direct interaction between receptors and pp60src, and raise the intriguing possibility that some of the phenotypes of transformed cells may be manifested by the posttranslational modification and activation of cellular receptors. In addition to enhanced growth properties, cells transformed by v-src also exhibit a reduction in cell-cell communication through gap junctions (74, 75). Connexin-43 has recently been identified as a gap junction protein in fibroblasts and becomes phosphorylated on tyrosine in v-src-transformed cells (76).Since this phosphorylation is virtually stoichiometric, it has been hypothesized that connexin-43 phosphorylation is responsible for the observed impairment of cell-cell communication (77).

IV. Novel Strategies for the Identification of Substrates The advent of antiphosphotyrosine antibodies allowed the ready detection of more than 30 tyrosine-phosphorylated proteins in v-src-transformed cells (78-81). Distinguishing between fortuitous phosphorylations that are inconsequential to the substrate, and phosphorylations that are important for

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transformation andlor relevant to the function of the substrate, has proved to be an enormous task. Two strategies have been applied to achieve this goal: (1)a genetic analysis using transformation-defectiveand enzymatically active variant PTKs to correlate the phosphorylation of certain proteins with the transformed phenotype, and (2) the development of specific reagents to characterize and identify specific phosphotyrosine-containing proteins, followed by assessment of the role of tyrosine phosphorylation on protein function in normal and transformed cells.

A. Genetic Analysis of PTKs The search for physiologically relevant PTK substrates has been augmented by the availability of numerous mutants ofppWrc (8). Two classes of such mutations have been particularly useful in assessing the importance of candidate substrates: (1) nontransforming variants expressing unmyristoylated forms of ppWrCthat fail to associate stably with cellular membranes, and (2) mutants with defects in the SH3 or SH2 regions of ppGosrc. The src variants in SH3 induce a hsiform morphology in infected cells, whereas mutations in SH2 render the variant defective for transformation. There are several examples of cellular proteins, identified only by their relative molecular weights on one-dimensional SDS-PAGE, that become tyrosine phosphorylated in cells transformed by Rous sarcoma virus (RSV), but are poorly phosphorylated on tyrosine in cells infected with unmyristoylated variants of ppWrc, e.g., pp120 (78, 81,82; see Section IV,B,4). Other studies show that activated forms of ppWrCform stable complexes with two tyrosine-phosphorylated cellular proteins of M, 110,000 and 130,000. Studies of these complexes revealed that stable association requires activation of the kinase activity of ppWrc and the structural integrity of the SH2 and SH3 regions of pp60src. SH3 variants failed to bind or to tyrosine phosphorylate ppll0, and SH2 mutants fail to associate stably with ppll0 or pp130 (43,83). The correlation between transformation and tyrosine phosphorylation and/or association of these substrates with ppW" makes these proteins attractive candidates for transformation-relevant substrates. Although many other studies have attempted to correlate the tyrosine phosphorylation of cellular proteins with transformation by specific src mutants, the number of proteins that bewme tyrosine phosphorylated and the limits of resolution provided by one-dimensional electrophoresis have complicated a systematic analysis and identification of cellular substrates for oncogenic PTKs.

B. Monoclonal Antibodies to Tyrosine-phosphorylated Proteins An alternative strategy for the identification of F'TK substrates has been described by two groups (84, 85), both of which have isolated monoclonal antibodies recognizing specific tyrosine-phosphorylated proteins. The ap-

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proach taken in these studies was to use immunoaffinity chromatography on antiphosphotyrosinecolumns to purify phosphotyrosine-containing proteins from chicken embryo cells transformed by v-src. The eluted fractions, enriched for phosphotyrosine-containingproteins, were then used to immunize mice. Spleen cells from immune mice were fused to a nonsecreting myeloma cell line and the resulting hybrid cells were screened for the production of monoclonal antibodies. In one case, an ELISA-based direct-binding assay identified several hundred hybridomas that yielded positive signals when screened against phosphotyrosine-containing proteins (84). Of this population, approximately 50 antibodies were strongly reactive in Western blots with phosphotyrosinecontaining proteins. For final analysis, 15 different antibody-secreting cells were selected and cloned by limiting dilution. These antibodies reacted with three different phosphotyrosine-containingcellular proteins exhibiting approximate molecular masses of 215, 68-76, and 22 kDa (Table 11). In a second, independent study, both a direct ELISA-based screen and an indirect assay were used to screen approximately 790 hybridomas obtained in two separate experiments (85').Using the dual assay system, approximately 250 clones were scored as positive for reactivity to phosphotyrosine-containing proteins. Subsequent cloning of antibody-secreting cells and identification of reactive antibodies by immunoprecipitation and/or immunoblotting experiments reduced this collection of hybridomas to 32, which were then subdivided into seven groups based on the size of the polypeptides reacting with individual antibodies. The monocIonal antibodies immunoprecipitated tyrosine-phosphorylated proteins with molecular masses of 210, 130, 125, 120, 110,80-85, and 64 kDa (Table 11).Subsequent analysis of these proteins led to their biochemical characterization and in some cases the isolation and sequencing of cDNAs encoding individual proteins. The results of these studies are summarized below.

1. CELLULAR SUBSTRATES ASSOCIATEDWITH

FOCAL

ADHESIONS

Spreading of cells on a solid matrix, either glass or plastic, leads to the formation of specialized sites of cell-substratum contact, referred to as focal adhesions or, alternatively, focal contacts or adhesion plaques. Such structures are rich in cytoskeletal proteins (e.g., talin, vinculin, and a-actinin) that play a prominent role in forming and stabilizing interactions between actin filaments and the plasma membrane (70).Studies with monoclonal antibodies to tyrosine-phosphorylated substrates (Table 11)reveals that three such proteins-pp68-76, pp215/210, and ppl25-reside in the focal adhesions of normal cells. Immunostaining of uninfected chicken embryo cells with monoclonal antibodies showed that pp68-76 colocalizes with d i n and vinculin to focal

FIG. 1. Immunostaining of PTK substrates in normal chicken embryo cells. Chicken embryo cells were grown overnight on coverslips, fixed with paraformaldehyde, permeablized, and immunostained as previously described (92).Cells were immunostained with either monoclonal antibodies (Mabs) (approximately 10 pg/ml) directed to individual substrates (A-E; see Table 11) or a rabbit antibody to the C-terminal peptide of pp6Wrc (F). Incubation with primary mouse Mabs was followed by incubation with goat antimouse immunoglobulin G and incubation with FIE-conjugated donkey antigoat immunoglobulin G (5 pg/ml). Following incubation with rabbit antibody, coverslips were incubated with FIX-conjugated donkey antirabbit IgG (5 pg/ml). (A) Anti-ppl25FAK Mab; (B) anti-tensin Mab; (C) anti-paxillin Mab; (D) anti-ppll0 Mab; (E) anti-pp80/85 Mab; (F) anti-ppMW serum.

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219

adhesions (86; Fig. 1). It is also present in the focal adhesion of MadinDarby bovine kidney cells, but is absent from the cell-cell adherent junctions of these cells. Based on this characteristic localization within the cell, these proteins were given the name "paxillin" [derived from the Latin puxillus, meaning a small stake or peg (86)l. Paxillin is most abundant in chicken gizzard smooth muscle and other muscle tissues, and is absent from neuronal tissues. Purified paxillin exhibits a specific interaction with vinculin in uitro. This binding appears to be through the rod domain of the vinculin molecule (86). Interestingly, paxillin appears to be tyrosine phosphorylated in normal chicken embryo cells, and in the gizzard, cardiac, and skeletal muscle of 8day chick embryos, indicating that tyrosine phosphorylation may be a component of its normal cellular regulation (87). Furthermore, the high stoichiometry of tyrosine phosphorylation of paxillin in RSV-transformed chicken embryo cells (20-30%) makes it a candidate for an important substrate for ppW" (84). A second protein-designated pp215 or pp210 (Table 11)-appears to be the cytoskeletal protein, tensin, a component of focal adhesions (88). This conclusion is supported by several lines of evidence. First, antibodies to pp210 readily stain focal adhesions present in chicken embryo cells allowed to spread on glass coverslips or on fibronectin- or laminin-coated coverslips (Fig. 1).Further, monoclonal antibodies to pp210 readily immunoblot tensin immunoprecipitated from normal or RSV-infected chicken embryo cells using polyclonal antibodies to purified tensin. Conversely, polyclonal antibodies to tensin readily immunoblot a 210-kDa protein immunoprecipitated by monoclonal antibodies to pp210 (R. R. Vines and J. T. Parsons, unpublished results). Previous studies show that tensin is a component of focal adhesions and binds to vinculin and actin in uitro. In addition, antibodies to tensin stain Z-lines of skeletal muscle fibrils and cultured embryonic heart cells (88).Molecular cloning of cDNAs encoding a 90-kDa fragment of tensin revealed the presence of an SH2 domain (88).Thus tensin is not only a target for phosphorylation by protein tyrosine kinases, but may also directly interact with other tyrosine-phosphorylatedcomponents of focal adhesions via a phosphotyrosine-bindingregion. Like paxillin, tensin is also tyrosine phosphorylated in normal growing cells (88). The monoclonal antibody 2A7 (Table 11) is directed against a 125-kDa protein that is constitutively tyrosine-phosphorylatedin normal chicken embryo cells. Transformation by v-src leads to a three- to fivefold increase in tyrosine phosphorylation of pp125 (85; B. Cobb and J. T. Parsons, unpublished results). When this antibody was used to immunostain uninfected chicken cells, a characteristic staining of focal adhesions that mimicked the staining seen with antibodies to talin, vinculin, paxillin, and tensin was observed (Fig. 1).Molecular cloning and subsequent sequencing of cDNAs

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encoding pp125 identified it as a novel cytoplasmic protein tyrosine kinase (89).Based on the localization of this tyrosine kinase to focal adhesions, it has been termed pplSFAK,or focal adhesion kinase (FAK). Expression ofTrpEFAK cDNA fusion proteins in E. coli confirmed the intrinsic tyrosine kinase activity of pp125FAK(89). Further, immune complexes of ~ ~ 1 exhibit 2 5 autocatalytic activity and readily catalyze the tyrosine phosphorylation of exogenously added substrates (M. Schaller and J. T. Parsons, unpublished results). Several lines of evidence suggest that the activation of pplSFAKis triggered by interaction of integrins with their extracellular ligands. Aggregation of cell surface integrins with specific antibodies, or spreading of cells on fibronectin-coated tissue culture dishes, results in an increase in the amount of phosphotyrosine in proteins of approximately 120-130 kDa (90,91),at least one of which is pplSFAK(L. J. Kornberg and R. L. Juliano, personal communication; J. L. Guan and R. 0. Hynes, personal communication; M . Schaller and J. T. Parsons, unpublished results). Furthermore, thrombin activation of normal platelets in tjitro results in an increase of phosphotyrosine on a number of proteins, including pp12SFAK.Phosphorylation of pplSFAKis abrogated in platelets from patients with Glanzmann’s thrombasthenia, a disease associated with a defect in the platelet integrin gpIIb/IIIa (L. Lipfert, B. Haimovich, M. Schaller, B. Cobb, J. T. Parsons, and J. S. Brugge, unpublished data). These observations raise the intriguing possibility that pp1SFAKactivation may be linked to integrin function. The intracellular localization of pplSFAKand its observed activation by integrins suggest that it may be a component of a signal transduction pathway, responsible for the transmission of a signal or signals from cell surface integrins into the cell. Alternatively, pp125FAKmay be responsible for phosphorylating the components of fbcal adhesions and regulating the interactions of integrins with the cytoskeleton and/or with the extracellular matrix.

2. CYTOSKELETAL-ASSOCIATED SUBSTRATES Several related proteins of M, 80,000-85,000, collectively referred to as pp80/85, are another interesting component of the cytoskeletal matrix. Immunostaining of normal and RSV-transformed cells with monoclonal antibodies to pp80/85 revealed a striking difference in the subcellular localization of pp80/85 in normal and src-transformed cells. In normal cells, pp80/85 colocalizes with cortical actin in peripheral extensions as well as in perinuclear regions of the cell (Fig. 1). In v-src-transformed cells, pp80/85 localizes almost exclusively in rosettes (podosomes), sites of cell-substratum contact characteristic of cells transformed by PTKs (92). The unique subcellular localization of the pp80/85 proteins in normal cells, as well as its virtually quantitative colocalization with actin in rosettes (podosomes), have

~

~

PROTEIN TYROSINE KINASE SUBSTRATES

22 1

prompted an analysis of its actin-binding properties. In cytochalasin-B-treated normal chicken cells, pp80/85 quantitatively codistributed with the shortened actin filaments, indicating that pp8Ol85 may bind to the ends of F-actin or to actin-associated proteins. Additional experiments have demonstrated that pp80/85, present either in extracts of normal or transformed cells or synthesized by translation in uitro, stably associated with polymerized Factin (H. Wu and J. T. Parsons, unpublished results). Sequence analysis of cDNA clones encoding one of these proteins, pp85, indicated an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids. A second class of cDNA clones encoded pp80 and differed from the pp85 cDNA by the precise deletion of a single repeat. The two species presumably arise by alternative splicing 0. Hildebrand and J. T. Parsons, unpublished results). Both pp80 and pp85 contain a carboxyl-terminal SH3 domain and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The "multidomain" structure of pp80 and pp85 coupled with their colocalization with cortical actin and actin-binding properties in uitro suggest that these proteins may associate with components of the cytoskeleton and contribute to the organization and/or reorganization of actin filaments during transformation. 3. ppm" BINDING PROTEINS pp130 AND ppllo (ACTIN FILAMENT BINDINGPROTEIN)

Immune complexes of ppW" isolated from src-transformed cells contain two tyrosine-phosphorylated cellular proteins designated pp130 and ppl10 (43, 83). Monoclonal antibodies to pp130 and ppll0 have been used to characterize these proteins. As outlined in Section IV,A, these proteins associate with ppWrc through the SH3 and SH2 domains and these complexes are postulated to be important for transformation. pp130 is present on cytoplasmic membranes in pp60Prc-transformed cells, whereas ppl1O localizes on actin stress filaments in normal cells and redistributes to rosettes (podosomes) in src-transformed cells (83; D. Flynn and J. T. Parsons, unpublished results). ppll0 is the only protein believed to associated with SH3 domains. The analysis of cDNAs encoding ppll0 may shed light on the elements involved in protein-protein interactions mediated by SH3 domains. 4. pp120, AN UNIDENTIFIED PTK SUBSTRATE

pp12O was previously identified as a cellular protein whose tyrosine phosphorylation in RSV-transformed cells was dependent upon stable association of pp6os" with cellular membranes (see Section IV,A). Consistent with this observation was the discovery that pp120 is membrane associated

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(93). In addition, it was observed that pp120 is tyrosine phosphorylated in avian cells expressing the polyoma virus middle-T antigen, and in rodent cells stimulated by ECF and PDGF, but not by insulin. These results suggest that the tyrosine phosphorylation of pp120 is directly coupled to activation of oncogenic PTKs and endogenous FTKs, either growth factor receptors or ppW-"'" activated via polyoma middle-T antigen binding. Recently, cDNA clones encoding pp120 have been isolated (A. Reynolds, personal communication). Completion of the analysis of these sequences will provide vaiuable insight into the structure and function of this substrate, which may be an important component of both growth-factor-stimulated signaling pathways, and the pathway through which src mediates some facets of transformation.

V. Tyrosine Phosphorylation: Molecular Consequences As summarized above, the search for PTK substrates has revealed at least two classes of tyrosine-phosphorylated cellular proteins, enzymes that probably play a role in the signal transduction process (i.e., PLC,, PI,K, GAP, and MAPK) and cytoskeletal proteins, many of which are associated with subcellular structures that participate in the maintenance of cell shape and cell movement (i.e., microfilaments and focal adhesions). The molecular consequences of tyrosine phosphorylation by receptor and oncogenic PTKs are severalfold. As pointed out above, tyrosine phosphorylation is clearly involved in enzyme activation. Tyrosine phosphorylation also plays an important role in mediating protein-protein interactions and changes in intracellular localization. A well-studied example of this is p21rm-GAP. Phosphorylation of GAP on tyrosine apparently has no effect upon its ability to activate the GTPase activity of p2lraS in uitro (94). However, following activation of the PDGF receptor, cytosolic GAP rapidly translocates to cellular membranes, forming stable complexes with the receptor (95). Complex formation and translocation to the membrane occur concomitantly with tyrosine phosphorylation of GAP and may be essential for both the regulation of GAP activity and the formation of receptor signaling complexes. In other cells, EGF stimulation or transformation by oncogenic PTKs leads to the stable association of GAP with the tyrosine-phosphorylated proteins pp190 and pp62-64 (56, 57). The phosphorylation of pp190 and pp62-64 on tyrosines may regulate complex formation and consequently alter the catalytic activity of GAP with respect to ~21"". In this fashion, tyrosine phosphorylation of CAP-associated proteins may regulate the activity of GAP by inducing protein-protein interactions, in much the same way that ligandactivated receptors bind and presumably regulate GAP activity. Another example is PI,K wherein tyrosine phosphorylation of the reg-

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223

ulatory subunit of PI,K and/or its physical association with receptor PTKs, polyoma middle-T antigen, and somepncogenic PTKs result in its enzymatic activation (3) and an increase in PI,K reaction products (96-100). In this case, tyrosine phosphorylation may be the “signal” that initiates the relocation of PI,K to the membrane, bringing it into juxtaposition with its relevant substrates. We can only speculate about the consequences of tyrosine phosphorylation upon the organization and function of cytoskeletal proteins, using the precedents established by the analysis of receptor PTK substrates. An attractive hypothesis is that tyrosine phosphorylation of certain cytoskeletal proteins is a “signal” for the formation or breakdown of interactions among cytoskeletal proteins. Tensin, an actin-binding protein, is a component of focal adhesions and contains an SH2 domain. Thus tensin may associate stably with another protein that contains phosphotyrosine. There are three candidate proteins with which tensin may associate in normal cells, paxillin, zyxin, and tensin itself. Each contains low levels of phosphotyrosine under normal growth conditions (88, 89). Tyrosine phosphorylation of the components of focal adhesions may be an important step in the organization and structure of proper cell contacts. The focal-adhesion-associated kinase, pp125FAK, is an attractive candidate for the PTK responsible for regulating interactions among focal adhesion components. In cells transformed by PTKs, tensin and paxillin exhibit a substantial increase in phosphotyrosine content, whereas vinculin and talin contain relatively small amounts. A possible consequence of enhanced tyrosine phosphorylation on tensin may be the formation of aberrant cytoskeletal protein complexes, a process that may contribute to the pronounced alterations in cell shape observed in PTKtransformed cells. In addition to regulating protein-protein interactions within the cytoskeletal matrix, PTKs may also influence interactions between focal adhesion proteins and the extracellular matrix. For example, tyrosine phosphorylation of the PI-integrin subunit reduces its binding to talin or fibronectin in uitro. The site of tyrosine phosphorylation resides within the cytoplasmic tail, at or near the talin-binding site of P,-integrin, and may thus directly block association with talin. However the fibronectin-binding domain is extracellular, and therefore binding to fibronectin must be altered by a conformational change, directly or indirectly caused by tyrosine phosphorylation. Dissecting the interplay between receptor and nonreceptor PTKs, their substrates, and higher order signaling complexes remains an important challenge. There is little question that such signaling processes play an important role in maintenance of the architecture of the cell, programmed rearrangements of cell structure, cell motility, cell cycle, and gene expression.

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The identification and characterization of the cellular substrates described above provide the first step in understanding many of these intricate mechanisms.

ACKXOWLEDGMENTS We thank Randy Vines and Cheryl Borgman for their excellent technical assistance and Jane Cornelius for help in preparation of the manuscript. We also thank our colleagues, Bradley Cobb, Hong Wu,Janice Huff, Timothy Jamieson, Sarah Parsons, and Michael Weber for their many useful discussions. We thank Keith Bunidge and Christopher Turner for antibody to paxillin and Michael Payne for peptide antibody to ppG0F"r. This work was supported by a fellowship from the National Cancer Institute of Canada to M. D. S.,NIH Grants CA29243 and CA40042.

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Index

A Actin filament binding protein, protein tyrosine kinase substrates and, 221 Activators, bacterial adenylyl cyclases and, 41-43 Adenylyl cyclases, bacterial, see Bacterial adenylyl cyclases Amnity chromatography, enzymes in DNA precursor biosynthesis and, 183-185 Alkylation damage repair in genomes O6-alky1guanine, 112-114 N-alkylpurines, 114-116 cloning, 118-120 DNA methylation, 128-129 drug resistance DNA repair, 132-135 gene amplification, 135-136 history, 109-112 inducibility, 129-132 06-methylguanine-DNA methyltransferases (MGMT) properties, 116-1 18 regulation, 120-128 N-methylpurine-DNA glycosylase (MPG), 120-128 outlook, 136-137 06-Alkylguanine, alkylation damage repair in genomes and, 112-114 N-Alkylpurines, alkylation damage repair in genomes and, 114-116 Angiogenin, see Mammalian ribonuclease inhibitor Anti-idiotypic antibodies, enzymes in DNA precursor biosynthesis and, 185-186 Antibodies antisense oligonucleotides and, 158-160 enzymes in DNA precursor biosynthesis and, 185-186 protein tyrosine kinase substrates and, 216-222 Antisense oligonucleotides, 163-164 history, 143-144

internalization, 153-154 cell targeting, 158-160 poly(L-lysine), 155, 157-158 polyanions, 157-158 intracellular distribution, 160-161 SNAIGE concept, 145-146 compartmentalization, 151-152 limitations, 148-149 metabolic stability, 149, 151 sense oligonucleotides, 148 side effects, 153 synthetic ribozymes, 147-148 target choice, 149-150 target hybrids, 152-153 triple helix, 145, 147 VSV model, 156, 161-163 ATP-binding sites, bacterial adenylyl cyclases and, 56-61 5-Azacytidine, alkylation damage repair in genomes and, 129

B Bacillus anthracis, bacterial adenylyl cyclases and, 44-49 Bacterial adenylyl cyclases, 31-32, 61-62 activators, 41-43 ATP-binding sites, 56-61 Bacillus anthracis, 44-49 Bordetella pertussis, 44-49 cya gene, 35-36 cyclic AMP regulation of levels, 34-35 transcription regulation, 32-34 inhibitors, 43-44 Mycoplasmu, 44 Rhizobium, 49-53 sequences, 53-56 sugar transport, 36-41 Bacterial cells, enzymes in DNA precursor hiosynthesis and, 186-187 229

230

INDEX

Bordeteila Irt'rtussis. bacterial adenylyl cyclaser and. 44-49

C Chromatography, enzymes in D N A precrirsor biosyntliesis and, 183-185 cya gene, ljitcterial adenylyl cyclases and. ;35-36 Cyclic AM P. hactc.rial adenylyl cyclases and regulation of levels, 31-35 transcription regulation, 32-34 Cytoplasm. protein tyrosine kinase sulrstrates and. 214-215 Cytoskcleton. protein tyosine kinase substrates inid, 214-215, 220-221

D

bacterid cells, 186-187 dNTP organizition, 171-177 dNTP synthesis in eukaryotic cells, 187 compartmentation, 188-196 DNA replication, 199-200 multienzyrne aggregates, 196-198 replication sites, 198-199 organization, 167-168 DNA metabolism, 170-171 patterns, 168-170 replication, 181- 182 &nit). chromatography, 183-185 antibodies, 185-186 genetic evidence, 182-183 T-l dNTP synthetase, 177 genetic specificity, 178-179 kinetic coupling, 177-178 properties, 179-181 Escherichiu coli bacterial adenylyl cyclases and, 32-36,

38-44 I)eoa!-ril)oiiiiclrotidc.s, enzymes in D N A

precimor I>iosynthesisand, 188-189. 19s- 196 DNA precursor biosyntbesis, enzymes in. see k:nzynies in D N A precursor biosynthesis DNA repair. alkylation daniage repair in genome5 and, 132-135 dNTP, enzyines i n DN.4 precursor hiosynthesis and compart mentat ion, 188- 196 multienzyme aggregates. 196;-198 organization. 171- 177 replication. 198-200 synthesis i n eukaryotic cells, 187 dNTP synthetase. T4, D N A precursor biosynthesis anti, 177-181 Drug resistance, alkylation damage repair i n genomrs and D N A repair, 132-135 gene amplificatictn, 135-136

enzymes in D N A precursor biosyntliesis and, 171-177 Eukaryotic cells. enzymes in D N A precursor hiosynthesis and, 187-200

F Focal adhesion, protein tyrosine kinase substrates and. 217-220

G Genomes, alkylation damage repair in, see Alkylation damage repair in genomes Clycosylase gene, alkylation damage repair in genomes and, 134 GTPase-activating protein, protein tyrosine kinase substrates and, 215

E

H

Enzymes, protein tyrosine kinase substriates and, 208, 210 Enzymes in f)N..\ precursor biosyntliesis,

Hybrids alkylation damage repair in genomes and, 1%-127 antisense oligonucleotides and, 152-153

200

231

INDEX

0

Hydroxyurea inhibitors, enzymes in DNA precursor biosynthesis and, 194-195

I Inhibitors bacterial adenylyl cyclases and, 43-44 enzymes in DNA precursor biosynthesis and, 194-195 mammalian ribonuclease, see Mammalian ribonuclease inhibitor Initiation of transcription in RNA polymerase 11, see RNA polymerase I1 transcription initiation

L Liposomes, antisense oligonucleotides and,

158-160

Oligonucleotides, antisense, see Antisense oligonucleotides Oncogenes alkylation damage repair in genomes and,

127-128 protein tyrosine kinase substrates and,

211-215

P Phosphatidylinositol 3-kinase, protein tyrosine kinase substrates and, 211, 214 Phosphorylation, protein tyrosine kinase substrates and, 208, 210-211, 216-224 Phosphotyrosine, protein tyrosine kinase substrates and, 207-208 Poly(L-lysine), antisense oligonucleotides and, 155, 157-158 Polyanions, antisense oligonucleotides and,

157-158

M Mammalian ribonuclease inhibitor, 1-2, 24-

25 biologic role, 20-24 inhibitory properties binding site, 12-20 constants, 10-12 experimental applications, 20 mode, 12 properties, 2-6 structure, 6-10 06-Methylguanine-DNA methyltransferases (MGMT), alkylation damage repair in genomes and activation, 128-129 properties, 116-118 regulation, 120-128 sequences, 128-129 N-Methylpurine-DNA glycosylase (MPG), alkylation damage repair in genomes and, 120-128 Monoclonal antibodies, protein tyrosine kinase substrates and, 216-222 Mycoplasm, bacterial adenylyl cyclases and, 44

Promoters, RNA polymerase I1 transcription initiation and, 94-98 Protein, enzymes in DNA precursor biosynthesis and, 183-186 Protein tyrosine kinase substrates, 205-207 detection of proteins, 207-208 identification, 215-222 oncogenes, 211-215 phosphorylation, 222-224 receptors, 208-211

R Replication, enzymes in DNA precursor biosynthesis and, 181-186, 189-191, 198-

200 Rhizobium, bacterial adenylyl cyclases and, 49-53 Ribonuclease inhibitor, mammalian, see Mammalian ribonuclease inhibitor Ribozymes, synthetic, antisense oligonucleotides and, 147-148 RNA polymerase I1 transcription initiation,

67-68 domains, 69-75

232

INDEX

motifs, 98-100 promoters, 94-98 repression, 102-105 structure, 68-69 transcription factors, 75-76, 93-94 activation, 100-102 TFIIA, 89-93 TFIIB, 81-83 TFIID, 75-81 TFIIE, 86-89 TFIIF, 83-86 TFIIH, 89

S Sequences alkylation damage repair in genomes and, 128-129 bacterial adenylyl cyclases and, 53-56 SNAIGE concept antisense oligonucleotides and, 145-146 limitations, 148-153 synthetic ribozymes, 147-148 triple helix, 145, 147 sense oligonucleotides and, 148 Substrates, protein tyrosine kinase, see Protein tyrosine kinase substrates

Sugar transport, bacterial adenylyl cyclases and, 36-41

T T4 dNTP synthetase, DNA precursor biosynthesis and, 177-181 Tolerance, alkylation damage repair in genomes and, 134-135 Transcription, bacterial adenylyl cyclases and, 32-34 Transcription initiation, of RNA polymerase 11, see RNA polymerase I1 transcription initiation Transferase repression, alkylation damage repair in genomes and, 127-128 Triple helix, antisense oligonucleotides and, 145, 147 Tyrosine kinase substrates, protein, see Protein tyrosine kinase substrates

V Vaccinia virus, enzymes in DNA precursor biosynthesis and, 199-200