INTERACTIONS BETWEEN POLYOMAVIRUS LARGE T AND THE VIRAL REPLICATION ORIGIN DNA: HOW AND WHY

by

Yu-Cai Peng

Department of Microbiology and Immunology McGill University, Montreal April, 1999

A thesis submitted to the Faculty of Craduate Studies and Re3earch in partial fulfullmeat of the requirements of the degree of doctor of philosophy

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. TABLE OF CONTENTS

Page

Tableofcontents...... I Abstract ...... VI Resumé...... VI11 Acknowledgements ...... X Claim of contribution to kaowledge ...... XI Listoffigures ...... XllI Guidelines regarding doctoral thesis ...... XV

CHAPTER 1. INTRODUCTION ...... 1

1. Overview: Life cycle of polyomavirus and simian 40 ...... 1

II .A most multifunctional : large T antigen...... 1. General properties...... A) Posttranslational rnodifcations ...... B) Nuclear locaiization ...... C) Oligomerization ...... 2 . Functions in viral DNA replication ...... A) Originsfor viral DNA repiicatbn ...... B) Large T antigen and viral DNA replication ...... C) Regulation of DNA replication by of large T antigen . D) Replication factors interacting with large T antigen ...... i) ...... ii) DNA polymerase a: primase ...... iii) Topoisomerase I ...... E) Zhc-f»>gerand Dnd domains on large T antigen ...... i) Zinc-jnger domain ...... ii) Dnal domain ...... 3. Functionfi in regulation of ...... A) Regulation of viral ...... B) Interactions between large T antigen and ~ranscriptionfactors ...... 32 4 . Functions in immortalization and transformation of infected cells ... 34 A) TranF/orming activiy of T ...... 35 B) Cellular targetsfor large T antigen implicated in its transforming uctiviîy ...... 38 i) p ...... 38 ii) pRb/p107/p130 ...... 39 iii) p300/CBP ...... 42

III .DNA Binding by large T antigens ...... 1. Origin specific DNA binding ...... 2 . Nonorigin DNA binding ...... 3. DNA-binding domains on large T antigen ...... 4 . Formation of large T antigen hexamers in the core origin ...... 5. DNA dbtortioiis associated with large T antigen binding andoligomeriution ...... 6. Conditions that affect DNA binding by large T antigen ...... A) nucleotides ...... B) phosphorylation ...... C) pH ......

IV . Production of large T antigens for in vitro analysis ...... 67 1. Expression with an adenovirus vector system ...... 67 2 . Expression in E. coli ...... 69 3 . Expression with a baculovirus system ...... 70 4 . Expression in yeast: Pichia pastoris system ...... 71

CHAPTER 2 . MATERIALS AND METHODS ...... 75

1. Ce11 cultures ...... 75 1. Strains ...... 75 2. Media and growth conditions ...... 76 3. Yeast and bacterial transformation ...... 77 4. Screening for yeast transformants expressing large T antigen ...... 77

II. Nucleicacids ...... 78 1. DNA extraction and enzymatic manipulations ...... 78 2 . PCR ampliIication ...... 79 3. PIasmid constructs ...... 80

III. Protein analysis ...... 83 1. Western blottiag and silver staining ...... 83 2 . Immunoaffinity column prepamtion ...... 83 3. Expression and purification of polyomavirus large T antigen ...... 84

IV. Assays for protein-DNA interactions ...... 87 1. Filter binding assay ...... 87 2. DNase 1 footprinting analysis ...... 88 3. Gel electrophoresis mobility shift assay ...... 89 1. DNA immunoprecipitation assay ...... 89 5 . Glycerol gradient centrifugation ...... 90 6. Duplex DNA fragment unwinding assay ...... 91

CHAPTER 3. Production of active polyomavirus large T antigen inycastPlcliiapmtoris ...... Absfract ...... Introduction ...... Results ...... lntegration of polyomavhs large T antigen gene into yeast Pichia pmris and expression of the large T antigen ...... Specific DNA-binding and purification of the yeast-derived large T antigen ...... Purified large T antigen unwinds duplex DNA fragment ...... Discussion ......

III CHAPTER 4. Polyomavinis large T mtigen bhds cooperatively to its multiple binding sites in the viral ongin of DNA repücation .. Connecting text ......

Introduction ...... ~~...~.~.~.~~.~ Results ...... Specific origin-binding activity of polyomavirus large T antigen is enhanced at pH 7 and below ...... Large T antigen-DNA complexes are stable at pH 6 to 7 but are unstable at pH 7.6 ...... ATP does not affect DNA binding at pH 7 or below but stabilizes a fraction of large T antigen-origin DNA complexes at high pH ...... Stabilization of complexes by ATP depends on the presence of site 112 Binding of large T antigen to origin DNA fragments containing one or more adjacent binding sites ...... Binding of large T antigen to DNA targets containing mutated binding sites ...... DNase 1 footprinting of mutant in the presence of large T antigen ...... ATP specifically enhances protection against DNase 1 digestion of the central 1O to 12 bp of site 112 ...... Discussion ......

CHAPTER 5. Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfucd complexes ...... 145 Connecting text ...... 146 Abstract ...... 147 Introduction ...... 148 Results ...... ,...... 150 Gel mobility shift assay of large T antigen-DNA complexes at pH 6: sensitivity and specificity ...... 150 Application of gel mobility shift assay to crude lysates of cells expressing large T antigen ...... Glycerol gradient analysis of large T antigen-DNA complexes ...... Influence of binding sites on gel retardation ...... Discussion ......

CHAPTER 6. SUMMARY AND CONCLUSIONS ......

1. Expression of large T antigen in Pichia pasioris ...... II . pH effect on origin DNA binding by large T antigen ...... III . Applications of the pH effect on large T antigen DNA binding ..... IV . ATP effect on ongin DNA binding by large T antigen ...... V . Cooperative binding of large T antigen to viral origia of DNA replication ...... VI . Formation of hexamers of large T antigen at site 1/2 ...... ABSTRACT

Polyomavirus large T antigen is the major regulatory protein in the polyomavirus life cycle. It binds to multiple G(A/G)GGC pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus . The nature of interactions beiwern large T antigen and the viral origin of DNA replication is not fully understood. We set out to produce large T antigen protein in the methylotropic yeast Pichiu pasloris by placing the large T antigen gene downstream of the strong alcohol oxidase (AOXI) . Large T antigen was purified by immuno- finity chrornatography by using a monoclonal antibody.

While optimizing the conditions for binding of large T antigen to viral ongin

DNA, we discovered that binding was substantially stronger at pH 6 to 7 than at pH 7.4 to

7.8. a range ofien used in DNA binding assays. We showed that increased binding at low pH is due to increased stability of protein-DNA complexes, and that large T antigen molecules self-associated at low pH, forming massive complexes. ATP increased binding of large T antigen to ongin DNA by about 2-fold at pH 7.8, but had no detectable effect at pH 7 or below.

Enhanced, stable DNA binding by large T antigen to viral origin DNA at pH 6 enabled us to develop a novel gel mobility shift assay ushg unfixed protein-DNA complexes. We demonstrated that this assay is very sensitive and highly specific. This method cm be used both for detection of large T antigen in crude ce11 lysates and for quantitation of binding of purified large T antigen to target DNAs under various conditions. Using a series of point and deletion mutants in the virai ongin of DNA replication, we demonstrated that binding of large T antigen to sites I/2, A, B, and C is cooperative. Binding of large T antigen to one site stimulated binding to other sites 20 to

100 bp distant, and binding to inherently weak sites was strengthened if two or more such sites were present on the same DNA molecule. These findings suggest that large T antigen molecules bound to DNA interact with each other to mutually stabilise their binding.

ATP was shown to stabilise large T antigen-DNA complexes against dissociation only if the DNA contained site 112. ATP specifically enhanced protection against DNase

I digestion in the central 10 to 12 bp of site 1/2, where hexamers are believed to form and begin unwinding DNA. We propose a mode1 in which large T antigen molecules bound to sites 1/2, A, B. and C on origin DNA form a compact protein-DNA complex via mutual interactions; large T antigen molecules bound to sites A, B, and C are mobilised and "handed over" to site 112, where ATP stimulates their assembly into hexamers. L'antigène grand T est la principale protéine régulatrice du cycle de croissance du virus polyome. Cette protéine reco~aitet se lie aux pentanucléotides G(A/C)GGC présents en plusieurs exemplaires dans les sites 112, A, B et C situés aux environs et a l'intérieur de l'origine de réplication (on) de l'ADN viral. La nature des interactions entre la protéine régulatrice et l'on n'est pas encore entièrement comprise. Nous avons décidé de faire produire l'antigène grand T à la levure méthylotropique Pichia pastoris en insérant le géne d'intérêt en aval d'un promoteur fort (promoteur AOXl de l'alcool oxydase). La protéine était ensuite purifiée par chromatographie d'affiinité à l'aide d'un anticorps monoclonal.

Durant l'étude d'optimaiisation de la liaison de I'antigène grand T à I'ori. nous avons découvert que la fixation était plus forte à des pH compris entre 6 et 7 qu'à des pH compris entre 7.4 et 7.8, domaine plus couramment utilisé pour les études de liaison à

1'ADN. Le fait que ['interaction soit meilleure à bas pH s'explique par une plus grande stabilité des complexes nucléoprotéiques en milieu acide. La présence d' ATP améliorait la fixation de l'antigène grmd T a l'on d'environ deuv fois son efficacité normale a pH 7-

8, mais n'avait aucun effet détectable pour des pH inférieurs a 7.

L'obtention d'une liaison plus stable entre l'antigène grand T et l'on nous a permis de mettre au point un protocole d'électrophorèse différentielle car la fixation de la protéine à l'ADN modifie la mobilité des complexes nucléoprotéiques. Nous avons démontré que cette technique est très sensible et très spécifique. Elle peut être utilisée pour la détection d'antigène grand T purifié ou dans des lysats brut, ou pour la quantification de l'efficacité de liaison de la protéine à différents ADN dans différentes conditions.

En utilisant une sene d'on modifiées par ponctuelles ou par délétions, nous avons démontré que la liaison de l'antigène grand T aux sites 1/2, A, B et C est coopérative. La liaison de molécules d'antigène gmd T 5 l'un de ces sites facilite 13 liaison d'autres molécules aux autres sites éloignés de 20 à 100 nt. De plus, l'affinité faible de l'antigène grand T pour certains sites était renforcée si deux ou plus de ces sites se trouvaient sur la même molécule d'ADN. Ces résultats suggèrent que les molécules d'antigène grand T liées à l'ADN ont des interactions mutuelles leur permenant de se stabiliser et de renforcer leur liaison à l'ADN.

Nous avons montré que L'ATP ne stabilise les complexes ADNlantigéne grand T que si le site 1/2 est présent sur la molécule d'ADN. L' ATP engendrait une protection spécifique au niveau des 10 à 12 pb centrales du site I/2 contre l'hydrolyse par la DNAse

1. Or c'est à l'intérieur du site 112 que les hexameres sont supposés se former et que le déroulement de l'ADN commence. Nous présentons un modèle dans lequel les molécules d'antigène grand T se lient aux sites IR, A, B et C de l'ori et forment un complexe

ADN/protéine grâce à des interactions mutuelles entre les molécules. Ensuite, les molécules de l'antigène grand T liées aux sites A, B et C seraient transferrées vers le site

112 où lTATPstimule la formation des hexamères, First, 1 would like to thank my parents and my famiiy for their unconditional love and support throughout my studies. Second, 1 would like to thank my supervisor, Dr.

Nicholas H. Acheson. for giving me the opportunity to enter graduate studies, for providing excellent advice and direction with my project, and for patience while supervising me. Third, 1 would like to acknowledge the McGill Max Stem recruitment fellowship hnd, the F. C. Harrison Fellowship fund, the Medical Research Council of

Canada, and the Faculties of Graduate Studies and Research and of Medicine of McGill

University for providing generous financial support.

1 would like to th& Dr. Greg Maczynski, Dr. Bernard Turcotte, and Dr. Michael

Dubow for discussion and advice about my project, and Dr. Dalius Briedis for allowing me to use equipment for yeast . 1 thank Marie-Claude Ouimet for technical assistance and Elisa Fromm for help with of my abstract into French. 1 thank

Dr. Noelle-Ann Sunstrorn for the constniction of deletion mutant plasmids which 1 used as PCR templates to make DNA fragments containing single or multiple large T antigen binding sites, and David Luckow for help in screening Pichia postoris transformants.

1 thank former members of the laboratory, Dr. Celestino Di Flumeri and Dr. Jaw-

Ming Chemg, for their advice and help in experiments. Special th& to Dr. Alfiedo

Staff for friendship, encouragement, and advice. Thanks to Felix Sieder for fnendship and priceless help with cornputer problems. CLAiM OF CONTRIBUTIONS TO KNOWLEDGE

Polyomavirus large T antigen was successfully expressed in Pichia pastoris. Two

strains of yeast cell, E-3 and E-5 1, which give relatively high expression, were

obtained. Expression was determined to reach optimal level after incubation in

nethmol-containhg medium for 60 hours.

A strong pH effect on viral origin-specific DNA binding by large T antigen was

discovered. Optimal binding occurs at pH 6-7 and binding decreases sharply above

pH 7.4.

A novel gel mobility shift assay based on enhanced and stable DNA binding at pH 6

was developed for detection of large T antigen. This method is specific and sensitive,

and cm be applied to pwified large T antigen or to crude ceIl lysates from different

sources.

Large T antigen was show to self-associate at pH 6-7,resulting in large complexes.

The effect of ATP on specific DNA binding by large T antipen was determined under

different pH conditions. Binding was stimulated by the presence of A?P at pH 7.8, as

shown by previous studies; but ATP had no detectable effect at pH 7 and below. ATP

was show to stabilize large T antigen-DNA complexes at hi& pH.

Large T antigen was demonstrated to bind cooperatively to sites 112, A, B, and C in

polyomavirus origin of DNA replication. Binding of large T antigen to one site

aimulated binding to other sites 20 to 100 bp distant, and binding to inherentiy weak

sites was mengthened if two or more such sites are present on the same DNA

molecule. 7. The stimulation of large T antigen binding to the origin region by ATP requires site

1/2 and is specific for the centrai 10-1 2 bp of site 1/2. A single hexamer mode1 was

proposed to account for the effect of ATP on site 112.

8. We proposed that large T antigen molecules bound to multiple sites on origin DNA

interact with each other to fom a compact protein-DNA complex; and molecules

bound to site A, B, and C may be "handed over", through protein-protein interactions,

to site 1/2, where ATP stimulates the assembly of hexamen.

XII LIST OF FIGURES

Page

Figure 1. Genomic organization of polyomavirus and SV40 ...... 3

Figure 2. The domain structures of SV40 and mouse polyomavirus 1argeTantigens ...... 8 Figure 3. Nucleotide sequences containing yomavirus and SV40 core-oris and large T antigen binding sites ...... 14 Figure 4. Sequence cornparison of the DNA binding domains of polyomavims and SV40 large T antigens ...... 51 Figure 5. Cornparison of structural modifications of polyomavirus core-ori DNA by polyomavirus large T antigen and SV40 core-ori DNA by SV40 large T antigen ...... 58 Figure 6. A mode1 showing steps in the binding of SV40 large T antigen to the central palindrome of core-ori that are enhanced by phosphorylation of Thr-124. and ATP ...... 65 Figure 7. Construction and sequence of plasrnid pHIL-D2-PyLT ...... 98

Figure 8. Specific binding of yeast-derived polyomavirus large T antigen to the intergenic region of polyomavirus DNA ...... 100 Figure 9. Analysis of immunoaffinity-purified large T antigen by Western blotting and silver staining ...... 101 Figure 10. DNasr 1 footprinting reveals protection of large T antigen binding sites by yeast-derived large T antigen ...... 103 Figure II. Yeast-denved large T antigen unwinds duplex DNA Fragment . . . . 104

Figure 12. Effect of pH on DNA-binding activity of polyomavirus large T antigen ...... 115 Figure 13. Stability of DNA-protein complexes subjected to electrophoresis at pH 6.0 or pH 7.6...... 118 Figure 14. ATP stimulates DNA binding at pH 7.8 but not at pH 7 or below . . 120 Fipre 15. Kinetics of dissociation of protein-DNA complexes upon dilution from low to high pH in presence or absence of ATP ...... 123 Figure 16. Binding of large T antigen to DNAs containing combinations of adjacent binding sites ...... 125 Figure 17. Binding of large T antigen to DNAs containing mutated binding sites...... 128 Figure 18. DNase I footprinting of DNAs containing mutated binding sites . . . 131

Figure 19. ATP enhances protection of the central portion of site 112 against DNase 1 digestion ...... 134 Figure 20. Mode1 for cooperative binding of large T antigen to origin DNA . . 137

Figure 21. "Handover" mode1 for assembly of hexamers at replication ongin. . 144

Figure 22. Gel mobility shift of large T antigen-DNA complexes subjected to electrophoresis at pH 6.0 ...... 151 Figure 23. Gel rnobility shift assay is specific for DNA containing binding sites for large T antigen ...... 152 Figure 24. Use of gel mobility shifi assays with crude lysates of cells expressin2 large T antigen ...... 154 Figure 25. Glycerol gradient andysis of large T antigen-DNA complexes formed at pH 6,7, or 7.6 ...... 156 Figure 26. Binding of large T antigen to DNAs containing mutated binding sites ...... 159 Figure 27. Oligomeriwtion and DNA binding of polyomavinis large T

XIV GUIDELINES RECARDING DOCTORAL THESIS

The following excerpt has been included in order to conform to the Faculty of Graduate

Studies and Research thesis reguiations:

:M4!VL'SCRIPTS .4YD .4 LTN0RSH.P

Candidates have the option of incliiding, as part of the ~hesis.the text of one or more papers szibmitteed, or to be subrnitted, for publication, or the clearly-duplicated text of one or more pziblished papers. These texts must be borind together as an inregml part of the thesis.

If this oprion is chosen, connecting trris tltat provide logical bridges between the difjferent pprrpers are mundatory. The thesis must be written NI such a way thar ir is more rhan a rnere collecrion of manuscriprs; in orher words, results of a series of paperr musr be integrareci.

The thesis mzrst conform to ail other requirements of the 'Guidelines for Thesis

Preparotion". The thesis must include: a table of contents, an absnacr in Engiish and

French, an introduction ivhich clearly stutes the rational and objectives of the srudj, a comprehensive review of the literature, and afinal conclusion and summary; Additional material musr be provided (e.g., in appendices) in suflcient detail to ai10 w a clear and precise judgement to be made ofthe importance and originaliy of the research reported in the thesis.

In the case of manciscripts CO-aufhoredby the candidate and others. the candidate is required io make un erplicir sfutemen? in the thesis us tu who confributedtu such work and to what Ment. The supervisor mzîst uttest to the accuracy of rhis sratement ut the doctoral oral defence. Since the task of the examiners is made more diflcult in rhese cases. ir is in the candidate's interest to clearly speci' rhe responsibilities of al2 the authors of the co-acrthored papers.

In accordance with the above guidelines, I hereby state that three manuscripts have been included as the main body of this thesis and appear in chapters 3. 4. and 5. As required. this thesis includes a Table of Contents, an abstract in English an French. a statement of the rationale and objectives of the study, a litenture review. a final conclusion and sumxnary . and a list of references. Regarding multi-authored published material. the specific contributions of CO-authorsare presented below:

Chapter 3 of this thesis is based on the text of a published manuscript entitled:

"Production of active pol yomavirus large T antigen in yeast Pichia pertoris", by Yu-Cai

Peng and Nicholas H. Acheson. This manuscript was published in Vins Research (1997)

49:4 147. David Luckow helped to screen Pichia pastoris transformants. Marie-Claude

Ouimet cultured the hybndoma cells for the production of monoclonal aatibody F5. Al1

XVf other experirnents were designed in consultation with Dr. Acheson and performed by myself. The manuscript was dnenby myself and edited by Dr. Acheson.

Chapter 4 of this thesis is based on the text of a published manuscript entitled:

"Polyornavinis large T antigen binds cooperatively to multiple binding sites in the viral origin of DNA replication", by Yu-Cai Peng and Nicholas H. Acheson. This manuscript was published in Journal of Yirology ( 1998) 72 :73 30-7340. Noelle-AM Sunstrom constnicted the deletion mutant plasmids, and John Bertin constructed the point plasmids. which were used as PCR templates to make DNA fragments containing single or multiple binding sites. Al1 experiments were designed in consultation with Dr.

Acheson and performed by myself. The manuscnpts was written by Dr. Acheson and myself.

Chapter 5 of this thesis is based on the test of a published manuscript entitled:

"Enhanced binding to origin DNA at low pH enables easy detection of polyomavims large T antigen by gel mobility shift assay of unfixed complexes", by Yu-Cai Penp and

Nicholas H. Acheson. This manuscript was published in Joiirnal of Virologicai iblethods

(1999) 78: 13-19. All experiments were designed in consultation with Dr. Acheson and performed by myself. The manuscript was written by myself and edited by Dr. Acheson. CHAPTER 1. INTRODUCTION

1. Overview: Life cycle of polyomavirus and simian virus 40

The papovavinis family comprises two vims subgroups: the polyomaviruses and the papillomaviruses (for review, sec? Tooze, 198 1). The polyomavirus subgroup consists of a dozen members and includes the type species, murine polyomavinis (Gross, 1953), simian virus 40 (SV40) (Sweet and Hillernan, 1960), and two human isolates, JC virus

(Padgett, et al., 1971) and BK vims (Gardner, et al., 1971). The term "polyomavinis" therefore has two meanings: one refers to the polyomaviw subgroup, and the other refers to the . The polyomaviruses display species specificity for their replication, but otherwise share a common architecture, genome organization, and replication cycle. Polyomavinis replicates in mouse cells, whereas SV40 replicates in simian cells and to a lesser extent in human cells (for review. see Cole, 1996). The host range specificity may be related to distinct viral recepton on the host ce11 surface (Basak et al., 1992; Bolen et al., 198 1; Bolen and Consigli, 1979; Breau et al., 1W), and specific interactions between viral and cellular factors (Pipas, 1985; Schneider et al., 1994). The polyomaviruses can bortdize primary ce11 cultures, transfomi established ce11 lines, and induce tumors in animds (for review, see Tooze, 198 1). These characteristics, as well as their ease of growth in cells cultivated in vitro, have made these vinises modei systems for a wide variety of studies in oncolog~and molecular biology, especially for understanding the molecular basis of oncogenesis, ce11 growth regdation,

1 eucaryotic DNA replication, regulation of gene expression, and splicing.

Both SV40 and polyomavirus are small, nonenveloped vimes with icosahedral that contain three virus-encoded proteins, VPl, VP2, and VP3. Viral DNA within the and inside the ce11 is complexed with cellular histones H2A, H2B, H3, and H4 to form a mi ni chromosome"^ stnicturaily indistinguishable from that of the ce11 except for the absence of histone H1 (Griffin, 1982).

SV40 and polyornavirus contain circular double-stranded DNA about

5300 bp in length. The early region of the genome is transcribed and expressed during the first 10-1 5 hours after the virus enters the cells, but it continues to be expressed at later times; the late region is expressed efficiently only after viral DNA replication begins. Diagrams of the organization of polyomavims and SV40 genomes are shown in figure 1. Transcription extends bidirectionally from initiation sites near the replication origin, with early and late rnRNAs being transcribed fiom opposite strands of the viral genome. In between the early and late transcription initiation sites is a non-coding region called the viral regulatory region or intergenic region. This region contains transcriptional promoters, enhancer elements, and the viral origin of DNA replication

(Eckhart, IWO).

Four strains of polyomavinis have been used for a variety of studies. Strain A2

(Soeda et al., 1980) has a genome size of 5291 bp (Fig. 1A). Strains A3 (Friedmann et ai., 1979), AT3 (Skames et al., 1988), and CSP (Jarrett, 1966; Rothwell and Folk, 1983) differ fkom strain A2 by insertions and deletions in the intergenic region, as well as by a number of difierences in protein coding regions (see below). However, the nucleotide

2 Figure 1. Genomic organizatioa of polyomavirus and SV40. The ongin of replication and transcriptional regulatory region is at the top. Early transcription extends counterclockwise and late transcription extends clockwise fkom the origin. The coding regions for viral proteins are boxed (dark thick lines), while untranslated regions are depicted by continuous lines and introns by doaed lines. Nucleotides are indicated on the inner circle. A) Mouse polyornavirus (nnin Al). Reading fnmes from for large T, middle T, and small T antigens as well as late proteins VPl, VP2, and VP3 are shown. 8) SV40. Reading frames for large T and small T antigens, and capsid proteins VP1, VP2, and VP3 are shown. A fourth , called the agno- protein, is also shown.

sequences of these viral strains are greater than 95% identical. Strain CSP (Crawford small-plaque) was noted originally for its capacity to induce readily detected tumor- specific transplantation antigen in hamster cells (Jarrea, 1966).

Early transcription gives rise to viral regulatory proteins, the tumor or T antigens, so called because they can be detected with antisera derived from animals bearing tumors induced by these vinises. In the case of SV40 and other primate polyomaiiruses, two T antigens are made: large T and small T antigens, designated on the basis of size. Recent studies suggest that there is another 17 kD T antigen encoded in the SV40 genome, expressed from a third species of SV40 early mRNA (Zemhn et al., 1993). Murine polyomavirus and closely related vimses encode four T antigens-large T, middle T, small T. and tiny T antigens. The smallest polyomavirus antigen. tiny T antigen, was only recently identified (Riley et al., 1997), and is not shown in Fig. 1. It shares the first 79 amino acids with the other three T antigens and has 6 additional arnino acids in the C- terminus. Al1 the T antigens of each virus share N-terminal sequences and contain different C-terminal regions. The mRNAs encoding them are denved from a common pre-mRNA by alternative splicing (Berk and Shq, 1978; Kamen et al., 1979; Treisman et al., 1981a). Similarly, viral capsid proteins VPl, VP2, and VP3 are produced by alternative splicing fiom a common late pre-mRNA (Flavell et al., 1979; Haegeman and

Fiers. 1978a. 1978b; Treisman and Kamen, 1981).

Although SV40 and polyomavirus are able to infect a wide range of mammaiian ce11 lines. the response to infection can be productive (lytic) or nonproductive. The early phase begins with attachrnent of virus to cells, penetration of the virion, and its migration

4 to the nucleus. The route and mechanism for the entry of virions into the nucleus is not clear. Presumably, it is mediated by the recognition of the nuclear localization signal which resides in VP1 (Brady and Salzman, 1986; Chang et al., 1992; Forstova et al.,

1993; Moreland and Garcea, 1991; Wychowski et al., 1986). The viral DNA is transported into the nucleus and made available for transcription (for review, see

Acheson, 198 1). During the early phase of infection, the virai T antigcns are produccd, and they affect the host ce11 by stimulating the production of enzymes required for cellular DNA replication, thereby preparing the ce11 for replication of vin1 DNA

(Schlegel and Benjamin, 1978; Tjian et al., 1978). These viral early proteins also stimulate resting cells to re-enter the ce11 cycle (for review, see Acheson. 1981). The late stage of infection extends from the onset of viral DNA replication to the end of the infection cycle and involves the replication of viral DNA, expression of the viral late genes encoding the capsid proteins, the assembly of progeny virus particles in the nucleus of the infected cells, the release of virus, and cell death (for review, see Cole. 1996).

Nonproductive infections result when viral DNA replication cannot take place in the infected cell. For viral DNA replication to occur, SV40 or polyomavirus must produce adequate levels of large T antigen, the only needed for replication, and this protein must interact with cellular replication factors and the viral origin of DNA replication. nierefore, mutation of either the viral or large T antigen can lead to a nonproductive infection, whether it is in permissive ce11 or not (for review, see Cole, 1996). In nonproductively infected cells, the infection cycle begins normally.

The viral early mRNAs are produced and the viral T antigens can be detected. The early

5 proteins exert a variety of effects on the host cell. Since the polyomavimes require the host ce11 DNA replication machinery, they replicate only during ; T antigen stimulates cells to move through the ce11 cycle into S phase (for review, see Tooze, 1981).

When expressed in cells that are nonpermissive for viral replication, the viral T antigens also cause cells to acquire the properties of transformed cells: growth in the absence of high concentration of fetal calf serum, rounded morphology, multilayered growth, etc.

Usually, these transformed properties are manifested for only a few days, because the viral genome is lost from the cells. However, if the viral DNA becomes integrated into the host ce11 genome and the integration pemits continued expression of SV40 large T antigen or polyomavirus middle T antigen. then the transformed phenotype will be expressed permanently, and the ce11 is said to have been transformed by the virus. This happens at a frequency of lower than 1/10' cells. In addition to being able to cause of cultured cells, these cm immortaiize primary cells that would normally undergo a limited number of ce11 doublings. Irnmortalization is a function of the large T antigens of SV40 and polyomavirus (for review, see Tooze, 198 1).

II. A most multifunctional protein: large T nntigen

The large T antigens of polyomavirus and SV40 have been studied extensively for more than three decades, as key factors in the initiation of viral DNA replication and in interactions with cellular signaiing rnechanisms. Large T antigens have multiple functions. As the only viral protein required for DNA replication, large T antigen binds

6 to and unwinds viral DNA at the replication origin, then plays important roles in the initiation and progression of viral DNA replication via its multiple enzymatic activities

(for review, see Hassel1 and Brinton, 1996). Binding of large T antigen to the origin also regulates viral gene expression (Cogen, 1978; Dailey and Basilico, 1985; Farmerie and

Folk, 1984; Kem et al., 1986; Rio et al., 1980; Rio and Tjian, 1983), and can block passage of LIA polymerase II molecdes trwersing the viral DN.4 replication origin

(Bertin et al., 1992, 1993; Brabant and Acheson, 1995). In infected cells, large T antigens interact with several ceilular proteins that turn on DNA replication and ce11 cycling, leading to ce11 immortalization or transformation (Manfredi and Prives, 1994).

1. General properties

The large T antigens of al1 polyomaviruses are closely related and contain identical or similar sequences over much of their lengths (for review, see

Fanning, 1992; Fanning and Knippers, 1992; Pipas, 1992). Discrete functional domains located in similar regions of each protein are required to mediate various functions. The sizes and domain structures of polyomavirus and SV40 large T antigens are shown in Fig.

2. Polyomavirus large T antigen contains a region of 154 amino acids following amino acid 82 that has no homology to SV40 large T antigen sequences; the virai DNA encoding this portion of large T antigen is the portion of the polyomavirus genome that also encodes, in a different reading he,the unique portion of rniddle T antigen.

Conveaely, SV40, JC virus, and BK virus large T antigens contain approximately 70 amino acids at their carboxy-termini that have no homolou with sequences in

7 Figure 2. The domain structures of mouse polyomavinis (A) and SV40 (B)large T antigens. Amino acid numbenng is indicated on the top of each protein. Shown on both large T antigens are sequences that serve as nuclear localization signals (NLS) and sequences required for binding ATP, Zn*, viral origin DNA and cellular proteins pRB/p107/p130 (indicated by Rb). Also shown are the J domains which mediate interactions between large T antigen and hsc70, and two amino acid clusters where phosphorylation occun (depicted as "P" in two hatched boxes). Domains on SV40 large

T antigen required for ATPase activity, activity, host-range helper fiction activity (hr-hf). and transactivation of cellular genes, as well as domains required for interactions with cellular proteins DNA polymerase a:primase, topoisornerase 1, , p300. and TEP are also shown. Such domains on polyomavirus large T antigen have not yet been defined or do not exist (hr-hf and p53 binding). The size and location of each domain was determined according to data reviewed in Bullock (1997), Cole (1996),

Manfredi and Prives (1994), and Pipas (1992). polyomavirus large T antigen (for review, see Pipas, 1992). This carboxy-terminal domain of large T antigen is involved in host range and adenovirus helper function

(Heath et al., 1992; Pipas, 1985). Other domain structures will be discussed in the following sections when their functions are concemed.

SV40 large T antigen contains 708 axnino acids, and has a molecular weight of approximatrly 82 kD (before modification) (Tegtmeyer et al., 1975). Polyomavirus large

T antigen contains 782 (strain A3, AT3, and CSP) or 785 (strain A2) arnino acids, with molecular weights of approximately 88 kD (before modification) (Ito et al., 1977b). A2 and A3 large T antigens are identical in sequence with the following exceptions: amino acids Ser370-Val371 of A3 are replaced by Arg in A2; Asp495-Gly-Va1497 of A3 are replaced by Ala-Ser in A2; Asp648-Tyr-Leu-Asp65 1 of A3 are replaced by Th-Thr-Try-

Asn in A2; and Glu780-Tyr-Ser782 of A3 are replaced by Asp-Ile-Ala-Glu-Tyr-Thr-Val-

Tyr in A?. Large T antigen from the CSP strain has the same sequence as that from A3 except that amino acids Ser156, Tyr164, Cys259, Ser705. and Scr754 of A3 are changed to Pro. Pro, Gly, Ala and Ala respectively in CSP. It is estimated that a lytically infected ce11 cm contain up to 1O6 molecules of large T antigen (Cole. 1996).

A) Posttransiutional modif cations

The large T antigens are modified posttranslationaîly in severai ways. Both polyomavirus and SV40 large T antigens contain phosphorylation sites. Polyomavirus large T antigen is phosphorylated at two different regions, one between arnino acid residues 81 and 187 and the other between residues 257 and 285 (Bockus and

9 Schaffhausen, 1987a; Bockus and Schaflhausen, 1987b; Hassauer et al., 1986). SV40 large T antigen also has two clusters of phosphorylation sites, one close to the amino terminus between residues 106 and 124, and another at the carboxy terminus between residues 639 and 702 (Hassauer et al., 1986; Scheidtmann et al., 1982) (Fig. 2).

Polyomavirus large T antigen is phosphorylated on both and residues at a ratio of approximatdy 6 to 1 (Bockus and Schafhausen, 1 %Va). Pho~phorylation appears to be involved in the regulation of some of the biological activities of large T antigens (Chattejee et al., 1997; Mohr et al., 1987; Prives, 1990). In particular, phosphory Mon of ?Ir124 in SV40 large T antigen and Thr278 in polyomavims large T antigen, both located in a highly conserved region, just upstream of a nuclear localization signal and the DNA binding domain, has been shown to be critical for the function of large T antigen in DNA replication (Chatterjee et al., 1997; Li et al., 1997; McVey et al.,

1993, 1996). SV40 large T antjgen cm also be modified by O-glycosylation (at Serl11 and Ser 1 12) (Jarvis and Butel, 1985; Medina et al., 1998; Schmitt and Mann. 1987), acy lation (Klockrnann and Deppert. 1983), adenylation (Bradley et al.. 198J), poly(ADP)-ribosylation (Goldman et al., 198 l), and amino-terminal acetylation (Mellor and Smith. 1978). Little is know about the functions of any of these modifications.

B) Nuclear ~oca~ization

Found predominantly in the nucleus, large T antigen exists free in the nucleoplasm and is also associated with chromatin and the nuclear matrix (Staufenbiel and Deppert, 1983). Nuclear tmnsport of large T antigens is mediated by nuclear

1O localization signals (NU). The nuclear localization signal in SV40 large T antigen is located between residues 126 and 133 (Pro 126-Lys-Lys-Lys-Arg-Lys-Val-Asp)(Fig. 2)

(Kdderon et al., 1984a, 1984b; Lanford and Butel, 1984). Polyomavirus large T antigen contains two nuclear localization signals, one at a position analogous to the site of the

SV40 nuclear localization signal (Pro280-Lys-Lys-Ala-Arg-Glu-Asp),the other in a rrgion without homology to SV30 large T antigen (Vall89-Ser-a-Lys-kg-Pro---

Pro-Ala) (see Fig. 2). Either one of these two signals, in the absence of the other, is suficient to localize large T antigen to the nucleus (Richardson et al., 1986). SV40 large

T antigen with a mutated nuclear localization signal retains its ability to immortalize primary cells, initiate transformation, and transactivate E2F responsive promoters

(Tedesco et al.. 1993; Zhu et al.. 1991b); however. nuclear localization of polyomavirus large T antigen is required for productive association with pRb family members (Howes et al., 1996) and for irnmortdization (Larose et al.. 1991). Phosphorylation at sites adjacent to nuclear localization signals has been shown to modulate transport (Jans et al.,

199 1; Rihs et al., 199 1).

C) Oligornerization

Soluble large T antigens occur in multiple oligomeric forms, which differ in the degree of phosphorylation and in a number of biologicai activities (Dean et al., 1992;

Rder et al., 1987; Stahl and Knippers, 1987). Runzler et ai. (1987) separated three subclasses of large T antigen from SV40 infected monkey cells by zone velocity sedirnentation, which correspond to large T antigen monomers, dhers, and tetramers.

II Native gel electrophoresis displays oligomenc forms fiom dimer to hexarner as well as monomer (Dean et al., 1992; Wang and Prives, 1991a). It was demonstrated that ATP stimulates hexamer formation of poiyomavirus large T antigen in solution (Wang and

Prives, 199la) and SV40 large T antigen both in solution and on DNA (Dean et al.,

1987~;Dean et al., 1992; Mastrangelo et al., 1989). Double hexamer complexes of SV40 large T antigen on the viral origin of replication were observed by electron microscopy in the presence of ATP (Dean et al., 1987~;Mastrangelo et al., 1989). Other factors may also influence the oligomeric state of large T antigen. For exarnple, we showed that polyomavirus large T antigen tends to self-associate at pH 7 and beiow. but not at pH 7.6

(Peng and Achrson, 1998).

2. Functions in viral DNA replication

A) Originî for viral DN.4 replication

Initiation of polyomavirus DNA replication takes place within the origin (ori).

The genetically defined ori for SV40 or polyomavirus DNA replication is located in the noncoding region of the viral genome. Ori was defined by the construction and analysis of detailed sets of deletion and point substitution mutations and by the analysis of evolutionary variants of these viruses that cary deletions of sequences not required for replication (DePamphilis, 1993). Each ori comprises two functional components: a core region, referred to as core-ori (the minimal origin), and auxiliary regions. In vi~o,core- ori is required for replication under ail conditions and encompasses the sites where DNA replication initiates. The auxiliary regions increase the efficiency and ce11 type specificity

12 of viral DNA replication (Hassel1 et al., 1986).

The core-ori of each virus is about 65 bp in length and comprises three sets of sequences: an AIT-nch stretch at the late border; a central WC-rich palindrome; and a region at the early border referred to as the early palindrome in SV40, or the Pu/Py strand bias region in polyomavirus (Fig. 3) (for review, see DeParnphilis, 1993). The AIT-nch repion contins 14-17 base pairs including a string of 8 consecutive A's on one strruid, and is a natural site of DNA untwisting. The central 30-bp palindrome contains four pentanucleotide G(A/G)GGClarge T antigen binding sequences arranged as two inverted pairs. In the polyornavirus origin (Fig. 3A). the adjacent pentanucleotides in the central palindrome are separated by 2 nt, and the inverted pairs overlap by 2 nt. Adjacent pentanucleotides in the central palindrome of the SV40 origin are separated by 1 nt, and the inverted pairs are also separated by 1 nt (Fig. 3B). The G(A/G)GGC pentanucleotides outside the core-ori are oriented in the sarne direction within each large T antigen binding site, and are separated by 5-7 bp (in site III of the SV40 origin. three of the pentanucleotides are TGGGC) (Fig. 3). The arrangement of these pentanucleotide motifs may determine whether interaction of large T antigen with these sequences results in initiation of DNA replication or repression of early transcription (for review, see

DePamphilis, 1993). The PuPy strand bias region (in the polyomavinis ongin) or early palindrome (in the SV40 origin) is a domain where DNA is fmt unwound, and DNA replication initiates nearby this region, as shown by primer mapping experiments (Fig. 3)

(Hay and DePamphilis, 1982; Hendrickson et al., 1987).

There are two classes of auxiliary elements in the replication origins of SV40 and

13 Figure 3. Nucleotide sequences containhg polyomavirus and SV40 core-oris and large T antigen binding sites. Nucleotide nurnbers are indicated at ends of sequence blocks. Large T antigen recognition motifs G(A/G)GGC or T(A/G)GGC are marked by horizontal arrows above or below the sequences. A) Polyomavirus (strain AT3). Four distinct binding sites 112, A, B, and C are shown. The core-ori extends from site A through site 1!2, and inciudes a PulPy strand bias region, a central palindrome. and an

A/T-rich region. B) SV40. Sites 1, II, and III are shown. The core-ori is located around site II, and includes an early palindrome, a central palindrome, and an NT-rich region.

The three 21-bp GIC-rich repeats in site III are indicated. Also show in both viral sequences are the initiation sites for early transcription (early mRNA) as well as the corresponding TATA boxes (boxed and Iabeled). The initiation codon for translation

(ATG) of the early mRNA is shown in the begiming of each sequence (boxed). DNA replication initiation sites are indicated by thickened mows. (Adapted from Bertin et al.,

1993; Borowiec and Hurwitz, 1988; DePanphilis, 1993; and Pomerantz and Hassell,

1984). Early mRNA

TATA 1 Late strand

30 t I I 1 I TCTTTTGACMGTTGCCTCTGGAATGCCTCTCTTCTTTTTCTCCAGAGTM ACAAAACTGTTCAACGGAGACCTTACGGAGAAAAATT A DNA replication Central palindrome 1/5312 A/T-rich region I I I 11 1I GCGGAGGCCAGGGGCCCCCGGCCTCTGCTTAATACTAAAAAAAACAGCTG- - CGCCTCCGGTCCC GAGACGAATTATGATTTTTTTTGTCGAC I I I 1 (Early straod Late strand DNA replication

Central palindrome 5212 Early palindrome NT-rich region 35 5243/1 Il--Il ACTACTTCTOOAATAGCTCAGAGGCCGAGGCGGCCTCCGCCTCTGCATAAATAAAAAAAATTAGTCAII TGATCAAOACCTTATCGAGTCTCCGGCTCCGCCGGAGCCGGA~CGTA~TA~~TTTTMTCAGTu Early ,mRNA --a TATA

36 2 1-bp C/C-rich repeats 103 polyornavirus. One class comprises large T antigen binding sites. In SV40 these sites flank both borders of core-ori (sites I and III), while in polyomavirus this class of auxiliary elements lies on the early side of the polyomavinis core-ori (sites A, B, and C)

(Fig. 3). The aflïnity of large T antigen for these sites in vitro exceeds or equais that for sites within core-ori. Thus, this class of auxiiiary elernents may stimulate DNA replication by recruiting large T antigen to the vicinity of core-ori, facicilitating large T antigen-mediated origin unwinding (Gutierrez et ai., 1990; Peng and Acheson, 1998).

Deletion of site 1 in SV40 or mutation of sites A, B, and C in polyomavirus reduces viral replication, but the mutated viruses are still viable (Bertin et al., 1993; Guo et al., 1991;

Triezenberg and Folk, 1984).

The other category of auxiliary elements are adjacent to the late border of each core-ori. Thrse elements overlap the transcriptional motifs that make up the viral enhancer and promoten (Mueller et ai., 1988; Muller et al., 1988). Allviiiary eiernents of this type augment the replicative potential of the core-ori in vivo to a much greater extent than do those composed of large T antigen binding sites (DeParnphilis. 1988; Guo et al..

1989). Although quite different in sequence, these auviliary sequences (72 bp and 2 1 bp regions in SV40 and a and P regions in polyomavirus) are functionally redundant. The auxiliary domains of polyomavirus can substitute for those of SV40, and the SV40 72-bp enhancer, but not the 21-bp repeats (shown in Fig. 3), can also be used in place of the polyornavirus a and P enhancers (Cole, 1996). Interestingly, many different types of transcriptional elements can replace the polyornavirus auxiliary regions. Notably, binding sites for GAL4, a transcriptional activator fiom budding yeasts, can act as

15 auxiliary elements for polyomavirus DNA replication as long as GAL4 protein is provided in tram (Guo and DePanphilis, 1992). These transcriptional activators may stimulate viral DNA replication by relieving the repressive effects of nucleosomes

(Cheng and Kelly, 1989; Cheng et al., 1992), or by stimulating the binding or other activities of large T antigen at ori (Guo et al., 1989; Gutierrez et al., 1990). Some transcriptionai activators were dso show to intenct directly with replication protein A

(He et al., 1993; Li and Botchan, 1993), and therefore may fùnction by recniiting this protein to ori, helping large T antigen to unwind the DNA strands.

B) Large T antigen and viral DNA replication

Large T antigen binds to multiple sites in the inteqenic region on viral DNA via its DNA binding domain (Borowiec and Hunvitz, 1988b: Cowie and Kamen, 1984;

Dilworth et al., 1984; Myers et al., 198 1b; Ewen et al., 1991). Within core-ori, large T antigen monomers bind to each of the four pentanucleotide repeats (Mastrangelo et al..

1985). Additional large T antigen molecules bind to this complex and are assernbled into hexamers that straddle each side of core-ori ATP stimulates the binding of large T antigen to core-ori and is required for the assembly of hexarnenc large T antigen. A

"double hexamer" model was proposed for the large T antigen bound to the SV40 ongin.

In this model, bound large T antigen undergoes a conformational shift in the presence of

ATP, perrnitting the assembly of a bilobed double hexamer of T antigen at site II

(Borowiec and Hurwitz, 1988b; Dean et al., 1987~;Mastrangelo et al., 1989). The two hexamers are believed to move in opposite directions sirnultaneously after initiation of

16 viral DNA replication, providing a DNA unwinding activity at each replication fork.

Double hexamers have not been shown directly to assemble on polyomavinis DNA.

Large T antigen hexamen are circular structures that enclose the DNA like a wheel about an de,and each hexamer coven about 10 bp of the DNA helix (San Martin et al., 1997; Wessel et al., 1992). Although a single monomer of large T antigen can bind to a G(NG)GGC pentanucleotide, clearly not dl 12 molecules of large T antigen cm bind to a pentanucleotide in the assembled double hexarnedDNA cornplex. In the process of hexamer formation on DNA, ATP binding by large T antigen is necessary, but

ATP hydrolysis is not required, because nonhydrolyzable ATP analogs are efficient in promoting hexamer formation (Borowiec and Hunvitz, 1988b; Dean et al., 1987c;

Lorimer et al., 199 1; Mastrangelo et al., 1989; Reynisdottir et al., 1993; Wang and Prives,

1991a).

Large T antigen hexamers effect structural alterations in the core-ori, including unwinding of the early palindrome or POy strand bias region. and untwisting of the

A/T-rich element (Borowiec and Hunvitz, 1988b; Chen et al.. 1997; Dean and Hunvitz,

1991 ; SenGupta and Borowiec, 1994). Joining of single-stranded DNA-binding

replication protein A (RPA) to the large T antigedori complex facilitates more extensive

bidirectional unwinding of the ori by the intrinsic 3'-5' helicase activity of large T antigen (Dean et al., 1987b; Stahl et al., 1986). Once this initial unwinding has occurred,

large T antipn no longer binds specifically to viral DNA, since the ori region is no

longer double-stranded (Aubom et al., 1988). There are also reports suggesting that

SV40 origin DNA unwinding is accomplished by reeling the DNA through the double

17 hexamer complex rather than by independent progress of each hexamer bidirectionally fiom the center of core-ori (for review, see Weisshart and Fanning, 1996). Helicase activity of large T antigen is crucial for viral DNA replication, and requires functional

ATPase activity and the ability to bind to viral DNA. In vitro, large T antigen displays helicase activity with substrates lacking specific large T antigen binding sites (Wang and

Prives, 1991b). SV40 large T antigen also possesses RNA helicase activity (Scheffner et al., 1989) and can unwind DNA triple-helix or four-stranded DNA structures linked by

G-quartets (Baran et al., 1997; Kopel et al., 1996), but the importance of these activities is unknown. SV40 large T antigen hexamers are responsible for unwinding double-stranded

DNA fiom an intemal site (for review, see Borowiec, 1996). The association of the viral core-ori. the large T antigen hexamer, and RPA is referred to as the preinitiation complex. Hence, during the initiation of viral DNA replication, large T antigen plays a number of distinct roles: it alters the structure of ori DNA, unwinds DNA ahead of the replication fork. and nucleates the assembly of the preinitiation complex at core-ori.

Recruitment of DNA polymerase a:primase into the preinitiation complex converts it to an initiation complex. Following initiation, DNA synthesis moves bidirectionally away fiom the ori (Li and Kelly, 1985). Replication fork movement is facilitated by the helicase activity of large T antigen, which translocates dong the DNA.

The cellular replication factor C (WC)binds the 3' end of nascent DNA strands and facilitates the binding of proliferating ce11 nuclear antigen (PCNA) and DNA polymerase

6 (Lee et al., 1991; Tsurimoto and Stillman, 199 1). This complex extends both the continuous leadhg strand and the Okazaki fragments on the lagging strand. The actions

18 of RNase H and DNA ligase I are required for removal of primers and ligation of Okazaki fragments (Waga et al., 1994). Also required are topoisornerases 1 and II, both of which unwind the positive supercoils that accumulate ahead of the replication forks (Yang et al.,

1987), whereas topoisornerase II is needed for the decatenation (separation) of the progeny DNA molecules after replication is completed (Yang et al., 1987). There is no specific termination site for virai DXA replication; termination cm occur within any sequence located approximately 180' fiom the origin (Cole, 1996).

C) Regdation of viral DNA replicarion by phosphorylation of large T antigen

The activity of large T antigens is regulated both positively and negatively by phosphorylation (for review, see Fanning, 1992; Wang et al., 1993). For SV40 large T antigen. phosphorylation of threonine 124 positively influences the activity of large T antigen by increasing its ability to assemble into double hexamers in the core ori and bidirectionally unwind DNA (McVey et al., 1993. 1996). However. the helicase activity of large T antigen is unaffiected by this modification (McVey et al., 1993). The cyclin- dependent kinase, cdc2. in association with cyclin B, phosphorylates TM24 in vitro

(McVey et al., 1993). SV40 Large T antigen expressed in bacteria is not modified at

Th124 and is inactive for in vitro replication unless preincubated with cdc2 (McVey et al., 1989a). Interestingly, the S-phase cyclin-dependent kinase, cyclin Ncdk2, also phosphorylates large T antigen at Thr124, suggesting that this kinase may be a physiologically relevant kinase that regulates large T antigen activity (Adamczewski et al., 1993). On the other hand, enymatic dephosphorylation of some serine residues

19 (Ser 120, Ser123, Ser677, and Ser679) in purified large T antigen by alkaline phosphatase or the catalytic subunit of phosphatase 2A enhanced specific binding to SV40 DNA and initiation of viral DNA replication in vitro, implying that phosphorylation of these residues dom-regulates SV40 large T antigen activities (Fanning, 1992; Scheidtmann et al., 1991 ). Although phosphory lation of Ser 120 and Ser 123 negatively regulates the replicatise capacity of large T antigen in vitro (Cegielska et al., 1994), mutation of these residues to alanine resulted in an inactive large T antigen in vivo (Schneider and Fanning,

1988). These findings imply that phosphorylation-dephosphorylation of the these sites plays a regulatory role during the virus life cycle.

A cornparison of the phosphorylation sites on polyomavinis and SV40 large T antigens reveals both similarities and differences. Both large T antigens contain two distinct clusters of phosphorylation sites, and in each case, one of them is located within the vicinity of a nuclear localization signal (NLS)and the DNA-binding domain (Fig. 2), regions that are highly conserved between the two large T antigens. The second cluster of phosphorylation sites within polyomavirus large T antigen is located Merupstream within the amino-terminal region of the polypeptide, while in SV40 large T antigen, the second phosphorylated region lies near its C-terminus. In addition, several Ser-Gln-Ser sequences are found in both the C-terminal phophorylation region of SV40 large T antigen and between amino acids 257 to 285 of polyomavinis large T antigen. In vitro studies showed that limited treatment of polyomavirus large T antigen with calf intestinal alkaline phosphatase (0)stimulated the ability of the viral protein to rnediate replication of constructs containing the viral replication origin in vi~o,while higher

20 concentrations of CIAP caused a rnarked diminution of this replication fùnction (Wang et al., 1993), suggesting that polyomavirus large T antigen is regdated by both repressing and activating phosphates. Further studies revealed that Thr278, which is the homolog of

Thri24 in SV40 large T antigen, is an important site on polyomavirus large T antigen for phosphorylation by cyclin-dependent kinases. Loss of this site leads to various defects of large T antigen in mediating ori DNA replication both in vivo and in vitro (Li et al., 1997;

Chatterjee et al., 1997).

D) Replicalion fac fors in feracting with large T antigen

Since the development of an in virro system capable of faithfully replicating SV40 and polyomavirus DNA (Li and Kelly, 1984! 1985; Murakami et al.. 1986a). numerous cellular replication proteins have bren identified. To date, a minimum of ten cellular proteins are known to be required together with large T antigen and ori-bearing plasmids to replicate SV40 and polyomavirus DNA in vitro (for review, see Hassel1 and Bnnton,

1996). Some of these replication factors interact directly with large T antigen. stabilizing the DNA replication complex and defining to a certain degree host specificity.

i) Replication protein A

Replication protein A (MA) was originally identified as an essential single- stranded DNA binding protein required for SV40 DNA replication in vitro (Wobbe et al.,

1987; Wold and Kelly, 1988). It functions in concert with large T antigen and DNA polymerase a:primase to initiate DNA replication at ori. RPA binds to large T antigen in

21 a partially species-specific manner. For example, SV40 large T antigen binds to vertebrate sources of RPA, but not to yeast RPA (Brown et al., 1993; Dornreiter et al.,

1992; Melendy and Stillman, 1993). This possibly accounts for the fact that al1 mammalian sources of RPA tested functionally substitute for human RPA in SV40 DNA replication, albeit to differing extents (Hassel1 and Brinton, 1996). RPA also binds in vitro to the activation domains of some transcription activators, includinp that of GAL4,

VP16, and p53 (He et al., 1993; Li and Botchan, 1993). The physiological relevance of these finding is suggested by the observation that transcription activators are required for efficient replication of' SV40 and polyomavirus DNA in vivo (Guo and DePanphilis,

1992). Binding of transcription activators to the ori auxiliary elements may recruit ancilor stabilize the interaction of RPA with the preinitiation complex in the core-ori.

Altematively, the function of RPA at this site may be stimulated by direct contact with transcription activaton. RPA also interacts physically with DNA polymerase a:primase

(Domreiter et al., 1992).

ii) DiVA polymerase mprimase

DNA polymerase a:primase is the only DNA polymerase in mammalian cells capable of initiating DNA synthesis de novo through the synthesis of RNA primers

(Wang, 1991). Human DNA polymerase a:primase is composed of four subunits of molecular mass 180, 70, 58, and 48 kD; the subunits of the mouse protein are simila. in size (180, 68, 54, and 46 kD) (Wang, 1991). DNA polymerase activity is intrinsic to the 180 kD subunit, whereas primase activity resides in the 48 kD subunit. The 70 and 58 kD

subunits associate with 180 and 48 kD subunits respectively and seem to stabilize their

activities (Copeland and Wang, 1993).

The replication of SV40 and polyomavirus is highly species-specific. SV40 DNA

replication takes place in monkey or human cells, but not in mouse cells. In contrast,

mouse cells, but not primate cells, support the replication of polyornavinis DNX (Bennett

et al., 1989). By using cell-free replication systems, it was demonstrated that addition of

purified mouse DNA polymerase a:primase to human ce11 extracts renders them

permissive for polyomavirus DNA replication, and correspondingly, addition of the

hurnan enzyme to mouse ce11 extracts allows the mouse extracts to support SV40 DNA

replication (Murakami et al.. 1986). These experiments suggest that DNA polymerase

a:prirnasc is the primary host determinant for species-specific viral DNA replication.

Further fractionaiion revealed that the pnmase subunits (a heterodimer of the 54 kD and

46 kD subunits) from mouse DNA polymerase a:primase enabled hurnan extracts to

replicate polyomavinis DNA (Eki et al., 1991). and extension of these experiments

showed that the mouse 46 kD primase subunit is sufficient to rnediate species-specific replication of polyomavirus DNA in vitro in human ce11 extracts (Bruckner et al., 1995).

Surprisingly, similar experiments with reconstituted DNA polymerase a:primase enzymes of mixed human-mouse origin showed that SV40 DNA repiication in vifro required the human 180 kD subunit (Stadlbauer et ai.. 1996). This observation may reflect differences in the fûnctionai interaction between the DNA polpeme and the

large T antigen of each vins. Recentiy, it was reported that polyomavinis large T antigen

23 was able to support replication of polyornavirus origin-containing plasmids in human ce11 lines (Sverhp et al., 1998), indicating that under certain conditions, polyornavirus large

T antigen must functionally interact with human DNA polymerase a:primase to permit

DNA replication.

In vitro studies also showed that the large T antigens of both SV40 and polyornavirus physically interact with several of the subunits of DN,4 polymerase a:primase (Bruckner et al., 1995; Domreiter et al., 1990; Moses and Prives, 1994;

Schneider et al., 1994). In SV40 large T antigen, two discontinuous regions are required to bind to the human DNA polymerase (Fig. 2), but it is not known whether each region binds to a different subunit (Dornreiter et al., 1990). The binding of SV40 large T antigen to human DNA polymerase a:pnmase increases both the pnmase and DNA polymerase activity of the holoenzyme (Collins and Kelly, 1991). Large T antigen is thought to bridge the interaction between DNA polymerase u:primase and the DNA tempiate. thereby facilitaiing DNA replication (Collins et al., 1993). On the other hand,

DNA polymerase a:primase stimulates the ATP-dependent binding of SV40 large T antigen to the SV40 origin of replication (Murakami and Hurwitz. 1993b). As mentioned above, DNA polymerase a:primase also binds to RPA. and the association appears to be species-specific (Dornreiter et al., 1992). Thus, multiple and species-specific protein- protein interactions occur among the three major protein components (large T antigen,

RPA, and DNA polymerase a:primase) of the initiation complex, resulting in the stimulation of those enzymatic activities required for the initiation of DNA replication at ori. iii) Topoisornerase I

In 1993, it was reported that purified SV40 large T antigen prepared from different sources is able to relax negatively supercoiled DNA molecules (Mann, 1993;

Marton et al., 1993). The large T antigen associated reiaxing activity was stimulated by

Mg", but not by ATP, suggesting the involvement of an enzymatic activity similar to that of topoisomerase 1. However, this activity appears to be different hm cellular topoisorneme 1 in that it responds differently to salt concentrations and pH requirements

(Marton et al.. 1993). Antibodies reacting with large T antigen inhibited relaxation by preparations of large T antigen but not topoisomerase 1, whereas antibodies inhibiting relaxation by topoisomerase 1 had no effect on relaxation by large T antigen (Marton et al.. 1993). On the other hand, immunoprecipitation of large T antigen preparations by using antibodies against human topoisornerase 1 and iI failed to detect those proteins

(Mann. 1993). in another experiment, Large T antigen-associated topoisomerase activity

CO-sedimentedwith the hexameric fom of large T antigen (Mann, 1993). Therefore, the topoisomerase activity is either inherent to large T antigen or due to a cellular topoisomerase 1 tightly bound to large T antigen. Similar results were obtained with polyomavinis large T antigen (Marton et al., 1995).

Subsequent studies demonstrated that binding of SV40 large T antigen to human and calf thymus topoisomerase I was readily detected by using enzyme-linked

immunosorbent assays and immunoblots, and the interaction between large T antigen and

topoi~merase1 is much stronger than those between large T antigen and DNA

polymerase a:primase and RPA. In addition, binding sites for topoisomerase 1 and DNA

25 polymerase a:primase overlap, whereas the binding domain for RPA differs (Simrnons et al., 1996). Binding to large T antigen has been located to the N-terminus of human topoisornerase 1 (Haluska, Jr. et al., 1998). One obvious possibility is that the association of large T antigen with topoisomerase I prornotes the proper unwinding and relaxation of the DNA at replication forks. In fact, recent studies have shown that topoisomerase 1 stimulates SV40 large T ûntigen-mediated DNA replication and inhibits the ability of large T antigen to unwind DNA at nonorigin sites (Simmons et al., 1998b). On the other hand, the activity of topoisomerase 1 is also modulated by large T antigen during unwinding of the SV40 origin. Large T antigen activates topoisornerase I nicking at discrete sites and releases these nicked strands during unwinding (Simrnons et al., 1998a).

Some evidence indicates that a single molecule of topoisomerase I cm form a fùnctional complex with a double hexamer of large T antigen to simultaneously relax and unwind double-stranded origin-containing DNA (Simmons et al., 199th).

E) Zinc-finger and Dnddomains on large T antigen i) Zhc-finger domain

Located between the DNA binding dornain md the ATP binding1ATPase domain in SV40 large T antigen (between amino acid residues 302 and 320) is a zinc fmger structure (Fig. 2B) (Berg, 1986; Loeber et al., 1989). Mutation of the conserved amino acid residues in this structure makes the vhs inviable (Loeber et ai., 1989); therefore it is essential for viral DNA replication. Further studies showed that mutations in the dc finger domain greatiy decreased large T antigen binding to site II, while binding to site 1

26 was not significantly af5ected (Hoss et al., 1990). Mutant large T antigen bound to the central pentanucleotide domain of the core origin but failed to melt the adjacent early palindrome and to untwist the A/T-nch domain (Loeber et al., 1991). It was proposed that the zinc finger region contributes to protein-protein interactiors which are essential for the assembly of stable large T antigen hexamers at the origin of replication (Loeber et al., 199 1).

Polyomavirus large T antigen contains a zinc finger structure between residues

452 and 472. which is homologous in both location and sequence to the one fond in

SV40 large T antigen (Fig. 2). Site-directed mutagenesis showed that point mutations of the conserved residues blocked the ability of large T antigen to function in viral DNA replication. Mutant large T antigen retained the ability to bind origin DNA but showed a defect in formation of (Rose and Schaffhausen, 1995).

ii) DnaJ domain

Dnd proteins function as cofactors by regulating the activity of DnaK, a -70-kD

ATPase whose synthesis is induced under heat or cellular stress conditions. DnaK is able to bind and release polypeptides concomitant with ATP binding and hydrolysis. The

ATPase activity of DnaK is stimulated by Dnd. The .i domain of DnaJ chaperones, which is required for DnaJ function, is proposed to mediate complex formation with hsp70/DnaK family mernbers. Hsp70 and DnaJ function together in a complex to carry out a variety of biochemical activities including nascent , protein translocation across membranes, regulation of protein conformation and multi-protein

27 cornplex formation (for review, see Georgopoulos et al., 1994; Hartl, 1996).

Genetic analyses demonstrated that amino terminal sequences of large T antigens comprise a novel J domain (Cheetham et al., 1992; Kelley and Landry, 1994). The J domain sequence is conserved among the T antigens of dl sequenced polyomavinises

(Pipas, 1992) (Fig. 2). Recently, a nurnber of reports have firmly established that this J domûin shms functional homology with the J domain of Dnd rno1ecuIar chaperones.

Fint. an amino-terminal fragment of SV40 large T antigen that includes the J domain mediates binding of large T antigen to hsc70 (the mamrnalian counterpart of hsp70)

(Campbell et al., 1997) and stimulates the ATPase activity of both hsc70 and a cytosolic yeast hsc70, Ssalp (Srhivasan et al., 1997). Full length large and small T antigens also stimulate Ssalp ATPase activity, and this stimulation is partidly blocked by antibodies directed to the amino terminus of T antigen and by mutations within the J domain. The polyomavirus tiny T antigen, containing a J domain, also stimulates hsc70 ATPase activity (Riley et al., 1997). Second, Kelley and Grorgopoulos (1997) have shown that the J domain of SV40 large T antigen inserted into the corresponding domain of

Escherichia diDnaJ sustains bacterial growth and sensitivity to infection by bacteriophage Ah. Finaily, the J domain fiom the human HSJ-1 protein supports SV40

DNA replication when inserted into the corresponding region of large T antigen

(Campbell et al., 1997).

Successful infection by polyomavimes requires the ordered assembly and remangement of several different multiprotein complexes involved in DNA replication. gene expression, and virion assembly. T antigens with mutations in the J domain are

28 frequently defective for DNA replication (Peden and Pipas, 1992) and transformation

(Sheng et al., 1997; Stubdal et al., 1997; Zalvide et al., 1998). Aithough the mechanisrn(s) used by the T antigens to effect these diverse processes has been obscure, the data mentioned above and reports fiom a nurnber of laboratones indicate that a class of proteins known as molecular chaperones rnay coordinate many aspects of polyornavims infection. There is a suiking parallel between the replication of polyomaviruses and bacteriophage h replication, which requires viral proteins O and P to recruit host cellular proteins DnaJ and DnaK to a multiprotein complex at the origin of replication. By hydrolyzing ATP, DnaJ and DnaK free the DnaB helicase from its stable association with the preinitiation complex and alter the conformation of one or more rnernbers of the complex (for review, see Georgopoulos et al., 1994). Large T antigen has evolved a more efficient strategy of DNA replication through an intrinsic J dornain to associate directly with a pmer protein, and this mechanism rnay be used for viral functions in addition to DNA replication, Le., transcriptional control and virion

assernbly (for review, see Brodsky and Pipas. 1998). In a mode1 proposed by Brodsky

and Pipas (1998) for T antigen chaperone action on a muitiprotein complex. T antigen

recruits a target protein or into a temary complex consisting of the

target. T antigen, and hsc70. A conformational change on one or more components of the

target complex is then effected by using energy derived from ATP hydroiysis by hsc7O.

Functions in regulation of transcription

Regulation of viral gene expression

29 The early and late transcription units on the circular polyomavirus DNA genome are transcribed by RNA polymerase II in divergent directions fiom the regulatory region, where both transcriptional promoters and enhancers are located. The early promoters of polyomaviruses are autoregulated by their large T antigens. In cells infected with mutants affecting large T antigen, early mRNAs are overproduced and the rate of synthesis of large T antigen is elrvated in parallel (Khoury and May, 1977; Rêed et al.,

1976; Cogen, 1978; Fenton and Basilico, 1982). In vitro studies showed that regulation of SV40 early transcription is dependent on large T antigen binding sites (Hansen et al.,

1981 ; Rio et al., 1980; Rio and Tj ian, 1983). indicating that large T antigen autoregulates synthesis of its own mRNA. The SV40 binding sites 1 and II (Fig. 3B) are located dowstrearn of. or overlap, the early rnRNA start sites used shortly after infection (Rio and Tjian. 1983). SV40 large T antigen binding to viral DNA in vitro blocks the assembly of a functional transcription complex. thereby repressing early transcription

(Myers et al.. 198 1a; Wildeman, 1988). Binding of polyomavirus large T antigen to the major binding sites located within the early promoter region (sites B and C) results in subsequent repression of early gene expression (Dailey and Basilico, 1985; Farmene and

Folk. 1984). Other mechanism of autoregulation may involve direct repression by large

T antigen of enhancer-binding transcription factors to reptess enhancer functions

(Mitchell et al., 1987).

The onset of viral DNA replication brings about a shift in the pattern of viral transcription. With the downregulation of early transcription by large T antigen there is a dramatic increase in transcription to produce the viral late mRNAs. Two primary

30 mechanisms are involved in the production of high levels of the viral late mRNAs. One

is the amplification of templates for late transcription by viral DNA replication (for

review, see Tooze, 198 1). Inducing replication of the viral genome to a high template copy nurnber by large Y antigen also enables the titration of cellular repressors of the late promoter (Wiley et al., 1993). The other is transactivation of transcription fiom the viral

latr promoter by large T antigen (Brady and Iihoury, 1985; Kem et ai., 1986). Studies with origin-defective viral DNA molecules indicate that genome amplification is not required for activation of the late promoter by large T antigen (Brady et al., 1984; Keller and Alwine, 1984). Mutations that prevent large T antigen from entenng the nucleus or that disrupt the origin result in activation of the late promoter (Wildeman, 1989).

Transactivation of the late prornoter by large T antigen is independent of binding of large

T antigen to a specific DNA sequence (Brady et al., 1984). but requires sequences within the 71-bp enhancer region, including a unique sequence element located at the junction of the two 72-bp repeats (Gmda and Alwine, 199 1). One factor able to bind to these

sequences is the cellular transcription enhancing factor 1 (TEF-l), which is able to form a

complex with large T antigen (Casaz et al., 199 1; Gmda et al., 1993). However, large T antigen can also transactivate late transcription through other pathways independent of

TEF-1 (Casaz et al.. 1995). The region of large T antigen involved in activation of the

late promoter maps to the amino-terminal half of the protein (Zhu et al., 1991b). On the other hand, stimulation of late transcription by polyomavirus large T antigen seems to be

more dependent on viral DNA replication (Cahill et al., 1990; Liu and Cdchael, 1993).

Sequence elements near the 5' end of polyomavirus late mRNAs were shown to be

3 1 required for proper processing, transport, or stability of late mRNAs, because RNAs lacking critical sequences near the 5' end did not accumulate as mRNAs (Lanoix et ai.,

199 1). It was concluded that the control of late mRNA production depends in part on the choice of transcription initiation sites at the late promoter (Lanoix et al., 1991).

Bj inreracrions benveen large T antigen and the rranscription machinery

Large T antigen is a promiscuous transactivator. It can activate transcription fiom a variety of different promoters other than the viral late prornoter, including ones with either strong or weak TATA box sequences and a variety of upstream activator binding sites (Gilinger and Alwine, 1993; Rice and Cole, 1993). Large T antigen activates transcription through interactions with a nurnber of transcription factors. Besides the above mentioned TEF-1, SV40 large T antigen also interacts with the TATA binding protein (TBP) of transcription factor TFIID, the general transcription factor TFIIB, the activator protein Spl, enhancer binding protein AP-2. and the 140 kD subunit of RNA polymerase II. The first identified transcription activator whose activity is inhibited by the presence of SV40 large T antigen was AP-2. Direct protein-protein interaction between large T antigen and AP-2 blocks sequence-specific DNA binding by AP-2

(Mitchell et ai., 1987). TBP was reported to bind several hgments of large T antigen containing amino acid residues in the N-terminus (Berger et al., 1996; Damania and

Alwine, 1996; Gruda et al., 1993; Johnston et al., 1996). The residues responsible for

TE3P binding within these fiagrnents have not been localized in detail, but substitution of amino acid residues 173 and 174 in a fragment spanning residues 133 to 249 inactivated

32 binding to TBP (Fig. 2) (Johnston et al., 1996). The same fragment (including amino acids from 133 to 249) also binds TFIIB, Spl, and the 140 kD subunit of RNA

polymerase in addition to TBP, but it cannot bind concunently to more than one of these transcription factors (Johnston et al., 1996), indicating that SV40 large T antigen probably does not transactivate via concurrent interaction with multiple components of

the preinitiation complex. Transactivation by large T antigen may primarily occur by

rernoving or preventing the binding of factors that inhibit the formation of preinitiation complexes (Johnston et al., 1996). In line with this hypothesis is the observation that the

N-terminal region of large T antigen binds to the DNA binding domain (TEA) of TEF-1,

which activates transcription in vitro from the SV40 late promoter (Berger et al., 1996).

In this case, TEF-1 appears to function as a repressor of late transcription.

Another set of evidence indicating that large T antigen interacts with the transcription complex came fiom nuclear run-on experiments with polyomavirus. In

polyomavirus. the late polyadenylation signal. about 2 kb downstream of the late

transcription start sites, is not eficiently recognized, and many RNA polyomerases

continue transcription around the circula DNA without termination. As a result, some

RNA polymerases read through the origin and extend transcription for additional rounds

through the late region, generating large transcripts that contain tandem repeats of the

sequence of the entire virai genome (Acheson, 1978, 1984). Under some conditions,

RNA polymerases can traverse the cucular viral genome up to 12 times before

terminating (Hyde-DeRuyscher and Carmichael, 1990). One consequence of the lack of

efficient termination of transcription on the late DNA strand is that a significant nurnber

3 3 of RNA polymerases read through the replication origin. To examine readthmugh transcription, viral transcription complexes were isolated from polyomavirus-infected cells during the late phase of productive infection. RNAs were labeled in vitro by incorporation of "P-labeled ribonucleoside triphosphates, and analyzed by hybndization to unlabeled, single-stranded DNA fragments (Skarnes et al., 1988). It was discovered that there is a substantial excess accumulation of RNA polymerasrs in viral transcription complexes on the Iate DNA strand just upstream of the core ongin, indicating bat RNA polymerases are "stalled" there in vivo (Bertin et al.. 1992; Skarnes et al., 1988).

Subsequent studies showed that stalling by RNA polymerase II in the polyomavirus intergenic region is dependent on the presence of functional large T antigen and on the integrity of binding site A, but not sites B or C (Bertin et al., 1992, 1993). RNA footprint analysis fùrther showed that the stalled RNA polymerases corne in close contact with large T antigen bound to site A (Brabant and Acheson. 1995). Therefore, large T antigen bound to site A, just upstream of the DNA replication origin, blocks the elongation of

RNA polymerase II through this region. Whether there is direct interaction between large

T antigen and transcribing RNA polymerase II is not known.

4. Functions in immortalizatioa and transformation of infected cells

The transfoming potential of DNA turnor vinises denves mainly fiom the ability of their encoded products to interact with cellular proteins. Early studies indicated that genetic information in the viral early region was necessary and sufficient for transformation (for review, see Tooze, 1981). It is now known that large T antigens

34 play role(s) in both the initiation and maintenance of the transformed state.

A) Transforming activity of T antigens

Polyomavirus large T antigen is required for transformation (Eckhart, 1969), but subsequent studies indicated that large T antigen expression is needed only for initiation but not for the maintenance of the tmsfomied state. The principal transforming grne

(oncogene) product of polyomavirus is middle T antigen; its presence is both necessary and sufficient to transform established cells in culture (Benjamin, 1970: Eckhart, 1977;

Lania et al., 1980; Rassoulzadegan et al., 1982; Treisman et al., 1981b). Middle T antigen is located primarily at the plasma membrane (Ito et al., 1977a; Segawa and Ito,

1982). where it associates with several cellular proteins. These include c-src and related src-family kinase members (Courtneidge and Smith. 1983). This association leads to the activation of the tyrosine kinase activity of these proteins. Also complexed with middle

T antipn and cmare the phosphoinositide-3 kinase (Auger et al., 1992) and a src homology 2 (SH3)-containing protooncogene product, shc. Both these proteins play roles upstream of ras in the signal transduction pathway (Campbell et al., 1994; Dilworth et al.,

1994). In addition, the same two subunits of protein phosphatase 2A that associate with the small T antigens were found to interact with middle T antigen (Pallas et al., 1990;

Walter et al., 1990). Mutants of polyomavinis unable to produce middle T antigen are defective for replication, persistence, tmnsforrnation, and tumor induction in mice

(Freund et al., 1992b). However, these mutants are able to replicate in some ce11 types grown in vitro (Benjamin and Goldman, 1975). In transformation, middle T antigen

3 5 resembles an activated growth factor receptor (Kaplan et al., 1989). The additional presence of large T antigen is required for transformation (immortalization) of pnmary rat embryo fibroblasts and induction of tumors in newbom rnice (Asselin et al., 1984;

Rassoulzadegan et al., 1983). Polyomavhs large T antigen can induce cellular DNA replication in the absence of other virus transfoning genes (Gjorup et al., 1994; Schlegel and Benjamin, 1978).

Much of the ability of polyomavirus large T antigen to stimulate ce11 cycle progression is co~ectedto its association with memben of the susceptibility gene (Rb) family. Large T antigen imrnortalizes pnmary cells in a manner dependent on its binding site for pRb, p107, and pl 30 (Fig. 2) (Freund et al., 1993a;

Larose et al., 1991). Large T antigen also can block ce11 cycle withdrawal and prevent differentiation of myoblasts, again dependent on Rb family binding (Maione et al., 1994).

Recently, it was found that the DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function (Sheng et al.. 1997). In a mode1 proposed for the role of the DnaJ domain in large T antigen interactions with Rb family members. large T antigen encounters an pRb-E2F cornpiex and binds to pRb. DnaK is recruited to the complex by the large T antigen J domain, and acts on pRb to release active E2F via hydrolysis of ATP (Sheng et al., 1997). In addition to complexes of large

T antigen and pRb, specific complexes of large T antigen and CUTLI were also detected in uterine leiomyomas and rnammary tumors in transgenic mice expressing polyomavirus

Large T antigen (Webster et al., 1998), suggesting that large T antigen may be involved in inducing these tumors by sequestering both CUTL 1 and pRb proteins.

36 SV40 large T antigen can, by itself, transform and imrnortalize primary and established cells in culture and induce tumors in rodents (Manfredi and Prives, 1994).

Regions of large T antigen involved in transformation are species- and cell-specific. For example, mutational analyses demonstrated that only the amino-terminal portion of large

T antigen (amino acids 1-12 1) is required to transform mouse C3H 10T1/2 cells, while sequences êxtcnding over much of large T antigen are required to transform REF-52 rat fibroblasts and many other established rodent ce11 lines (Srinivasan et al., 1989).

Mutational analyses also showed that the replication and transformation hctions of large T antigen can be genetically sepmted (Manos and Gluzman. 1984; Gluzman and

Ahrens, 1982). Transformation does not require any of the activities involved in viral

DN4 replication (such as DNA binding, ATP binding, ATPase activity, and helicase activity) and host rangehelper funftion. Large T antigen vansforms ceils by inactivating tumor suppressor proteins, such as p53, pRb/p107/p130, and pNO/CBP. Domains on

SV40 large T antigen have been identified responsible for binding to these proteins (Fig.

2). For example, pRb and pl07 bind to amino acids 99-1 18 (Moran. 1988), and p53 binds to regions spanning residues 351-450 and residues 533-620 (Kierstead and

Tevethia, 1993; Manfiedi and Prives, 1990; Zhu et al., 1991a). Sirnilady to polyornavirus large T antigen, the Dnal domain on SV40 large T antigen is also required to inactivate the pRb family proteins (Brodsky and Pipas, 1998; Zaivide et al., 1998). On the other hand, analyses of the domains of SV40 large T antigen required for immortalizing primary rodent cells gave conflicting results. Some studies demonstrated that the ability to bind p53 is required for immortalization of pnmary moue cells (Zhu et

37 al., 1991 a); othen indicated that the amino-terminal 147 amino acids cm immortalize

primary mouse or rat cells (Sompayrac and Dama, 1991). Deletion of the nuclear

localization signal showed that it is important for efficient immortalization of primary

cells (Tedesco et al., 1993).

Under many conditions, SV40 small T antigen is also required for transformation

(Slcigh et al., 1978), but sufficiently high lrvels of expression of large T antigen (Bikel et

ai.. 1987), the growth conditions of the infected cells (Seif and Martin, 1979), or cellular

genetic background may overcome this requirement. The small T antigens in both

poiyomavirus and SV40 are -rich proteins whose arnino-terminal 79-82 arnino acids are shared with large T antigen. Studies with mutants of SV40 and polyomavirus

that contain deletions within the region of small T antigen not shared with large T antigen

indicate that small T antigen is dispensable for the lytic cycle of these viruses in cuitured cells (Shenk et al., 1976; Staneloni et al., 1977). The small T antigens associate with two cellular proteins of 36 kD and 63 kD, the regdatory and catalytic subunits of protein phosphatase 2A (PP2A) (Pallas et al., 1990). This interaction is thought to inactivate the

mitogen-activated protein kinases ERKl and MEK1, resulting in growth stimulation

(Sontag et al., 1993).

B) Cellular targets for large T antigen implicated in its transforrning activiîy

i) pj3

p53 is a nuclear phosphoprotein which has been shown to bhd DNA in a sequence specific manner and activate transcription (Bates and Vousden, 1996; Hansen

38 and Oren, 1997). Studies have found that rearrangements, deletions, and missense mutations of the p53 gene are the most common genetic alterations in human cancer

(Hollstein et al., 1991 ; Levine et al., 1991). Wild type p53 is localized in the nucleus and is highly unstable in cells, with a half-life on the order of 20 min (Levine et al., 1991).

SV40 large T antigen, as well as adenovirus El B and human papilloma virus E6 proteins, bavc been sho~mto bind to p53 (Lme and Cnwf'ord, 1979; Levine et al., 1991). In the presence of SV40 large T antigen, p53 is stabilized such that its half-life becomes on the order of several hours (Reihsaus et al., 1990). The absence of expression of wild type p53 in a variety of human tumors suggests that expression of wild type p53 is incompatible with the growth of oncogenically transformed cells (Hollstein et al., 199 1).

However. transformed cells expressing SV40 large T antigen cm readily tolerate hi& levels of expression of exogenous wild type p53 (Mercer et al.. 1990). Interaction with

SV40 large T antigen inhibits the ability of p53 to bind to DNA. and therefore inhibits its ability to activate promoten (Farmer et al., 1992). Polyomavirus large T antigen does not interact with p53 directly, but it was reported that polyomavirus large T antigen can overcome p53-dependent growth arrest (Doherty and Freund. 1997).

io pRbip107/p130

Deletion or gross rearrangements of the retinoblastoma susceptibility (Rb) gene have been found associated with certain cancers and there seerns to be selection against expression of pRb protein in these tumors (Weinberg, 1991). Alteration of pRb is restricted to a certain subset of tumor types, and is not as wide-spread as that seen with

39 p53 (Weinberg, 1991). A number of viral oncoproteins, including polyomaviw and

SV40 large T antigens, adenovirus ElA, and human papillomavims E7 (DeCapno et al.,

1988; Ludlow and Skuse, 1995; Dyson et al., 1990), have been shown to form a stable complex with pRb via a consensus amino acid sequence Dm-L-X-C-X-E (residues 141-

146 on polyomavirus large T antigen) (Pipas, 1992; Pilon et al., 1996). The capacity of these various oncoproteins to tnnsform cells has been linked to their ability to bind pRb

(Chellappan et al., 1992; Lillie et al., 1986; Munger et al., 1989; Pipas, 1992; Pilon et al.,

1996). Expression of pRb can both repress and activate different promoter elements, resulting in growth suppression and decreases in tumorigenicity (Bookstein et al., 1990;

Robbins et al., 1990). Unlike most transcriptional regulators, pRb does not bind to DNA specifically. Instead. it forms complexes with the cellular transcription factor E2F (Cao et al., 1992). This binding plays a role in the transcriptional regulatory activity of pRb

(Wang, 1997). Association with large T antigens releases E7F, which then activates trmscription fiom a variety of promoters (Wang, 1997). pRb is phosphorylated in a ce11 cycle-dependent manner and SV40 large T antigen only binds to the hypophosphorylated form of pRb (Ludlow et al., 1989, 1990). It was suggested that hypophosphorylated pRb is the active form in the G1 phase of the ce11 cycle exerting a negative growth effect, because phosphorylated pRb cm not bind to E2F (Bates and Vousden, 1996). Upon phosphorylation of pRb, cells can progress into S phase (Ludlow et al., 1990; Wang,

1997). Studies showed that SV40 large T antigen cm stimulate the phosphorylation of pRb (Wang et al., 1991). Thus, large T antigens may inhibit pRb function by both direct binding and by phosphorylation. Recent studies demonstrated that the J domain of both

40 SV40 and polyomavinis large T antigens is required to functionally inactivate pRb

(Sheng et al., 1997; Zdvide et al., 1998).

Large T antigens of SV40, polyomavim, and JC virus, as well as adenovinis ElA and human papilloma virus E7 proteins, have been show to form complexes with pl07 via the same consensus domain that binds io pRb (Dyson et al., 1989a; Dyson et al.,

1989k Pilon et al., 1996). Substitution of any arnino acid within the core pRbiplO7- binding sequence of polyomavirus large T antigen abolishes both pRb and pl07 binding in vitro and immortalization activity in vivo (Pilon et al., 1996). Although pl07 shares sequence similarity with pRb in the regions interacting with SV40 large T antigen (Ewen et al., 199 l), the biological activities of p 107 have not been as extensively studied as pRb.

ïhe EZF transcription factor has been show to interact with p 107 independently fiom pRb (Cao et al., 1992), suggesting that, rather than being redundant or equivalent regulators, pRb and pl07 provide different aspects of E2F regulation, thereby influencing transcription of EZF-responsive genes at distinct stages of the ce11 cycle (Shirodkar et al.,

1992). SV40 large T antigen is able to disrupt complexes fonned between EZF and either pRb or p 107, as does the adenovinis E 1A protein (Chellappan et al., 1992).

Direct interactions between p 130 and a number of DNA morvirus oncoproteins have been reported. including large T antigens of SV40 and polyomavirus (De Luca et al., 1997; Desjardins et al., 1997; Knudsen and Wang, 1998; Stubdd et al., 1996), and adenovirus E 1A protein (Whyte et al., 1989). Sequence adysis revealed that p 130 has sequence homology with both pRb and p107, and is a rnember of the retinoblastoma gene family (Li et al., 1993). Deletion of the pl30 gene has been found in severai types of

41 tumors, supporting the idea that it is a tumor suppressor (Yeung et ai., 1993). Studies with mouse embryo fibroblasts lacking the pRb gene have suggested that pl 30 and pl07 are important targets for SV40 large T antigen-mediated transformation (Christensen and

Imperide, 1995; Zalvide and DeCaprio, 1995). SV40 large T antigen sequences have been found in some hurnan mesotheliomas, and it was suggested that the retinoblastoma family proteins, including pRb, p107, and p130, in those tmor cells have been inactivated by large T antigen (De Luca et al., 1997). Recent studies showed that inactivation of p 130 and pl07 is mediated by the J domain of SV40 large T antigen

(Stubdal et al., 1997). SV40 large T antigen has also been found to alter the phosphorylation state of pl30 and p 107 (Stubdal et al., 1996).

iii) p300/CBP

p3OO and the CREB-binding protein CBP are transcriptional CO-activatorsthat act with other factors to regulate gene expression and play roles in many ce11 differentiation and signal transduction pathways (Eckner et al.. 1994; Kawasaki et al., 1998; Irnhof et al..

1997; Snowden and Perkins, 1998). Both proteins have intrinsic histone- acetyltransferase activity and may act directly on chromatin. of which histone is a component, to facilitate transcription (Imhof et al., 1997; Snowden and Perkins, 1998).

They are also involved in growth control pathways, as shown by their interactions with p53 (Gu et al., 1997; Lill et al., 1997) and the viral oncoproteins E1A and SV40 large T antigen (Avantaggiati et al., 1996; Eckner et al., 1994; Eckner et ai., 1996). Binding of

ElA and SV40 large T antigen to p300 and CBP inhibits the transcriptional activation

42 fùnction of both proteins on some promoters (Arany et al., 1995; Eckner et al., 1996). p300 was originally identified as an adenovirus E1A binding protein (Whyte et al., 1989).

ElA and SV40 large T antigen interact with distinct isoforms of p300 (Avantaggiati et al., 1996), but with the same domain on p300 (Eckner et al., 1996). Interaction between

SV40 large T antigen and p300 was first implicated by the ability of the N-terminal 82 mino acids of large T antigen to complement in tram an EIA protein defective for interaction with p300 (Yaciuk et al., 1991). Further studies showed that a SV40 large T antigen mutant containing only the N-terminal 136 amino acids can bind directly to p300

(Rushton et al., 1997). In addition to inhibition of transcriptional activities of p300 and

CBP, SV40 large T antigen binding has also been show to alter the phosphorylation

States of both proteins (Eckner et al., 1996).

III. DNA binding by large T antigens

1. Origin specific DNA binding

As noted above and shown in figure 3, there is extensive hornology between the regions to which polyomavirus and SV40 large T antigens bind on their respective viral

DNAs, as determined by DNase footprinting and dimethyl sulfate protection (Cowie and

Kamen. 1984, 1986; Clark et al., 1983; DeLucia et al., 1983; Jones et al., 1984). Both proteins recognize a similar consensus pentanucleotide sequence 5'-G(A/G)GGC-3'

(Cowie and Kamen, 1984; Pomerantz et ai., 1983; Pomerantz and Hassell, 1984; DeLucia

43 et al., 1983; Jones et al., 1984) (Fig. 3) [SV40 large T antigen can ais0 weakly recognize a 5'-T(G/A)GGC-3' sequence (Fig. 38) (DeLucia et al., 1983; Jones et al., 1984)l. This

consensus sequence is present in nurnerous copies in the viral regdatory regions (Fig. 3).

Although the distribution of these pentanucleotides on the DNA of the two viruses is

different, both polyornavirus and SV40 large T antigens are capable of binding to the

regulatory region on the DNA of the other virus (Pomerantz and Hassell, 1981; Scheller

and Prives, 1985).

The involvement of this pentanucleotide in SV40 large T antigen binding io the

viral origin was first proposed by Tjian (1978). Application of the methylation

interference technique (Siebenlist and Gilbert. 1980) defined the sequences recognized by

both large T antigens and allowed the identification of specific guanine residues that are

essential for DNA binding (Cowie and Kmen, 1986; DeLucia et al., 1983; Tegtmeyer et

al., 1983). This method depends on the fact that dimethyl sulfate reacts with DNA by

rnethylating the N-7 position of guanine. With double-stranded DNA, an added methyl

group becomes situated in the major groove of the double helix, and such methylation

interferes with the binding of a DNA-binding protein which would normally recognize

the guanine (Jones et al., 1984; Siebenlist and Gilbert, 1980). Modified DNA molecules

bound by large T antigen were separated by immunoprecipitation from those which were

not bound. Methylated guanines in both the bound and the unbound fractions were

compared by the DNA sequencing method of Maxam and Gilbert (1977). These studies

showed that large T antigen was able to shield the guanine residues in the consensus

pentanucleotide fiom methylation by dimethyi sulfate (Tjian, 1978) and methylation of

44 the guanine residues in the sequence interferes with large T antigen binding (Cowie and

Karnen, 1986; DeLucia et al., 1983; Tegtmeyer et al., 1983). Point mutation studies also confirmed these results (Cowie and Kamen, 1986; Deb et al., 1987).

On the other hand, since large T antigen also regulates the activity of cellular genes and cellular DNA synthesis, it seems possible that there are alternative binding

sequences other than G(A/G)GGC for large T antigens. To ask this question, Bondeson et al. (1995) synthesized a set of double-stranded polynucleotides with a 3 1-bp random

sequence in the central part. These polymen were subjected to several cycles of binding

to polyornavinis large T antigen with intervening PCR amplification. Individuai polynucleotides with affinity for large T antigen were then isolated by cloning and their nucleotide sequences were determined. The results showed that the majority of the

polynucleotides contained two or three G(A/G)GGC motifs, suggesting that there are not alternative high affinity binding sequence for large T antigen (Bondeson et al.. 1995).

A single pentanucleotide is not likely the sole feature of the large T antigen binding sites important for recognition and binding. These sequences occur often in the

SV40 and polyomavirus pnornes, as well as in pBR322 DNA, but such DNA fragments

are not specifically bound by large T antigen. The afinity of a given DNA molecule for

the large T antigens is also strongly influenced by the number and spatial arrangement of the pentanucleotides present. The minimum number of G(G/A)GGC repeats required for the efficient binding of polyornavirus or SV40 large T antigen to DNA is two. This is

indicated by observations that two repeats are required for the efficient binding of large T

antigens to DNA fragments in an in vitro immunoprecipitation assay (McKay, 1981;

45 Pomerantz et al., 1983; Pomerantz and Hassell, 1984). Other studies showed that binding of polyomavinis large T antigen to DNA fragments containing two copies of the pentanucleotide was detectable but at a low level, while binding to DNA fragments containing three copies or more was much stronger, implying that cooperative interactions rnay occur among large T antigen molecules bound to pentanucleotides in each of the binding regions (Cowie and Karnen, 1986; Peng and Acheson. 1998).

The correlation between the spacing of the G(G/A)GGC sequences in the binding sites and the affinity of large T antigens for these sites is implicated in the differential afinities of large T antigens to different parts of the regulatory regions. For example, the affinity of SV40 large T antigen is greatesr for site 1 where 7 nt intervene between the

GAGGC pentanucleotides (Fig. 3). However, site II is bound poorly by large T antigen in the absence of ATP, and here adjacent pentanucleotides are separated by only 1 nt

(Myers et al., 198 la; Tjian, 1981). In DNA fragments selected by polyomavinis large T antigen binding from a population of random sequences. the G(A/G)GGC motifs are separated by between five and eight variable nucleotides (Bondeson et al.. 1995). When the distance between the first base in adjacent pentanucleotides is close to 10-12 bp, then the site is tightly bound by large T antigen. This arrangement of repeats would place them on the same face of the DNA helix, and therefore allow interactions between adjacent bound large T antigen molecules, which may be important for the cooperative binding .

The nucleotide sequence of polyomavkus ongin DNA that includes the large T antigen binding sites is illustrated in Fig. 3A. Early studies mapped the large T antigen

46 binding sites to the early side of the origin of DNA replication (Gaudray et al., 1981;

Pomerantz et al., 1983; Cowie and Karnen, 1984). DNase 1 protection studies later determined that four distinct sites were protected by large T antigen; they are denoted sites 112, A, B, and C (Cowie and Kamen, 1984; Dilworth et al., 1984). Sites A, B, and C of polyomavinis strain A2 contain 3, 2, and 4 pentanucleotides respectively in tandem arrangement. Site 1!? contnins 4 copies of the pentanucleotides in dyad symmetxy.

Strains AT3 and A3 have respectively a 10- and 1 1-bp deletion within binding site A which removes one GAGGC pentanucleotide; therefore, binding site A in these two strains contains oniy two GAGGC pentanucleotides (Friedmann et al., 1979; Skames et al., 1988). The affi~nityof large T antigen for site 1/2 is lower than that for sites A, B and

C (Cowie and Kamen, 1984). Spacing between adjacent consensus sequences may account for the different affinities, as mentioned above.

Three distinct sites within the non-coding region on the SV40 genome are protected by large T antigen as show by DNase footprintinp (Tjian. 1981). filter binding

(Borowiec and Hunvitz, 1988b). and methylation protection (Myers et al., 198 1b) assays; they are named sites 1, II, and III (Fig. 3B). Binding site I contains two GAGGC pentanucleotides and one TAGGC pentanucleotide; binding site II contains four GAGGC pentanucleotides in dyad symmetry; and binding site III has six (T/G)GGGC pentanucleotides in tandem. Binding of large T antigen to site I is stronger than binding to site II in the absence of ATP. Site [II has a low affinity for large T antigen.

2. Nonorigin DNA binding Although large T antigens pre ferentially interact with pentanucleotide-containing origin DNAs, they also bind with lower anity to nonspecific duplex DNA and single- stranded DNA (Gaudray et al., 1980; Joo et al., 1997; Lin et al., 1992; Oren et al., 1980;

Wang and Prives, 1991b; Wright et ai., 1984). Both large T antigens have been shown to bind to nonspecific single-stranded DNA or double-stranded DNA, as well as specific origin DNA immobilized on cellulose (Gaudny et al., 1980; Oren et al., 1980); howevrr, higher salt concentrations were required to elute large T antigens from origin-containhg

DNA than from calf thymus DNA. Electron microscopie studies demonstrated that, in the absence of salt, SV40 large T antigen binds randomly to rnany nonspecific DNA sites excluding a preference for particular DNA sequence or structural features (Wessel et al.,

1992a). In studies using cornpetition filter binding and the DNA binding immunoassay, it was shown that SV40 large T antigen did not bind specifically to either early or late single-stranded DNA containing specific binding sites (Aubom et al.. 1988). Moreover, large T antigen did not bind to the specific sequences present in single-stranded RNA,

RNAlRNA duplexes, or RNNDNA hybrids (Aubom et al., 1988).

Interactions with nonspecific DNA may be critical for the ability of large T antigen to locate the virai origins and for their helicase activity. Indeed, in the context of

SV40 large T antigen, al1 mutations in the DNA binding domain that reduced nonspecific binding to duplex DNA also prevented origin-specific binding (Lin et al., 1992; Sirnmons et al., 1990b). Polyomavinis large T antigen unwinds nonspecific DNA fragments more eficiently than does SV40 large T antigen because of its relatively higher binding affinity for swh DNA (Wang and Prives, 1991 b).

48 3. DNA-binding domains on large T antigen

Among the most extensively studied regions of large T antigen is the origin binding domain (for review, see Bullock, 1997; Fanning, 1992; Fanning and Knippen,

1992). In SV40 large T antigen, this domain has been mapped to amino acids 131 to 259

(Figs. 2A and 4) (Arthur et al., 1988; Clark et al., 1983; Paucha et al., 1986; Simmons,

1986; Strauss et al., 1987), although other pmtic data suygest that arnino acids beyond residue 300 are involved in modulating specific binding to viral DNA sequences (Arthur et al., 1988). A polypeptide consisting of residues 132 to 246 has also been shown to be an independent domain capable of binding efficiently to both binding sites in the SV40 origin in a sequence specific manner (McVey et al., 1989b). It ha been suggested that the origin DNA binding domain is the central domain through which al1 interactions with

DNA take place (Lin et al., 1992). Consistent with ths proposal. the ongin binding domain is sufficient for sequence-specific binding to the SV40 ongin region (Arthur et al., 1988: Joo et al., 1997; Paucha et al., 1986). This domain is also required for nonspecific binding to double stranded DNA (Lin et al., 1992; Wun-Kim and Simmons.

1990) and may be necessary for binding to single stranded DNA (McVey et al., 1989b;

Wun-Kim and Simmons, 1990). Mutagenesis studies also suggest a role for this domain in oligomerization events and DNA structural distonions, including melting and untwisting the SV40 origin; in other words, it is essentiai for DNA unwinding and helicase activities of large T antigen (Arthur et al., 1988; McVey et al., 1989b; Paucha et al., 1986; Simmons, 1986; Strauss et al., 1987; Wun-Kim and Simmons, 1990).

Simmons et ai. (1990% 1990b) have identified three subdomains (elements) in the

49 SV40 large T DNA binding domain which are critical for binding of the protein to the

recognition pentanucleotides at the viral replication origin. Two of these elements are

necessary for sequence specific binding of the origin and a third one is required for

nonspecific DNA binding. The authors hypothesize that the critical sequences for ongin

binding make up a motif of three major elements denoted A, B 1, and B2 (Fig. 4). The

central &ment, B 1, is necessary for nonspécific DNA interactions. Séquence specific

contacts to the G(NG)GGC pentanucleotides are made by elements A and B2 (Simmons

et al., 1990a, 1990b), which are close together in three dimensional space (Builock,

1997). Residues in B 1 may be involved in making the initial contact with the DNA; the

anchored DNA then slides along until the pentanucleotide sequences appear, at which

point elernents A and B2 also make contact (Simmons et al., 1990a). Binding to site II

may involve an additional element denoted B3 (Fig. 4) and amino acids 166-167 and 145-

247, since mutations within these regions showed altered binding to site II DNA only

(Simmons et al., 1WOa, 1WOb).

Recently, a fragment of SV40 large T antigen containing the ongin DNA binding domain has been purified (loo et al., 1997) and the solution stnicture of this domain was detemined by nuclear magnetic resonance spectroscopy (Luo et al., 1996). These studies revealed that the origin binding domain is a five-stranded antiparallel P-sheet, flanked by two a-helices on one side and one a-heliu and one 3,,-helix on the other side (Luo et al.,

1996). The results also confirmed that elements A and B2, as previously identified

(Simmons et al., 1990a, 1990b), form two closely juxtaposed loops that define a continuous surface on the protein. The addition of a duplex oligonucleotide containing

50 Figure 4. Sequence cornparison of the DNA binding domains of polyomavims and

SV40 large T antigens. Amino acid residues from 278 to 398 of polyomavhs (strain

A2) large T antigen (PyVA2LT) and residues from 124 to 246 of SV40 large T antigen

(SV40LT) are shown. nie sequences of both large T antigens are aligned to show the regions of homology. Identical residues are labeled by solid vertical lines, and similar residues are labeled by dashed vertical lines. Residue numbers are marked m both ends of each sequence block. The nuclear localization signals irnmediately upstrem of each

DNA binding domain are shadowed and labeled. Four major sequence elements of SV40 large T antigen are boxed and labeled. Elements A and B? are necessary for sequence- specific binding of the viral origin, while element BI is required for nonspecific DNA- binding (Simrnons et al., 1990a, 1990b). Amino acids in A and B2 make direct contact with the G(G/A)GGC pentanucleotide sequences (Luo et al.. 1996) and are nearly identical in al1 known papovavirus large T antigns (Sirnrnons et al., 1990a. 1990b). A fourth element, B3, is necessary for the binding of large T antigen to site II but not to site

1 (Simmons et al., 1990a). *;8 NLS PyVAZLT TPP~I~DPAPSDFPSSLTGYLSHA FPAFLVYS

1l:r:u,y.., -,,,,;,zf .,>,:;:;:;Y~;(,.,,.,, ,r,,v,.-/., ,p,,(, 1 1 qlll , , , , ~~~~ SV4 OLT TPR : ;VEDPKDFPSELLSFLSHAV LACFAIYT I I

318 355 I I PyVA2LT TKEKCKQLYDTI-GKFRPEFKCLVHYEEGGMLFFLTMTK

SV4 OLT TKEKAALLYKKIMEKYSVTFIS~SYNHNILFFLTPHRIlII II II: I :IlIII :

PyVA2LT HRVSAVKNYCSKLCR-SFLMCKAVTKPMECYQVVTAAPFQLITE 11 111 111.11 II 1 /I Il 11 1 SV4 OLT I~AQKLCTFSFLICKGVNKEYLMYSALiTRDPFSVIEE I the ongin recognition pentanucleotide GAGGC induces chernical shifi changes and slows amide proton exchange in resonances from this region, indicating that this surface directly contacts the DNA (Luo et al., 1996).

Within the ongin binding domain, element B 1 and certain residues in elements A and B2 were reported to be important for nonspecific DNA binding (Wun-Kim et al.,

1993). However, the purified origin binding dornain has limited nonspecific binding activity (Joo et al., 1997). Other studies demonstrated that, in addition to the origin binding domain, amino acid residues 269 to 522 are also required for nonspecific binding to double stranded DNA (Lin et al., 1992). This additional region is not an independent binding domain. Rather, it cooperates with the origin binding domain to give nse to wild type levels of nonspecific double stranded DNA binding (Lin et al.. 1992). Little is hown about the regions of large T antigen involved in binding to single snanded DNA.

The origin specific DNA binding domain on polyomavirus large T antigen has been mapped to a 116 amino acid stretch betwern residues 282 and 398 (Figs. 2B and 4)

(Sunstrom et al., 1991). When both large T antigens are aligned to rnauimize sequence similarity, Val132 of SV40 is aligned with Asp286 of polyornavirus and Glu246 of SV40 is aligned with Glu398 of polyornavirus (Fig. 4); these two regions share 45% direct arnino acid sequence identity (Sunstrom et al., 1991). Conservation within the DNA binding domain would be expected because both proteins recognize similar pentanucleotide sequences in DNA targets and each protein binds to the hi& affinity binding sites on the DNA of the other virus (Pomerantz and Hassell, 1984; Scheller and

Prives, 1985).

52 4. Formation of large T antigen hexamers in the core origin

It was observed that in the absence of ATP, SV40 large T antigen foms oligomers when bound to the SV40 ongin (Bradley et al., 1982; Fanning et al., 198 1;

Gidoni et al., 1982; Myers et al., 1981b; Wachter et al., 1985). Using scanning

transmission electron microscopy, Mastrangelo et al. observed monomers through tetramers bound to the core origin (and monomers through trimers bound to site Ij

(Mastrangelo et al., 1985). Tetramer binding to site II was proposed to reflect the binding of four large T antigen monomers to the four pentanucleotides (Mastrangelo et al., 1985).

Following pentanucleotide recognition, large T monomers may undergo a conformational change and assemble into hexamers at the SV40 origin, which is critical for the initiation of viral DNA replication. Electron microscopy studies revealed that in the presence of ATP, large T antigen assembles as a bilobed structure on the SV40 origin

(Dean et al., 1987c) and each lobe contains 6 monomers of large T antigen (Mastrangelo et al.. 1989). Atomic force microscopy studies (Mastrangelo et al.. 1994) and gel-based assays (Dean et al.. 1992: Parsons et al., 1991) have confimed that. in an ATP-dependent manner, SV40 large T antigen forms a double hexarner at the SV40 core origin. Related studies indicated that al1 three core origin domains are important in the assembly of large

T antigen into a functional double hexamer (Borowiec, 1992; Dean et al., l987a; Parsons et al., 1991).

The SV40 core origin can be divided through the middle of the centrai palindrome into functional early and late halves (Fig. 3) (Parsons et al., 1991). Hexamer assembly is thought to initiate in the eady hdf of the core ongin (the side near the early palindrome),

53 and formation of the early hexamer enhances the formation of the weaker binding late hexamer (Parsons et al., 1991). After the assernbly of the second hexamer on the late side of the core ongin, double hexamer formation is completed. In this model, the double hexamers are depicted as two regular, hexagonal rings surrounding the core origin.

Evidence that large T antigen surrounds the DNA includes the observation that double hrxarner Formation protects the entire core origin l'rom DNase I digestion (Borowiec and

Hurwitz, l988b; Deb and Tegtmeyer, 1987; Parsons et al., 1991). Recent studies Mer demonstrated that hexamers probably bind to DNA by topologically enclosing the DNA double helix within the central hole formed by the circular hexamer rather than by recognizing and interacting with specific nucleotides, and each hexamer covers approximately one tum of the DNA helix (San Martin et al., 1997; Wessel et al., 1992b).

It was suggested that large T aniigen binds the four pentanucleotides in site II in two pairs. Members of a pair were considered to be arrangd in a head-to-head orientation (McVey et al., 1996; Tegtmeyer et al., 1983). Owing to the structure of duplex DNA, the hvo pentanucleotides that form a pair would be located on the same face of the duplex. Subsequent proteidprotein interactions would then favor the formation of the double hexarners. loo et al. (1998) recently reported that double hexamer formation on the SV40 core origin requires only a subset of the available binding sites. In their gel retardation-l ,I O-phenanthroiin-copper ion footprinting experiments, synthesized DNA fragments containing either wild type SV40 core origin sequence or one or more of the pentanucleotides mutated were bound by high concentrations of large T antigen. After fixation, the protein-DNA complexes were isolated in gel retardation assays and were

54 digested within the acrylarnide matrix by the nuclease activity of 1,10- phenanthrolin- copper ion (Joo et al., 1997; Kuwabara and Sigman, 1987). When the resulting DNA fragments were eluted and andyzed on a sequencing gel, footprints of the protein-DNA complexes were obtained. The results demonstrated that, while a single pentanucleotide cm support single hexarner formation, double hexamer formation on the core origin requires only a pair of the four pentanucleotides (Joo et al., 1998). Since al1 four pentanucleotides in the wild type origin are necessary for extensive DNA unwinding and replication, it was suggested that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process (Bullock, 1997; Joo et al., 1998).

5. DNA distortions associated with large T antigen biading and oligomerization

Upon formation of double hexarners of large T antigen on the SV40 core origin, the DNA structure is distorted (Borowiec and Hunvitz, 1988a; Panons et ai.. 1990).

Both topological and chemical assays were used to monitor the structural changes.

Topological assays dernonstrated that binding of large T antigen initiates an untwisting

(an increase in the number of bp per helical turn) of origin containing duplex DNA by 2 to 3 tums (Borowiec et ai., 1991; Borowiec and Hunvitz, 1988a). This observation was explained, in part, by the discovery that large T antigen melts approximately 8 bp of

DNA (SV40 nts 5210 to 5217) within the early palindrome (Fig. 3) (Borowiec and

HunMtz, 1988a; Parsons et al., 1990). Furthemore, it was determined that the -1 7 bp

PST-rich tract becomes stnicnirally distorted, probably by untwisting the DNA, although the distorted region is not sensitive to S1 nuclease digestion and DNA remains essentially

55 double stranded. However, S 1 nuclease sensitivity indicates that the SV40 early palindrome is actually melted by large T antigen (Borowiec et al., 1991; Borowiec and

Hurwitz, 1988a; Parsons et al., 1990; SenGupta and Borowiec, 1994). The use of chemical modification assays has allowed the determination of which sequences within the A/T-rich and early palindrome regions are distorted by SV40 large T antigen in the presence of nucieotides (Borowiec and Hurwitz, 1 Ma; SenGupta and Bbrowiec, 1994).

Based on these studies, it was concluded that the importance of the early palindrome and

AIT-rich regions during initiation of SV40 DNA replication lies in their ability to undergo structural changes essential for subsequent unwinding and initiation

(Bhattacharyya et al., 1995; Borowiec, 1992; Borowiec and Hunvitz, 1988a; Parsons et al., 1990).

Melting of the early palindrome region could be induced by nonhydrolyzable analogues of ATP, such as AMP-PNP (Borowiec and Hwitz, 1988a). Therefore, local melting of the early palindrome region does not require ATP hydrolysis. Because the

DNA helicase activity of large T antigen is energy dependent. it was proposed that melting of the early palindrome by large T depends on an activity distinct from its DNA helicase activity (Parsons et al., 1990). However, complete distortion of the A/T-rich tract requires ATP hydrolysis, an indication that subsequent distortions of the SV40 origin are dependent on activation of the helicase activity of large T antigen (Borowiec and Hurwitz, 1988a).

Less is known about formation of hexamers of polyomavirus large T antigen at the core origin and its effects on DNA distortion. Replication ongins of both viruses are

56 similar and polyomavirus large T antigen shares many biochemical properties with SV40

large T antigen. It is likely that the initiation of polyomavinis DNA replication also

involves large T antigen hexamer formation and structural alterations at the core origin.

Indeed, polyomavirus large T antigen is very efficient at unwinding SV40 ongin- containing DNA, although SV40 large T antigen is not equally proficient in unwinding

polyomavirus DNA (Wang and Prives, 1991b). However, despite its ability to unwind

SV40 origin-containing DNA, polyomavirus large T antigen cm not mediate its replication in vivo or in vitro (Murakami et al., l986a, 1986b; Wang and Prives, 1991b).

Bhattacharyya et al. (1995) examined and compared viral origin DNA binding and the resulting structural changes induced by polyomavirus and SV40 large T antigens, by

DNase 1 footprinting and KMnO, modification assays. The results showed that the stnictunl changes in the polyomavirus origin induced by polyomavirus large T antigen included sites within both the AT-rich region and the early side of the core origin, consistent with what was shown for SV40 (Fig. 5). However, KMnO, modification assays demonstrated that polyomavirus large T antigen produces a more extensive and substantially diflerent distortion pattern in the polyomavirus core ongin than does SV40 large T antigen in the SV40 core origin (Fig. 5). In their experiments, KMnO, oxidizes nucleotides (primarily T residues) in the origin region that have become exposed by alteration induced by large T antigen binding in the structure of the double helix

(Borowiec et al., 1987). These oxidized residues cause chah termination by DNA polymerase, and the positions of termination are determined by cornparison with DNA sequencing reactions. This method thus can be used to identify the sites at which DNA is

57 Figure 5. Cornparison of structural modifications of polyomavirus core-ori DNA by polyomavirus large T antigen and SV40 core-ori DNA by SV40 large T antigen.

Core-ori sequences are aligned according to the center of origin palindromes. Horizontal arrows represent G(A/G)GGC pentanucleotide sequences. Vertical filled arrows indicate bases modified by KMnO, in the presence of polyornavirus (top) or SV40 (bottom) large

T antigen. In the presence of nucieotides, the structurai changes in the poiyomavirus core-ori induced by polyomavirus large T antigen include sites within both the AIT-rich region and the early side of the core-ori, similar to changes in SV40 core-ori caused by

SV40 large T antigen. In addition, polyomavirus large T antigen distorts the polyomavirus core-ori DNA inside the central palindrome. (Adapted fiom Bhattacharyya et al., 1995). Py Core Origin

R/Y PEN A/T 1/5297 94-+- I JIWJJJI JI AGTTGCCTCTGGAACCCTCTACAATGCCTCTCTTCTTTTTCTCCAGAGTMGCGGAGGCCAGGGGCCCCCffiCCTCTGCTTAATACTAAAAAAAAChGC- - TCAACGGAGACCTTCGGAGATGTTACGGAGAGMGAAAAAGAGGTCTCATTCGCCTCCGGTCCCCGGGGCCCGGAGACGAATTATGA~MGTCG 'l"r rlv~ 9 19 + 9 ~9

EP PEN A/T

SV40 Core Origin melted or untwisted. SV40 large T antigen was shown to alter the structure of SV40

DNA in a way that allows strong KMnO, modification of bases within the AT-rich and early palindrome regions, but protects the entire central palindrome region as well as some flanking sequences fiom KMnO, modification (Fig. 5). On the other hand, in the presence of AMP-PNP, polyomavirus large T antigen produced sites of distortion within the central paiindrorne of the polyomavirus core origin and at several sites within both the early and late regions that Bank the core origin (Fig. 5) (Bhattacharyya et al., 1995).

Therefore, significant differences in the interactions of polyornavirus and SV40 large T antigens with the core origins may account for the failuce of each large T antigen to support replication of the reciprocal origin DNA in permissive ce11 extracts.

6. Conditions thrt affect DNA binding by large T antigen

A) Mrcleotides

Binding of large T antigens to core origin DNA was show to be altered both qualitatively and quantitatively by ATP and other nucleotides (Borowiec and Hurwitz,

1988b; Deb and Tegtmeyer, 1987; Lorimer et al., 1991). In the presence of ATP, binding to site II by SV40 large T antigen was increased by 10- to 15-fold, while binding to site 1 was increased to a much lesser degree (Borowiec and Hunvitz, 1988b; Deb and

Tegtmeyer, 1987). Furthemore, DNase 1 protection analysis showed that ATP extended the DNase protection domain of large T antigen symmetrically outward from site II @eb and Tegtmeyer, 1987). Related experiments demonstrated that in the absence of ATP, large T antigen preferentidly protects the central palindrome region, and not the A/T-rich or early palindrome regions, fi-om DNase 1 cleavage (Borowiec and Hurwitz, 1988b;

Panons et al., 1991). Borowiec and HuNvitz demonstrated that, in the absence of ATP,

SV40 large T antigen binds with high occupancy to site 1 at O'C, 25'~~and 37'~but to site II only at O°C and X°C. At 37OC, the temperature essential for the initiation of SV40

DNA replication in vitro, ATP is required for the interaction of large T and site II

(Borowiec and Hwitz, I988b).

Studies of core ongin DNA binding by polyomavirus large T antigen also showed that ATP increased the binding of polyomavirus large T to the core ongin by up to 10- fold. and extended the protected region within the core origin (Lorimer et al., 1991). Our studies Merdemonstrated that the effect of ATP requires the presence of site 1/2 in the polyomavirus core origin; linle effect of ATP could be detected on binding to sites A, B, and C when site 112 was mutated (Peng and Acheson, 1998). ATP can be replaced by other nucleotides and nonhydrolyzable analogs of ATP. such as dATP, AMP-PNP. ADP, dCTP. and UTP, although each type of nucleotide stimulates DNA binding to various degrees (Borowiec and Hurwitz, 1988b; Lorimer et al.. 1991). The ability of AMP-PNP to enhance core origin binding by large T antigens indicates that hydrolysis of ATP is not necessary for this stimulation.

It was also shown that ATP stimulation depends on the presence of MgCl,

(Lorimer et al., 1991), which can be explained by the fact that ATP, as well as other

NTPs, associate with a Mg" ion. Magnesium may alter the configuration andlor charge of the phosphate groups on ATP and thus makes it a suitable substnte for large T antigen.

In fact. ATP done (without Mc) has been shown to inhibit SV40 large T antigen

60 binding to the viral origin DNA (Vogt et al., 1986); and magnesium salt alone (without

ATP and other salts) induces the formation of tightly packed large T antigen aggregates which bind to nonspecific DNA to fom many DNA branches and loops that emanate fiom the aggregated protein core (Wessel et al., 1992a).

Examination of ongin-specific DNA-large T antigen complexes by electron rnicroscopy reveaied that ATP induces the formation of double hexamers of SV40 large T antigen (Dean et al., 1987c), whereas in the absence of ATP, the largest polymers are trimes and tetramers (Mastrangelo et ai., 1985). These studies demonstrated that ATP binding regulates protein-protein interactions necessary for oligornerization and promotes additional interactions of large T antigen with the SV40 origin (Dean et al., 1992;

Mastrangelo et al.? 1989; Parsons et al., 1991). The nucleotide (ATP) binding dornains of polyomavirus and SV40 large T antigens are located in the carboxy-terminal portion of the molecule (Fig. 2) (Bradley et al., 1987). A tnincated SV40 large T antigen, which contains the DNA binding dornain but lacks the ATP binding domain, does not respond to ATP in origin DNA binding (McVey et al., 1996). Using zone velocity sedimentation and nondenaturing polyacrylamide gel electrophoresis, Wang and Prives (1 99 1b) determined that ATP and MgCl, together induce the assembly of polyomavirus large T antigen into hexamers, indicating that DNA is not necessary for these proteidprotein interactions. Active ATP hyârolysis was not required for induction of large T antigen hexarner formation in vitro (Reynisdottir et al., 1993; Wang and Prives, 199 la). Finally, in addition to ATP, the zinc fmger regions of both large T antigens are also needed for hexamer assembly (Loeber et al., 199 1; Rose and Schafniausen, 1995).

6 1 To compare the biological activities of large T antigen in a hexameric state with that of monomers, Dean et al. isolated hexameric large T antigen by glycerol gradient centrifugation and tested its functions in vitro (Dean et al., 1992). While monomeric large T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric large T antigen was inactive in these reactions. Lsolated hexamen incubated ai 37'C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37OC in the absence of ATP.

This dissociation restored the activity of these preparations in the DNA replication reaction. indicating that hexameric large T antigen is not pemanently inactivated, but merely assembled into a nonproductive structure (Dean et al., 1992).

B) Phosphorylation

DNA-binding activity and oligomenzation of large T antigen were shown to correlate with distinct states of phosphorylation (Scheidtmann et al., 1984).

Phosphorylation regulates both negatively and positively the specific DNA binding activity of large T antigens, hence their functions in the viral replication cycle as rnentioned in a previous section (for review, see Fanning, 1992; Fannine and Knippers,

1992; Prives, 1990). Partial dephosphorylation of SV40 large T antigen with calf intestinal alkaline phosphatase (CIAP), which removes phosphate groups fiorn but not , stirnulated specific DNA binding in viîm (Klausing et al., 1988; Mohr et ai., 1987). Similady, treatment of polyomavirus large T antigen with smailer amounts of

CIAP stimulated its specific binding to the polyomavirus replication origin; however,

63 treatment with larger amounts of CIAP caused marked inhibition of origin-specific

binding by the viral protein (Wang et al., 1993).

SV40 large T antigen purified from E. coli is in an underphosphorylated state and

is deficient in directing DNA replication (Mohr et al., 1989). However, large T antigen

produced in bacteria retains specific origin binding activity (Arthur et al., 1988; Mohr et

al., 1989). Thus, phosphorylation of large T antigen is dispensable for origin binding

activity. Full length large T antigen or an amino-terminai fragment, both produced in E.

di,were show to be substrates for the cdc2 protein kinase, resulting in phosphorylation

of the single threonine residue at position 124, a site known to be phosphorylated in vivo

(Scheidtmann et al., 1982). Such phosphorylated molrcules bound far more efficiently to

site 11 within the ongin of DNA replication than their unphosphorylated counterparts

(McVey et ai.. I989a, 1993, 1996). In addition. mutating a single threonine residue at

position 278 of polyomavirus large T antigen to alanine severely impaired its origin DNA

binding activity (Li et al.. 1997). M78in polyomavirus large T antigen is a potential substrate for cyclin-dependent kinases (CDKs) (Li et al., 1997; Chatte jee et al.. 1997).

Using mutants of a fragment of SV40 large T antigen (arnino acid residues 82 to

259) containing Th124 and the DNA binding domain (amino acids 13 1 to 259)

(Simmons et al., 1990a), as well as other large T antigen variants, McVey et al. (1996) fwthsr determined that Thr124 phosphorylation enhances the interaction of amino acids

89 to 259 with the core ongin of replication (Fig. 6). Phosphorylation, therefore, activates the minimal DNA binding domain of large T antigen even in the absence of domains required for hexamer formation. The same authors Mer showed that

63 activation is mediated by the DNA binding elements required for proper binding to site II in the minimal DNA binding domain (Simmons et al., 1990a). These elements, includbg amino acid 167, and amino acids 2 15 to 2 19 (domain B3 in Fig. 4), enhance binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleotide arrangements found elsewhere in the SV40 origin of replication (McVey et al.' 1996). It was proposed that phosphorylation induces confonational shifts in the minimal DNA binding domain of large T antigen and thereby enhances interactions among large T antigen subunits oriented by core origin pentanucleotides (McVey et al..

1996). A model was Mersuggested to account for reguiation by phosphorylation and

ATP of the process of double hexamer formation in the SV40 core origin (McVey et al.,

1996) (Fig. 6). According to this model, a conformational change induced by phosphorylation of Tl24 would foaer interactions among large T antigen subunits, therefore enhancing the binding of four subunits of large T antigen to the four specially arranged pentanucleotide repeats in the central palindrome as show by Mastrangelo et al.

(Mastrangelo et al., 1985). The subsequent effect of ATP causes Merconformational change of bound monomers and leads to double hexamer formation in the core ongin.

The two steps of conformational change are probably independent, because phosphorylation is not required for large T antigen hexarner formation, since nonphosphorylated large T antigen made in E. coli can form hexarners in solution

(Reynisdottir et al., 1993). Figure 6. A mode1 showing steps in the binding of SV40 large T antigen to the central palindrome of core-ori thnt are enhanced by phosphorylation of Thrl24, and

ATP. Black arrows on the DNA indicate pentanucleotide repeats. Brackets indicate

protein-protein interactions. A) Possible interactions of the minimal DNA binding domain of large T antigen with pentanucleotide repeats of core origin DNA in the absence of ThrlLl phosphorylation. The direction of pentanucleotides orients the DNA binding domains on large T antigen. Because large T antigen does not bind single

pentanucleotides well (Wright et al., l984), it is suggested that two large T subunits bind

to two pentanucleotide repeats (Parsons and Tegtrneyer, 1992). B) The effects of IIirlN

phosphorylation by cdcî kinase. Conformational shifts induced by phosphorylation

would foster interactions among large T antigen subunits when the subunits are properly

spaced and oriented by core ongin pentanucleotides. Therefore, in the absence of ATP

binding and hexamer formation (Parsons et al., 1991), phosphorylation would enhance

the binding of four subunits of large T antigen to four pentanucleotide repeats

(Mastrangelo et al., 1985). C) The assembly of double hexamers on origin DNA in the

presence of ATP. ATP binding causes a secondary conformationai change in large T

antigen subunits, which enables the subunits to fit into a hexamer structure (Mastrangelo

et al., 1989; Parsons et al., 199 1). (Adapted from McVey et ai., 1996).

Cl PH

The activity of many proteins is strongly pH-dependent. For exarnple, the DNA- dependent ATPase activity of the virus type 1 origin binding protein, encoded by the UL9 gene, is optimally active between pH 8.3 and 9.5 (Dodson and

Lehman, 1993); and the recA protein of E. coli catalyzes ATP hydrolysis at pH 6.2 better than it does at pH 7.5 (Bryant, 1988). The DNA binding activities of various proteins are also affected by pH. The same recA protein binds to linear duplex DNA at low pH, but when the pH is shifted above 6.8, the DNA can no longer be stably bound (Lindsley and

Cox. 1989). The single-stranded DNA binding activity of type 1 major DNA binding protein, infected-ce11 polypeptide 8 (ICP8), is maximal near pH 7.6, and is quite sensitive to both slightly acidic and slightly alkaline pH. as a pH of 8.5 reduced the observed binding by >90% (Ruyechan and Weir. 1984). An acid-soluble spore protein fiom Bacillus subtilis binds to DNA at optimal pH of 6.7; the binding decreases by about 30% when pH is shifted to 6.1 or 7.5, and little binding was observed at pH 8.5 (Nicholson et al., 1990).

SV40 large T antigen was reported to bind to nonspecific (calf thymus) DNA more redily at pH 6 than ai higher pH values (Dom et al., 1982; Montenarh and

Henning, 1982; Oren et al., 1980); however, cornparisons of binding to specific DNA at different pH values were not made in those studies. SV40 large T antigen also showed increased protection of specific DNA fragments against DNase 1 digestion when binding was carried out at O°C in a pH 7.0 bufYer containing 40 mM NaCl compared with 37°C in a pH 7.5 buffer with no added NaCl (Deb and Tegtmeyer, 1987); however, the distinct

66 effects of sait, temperature, and pH on DNA binding were not disthguished in that study.

Electron microscopic studies with SV40 large T antigen demonstrated that, at pH 6 and in the absence of sdt, large T antigen bound eficiently to nonspecific double-stranded

DNA, forming a dense nucleoprotein rod; whereas at pH 7.5 and in the absence of sait, the affinity of large T antigen to nonspecific DNA was reduced, and only a fraction of total large T antigen molecules \vas found in association with DNA (Wessel et al.,

1997a). It is likely that, at low pH values, positively charged amino acid side chahs become available for an electrostatic interaction with DNA in the absence of sdt. Most recent studies of specific DNA binding by polyomavims and SV40 large T antigens were carried out at pH 7.5 or above because DNA replication in vitro is stimulated under those conditions. However, before our work, no systematic studies of the effect of pH on binding to origin DNA had been cmied out.

IV. Production of large T antigens for in vittu analysis

The first step to define the biochemical propenies of large T antigens is to obtain purified protein. However, it has been dificult to puri@ large amounts of large T antigens for in vitro studies because of their low level of synthesis in Iytically infected and transformed ceils. To circumvent this problem, different protein expression systems have been employed to make large T antigen.

1. Expression with an adenovirus vector system

67 The adenovirus vector system was developed for expression of SV40 large T antigen as well as other foreign proteins at high levels by Thummel et al. (Thummel et al., 1981, 1983). Insertion of the SV40 large T antigen gene into an adenovirus vector resulted in adenovinis-SV40 hybrid viruses. Optimal production of protein was attained when SV40 large T antigen coding sequences were tnnscnbed from the adenovirus major iate promoter, and the hybrid large T antigen mRNA carried dmoa the entire adenovirus tripartite leader at its 5' ends (Thummel et al., 1983). Isolation of adenovirus-SV40 recombinant vimses depended on a strong biological selection for SV40 large T antigen, which has a helper function activity that allows human adenoviruses to grow efficiently on otherwise nonpermissive monkey cells. Biological characterization of SV40 large T antigen overproduced by the hybrid viruses indicated that ir is functionally indistinguishable from wild type SV40 large T antigen (Thummel et al., 198 1).

Mansour et al. (1985) placed the polyomavirus large T antigen coding sequences under the control of the adenovirus major late promoter for overproduction of this protein. Selection for recombinant adenoviruses that cany polyomavinis sequences was accomplished by including the SV40 large T antigen gene with its own promoter in the virus constructions and growing the recombinant viruses on monkey cells (Mansour et al., 1985). It was shown that the adenovirus-polyomavhs recombinant viruses produced

5-fold more polyomavirus large T antigen than do wild-type polyomavirus-infected mouse cells (Mansour et al., 1985). The disadvantage is that small amounts of SV40 large T antigen were CO-produced,and this may interfere with functionai aaalysis of polyomavVus large T antipn. To overcome this problem, a helper-free recombinant

68 adenovims that expresses polyomavims large T antigen was constmcted by inserting the

polyomavirus large T antigen gene into an adenovinis vector which lacks the EIA and

E IB transcription units; the recombinant virus can replicate in human 293 cells, which

provides EIA fictions in tram (Massie et al., 1986). Cornparison of the amount of

large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells

infected with recombinant viruses revealed at ieast a 5-fold greater yield of the protein on

a per ce11 basis in the latter system (Massie et al., 1986).

2. Expression in E. coïi

Bacterial systems have been used to produce SV40 large T antigen lacking the extensive posttranslational modifications that occur in mammalian cells. In one system, a

BglII site and a BamHI site were inuoduced at the 5' and 3' boundaries. respectively, of an intronless SV40 large T antigen gene; the DNA fragments were cloned into the polylinker oEpUC9 (Arthur et al.. 1988). Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac prornoter in

E. coli JM 103 cells. Large T antigens were e~chedby immunoaffinity chromatography from an E. coli ce11 lysate and furthet purified by liquid chromatography (FPLC)(Arthur et al., 1988). Purified large T antigen showed specific DNA binding activity, and the origin DNA binding domain of SV40 large T antigen was defmed by using mutant large

T antigens produced in this system (Arthur et ai., 1988).

I In another system used by Mohr et al. (1989), The coding sequences of SV40 large T antigen were cloned into a T7 expression plasmid (Studier and Moffatt, 1986) that was transformed into a strain of E. coli harboring a h. prophage containhg the gene for T7

RNA polymerase under the control of the lac W5 promoter. Induction of cultures with isopropyl f3-D-thiogalactoside results in the synthesis of large amounts of T7 RNA polymerase which in turn, directs transcription from a T7 promoter which drives the expression of large T antigen (Mohr et al., 1989; Studier and Moffatt, 1986). Large T antigens were purified kom induced cultures by irnmunoaffinity procedures. The yield of full-length large T antigen purified by this method was approximately 25 pg fiom 1 liter of induced cultures (Mohr et al., 1989). Various amounts of immunologically related, truncated proteins copurified with the Full-length polypeptide. Side-by-side pl elec trophoretic anal ysis of proteins purified from bacterial and mamrnalian sources revealed that E. coli large T antigen migntes slightly slower than large T antigen fiom

Hela cells, which was attributed to differences in the phosphorylation of the proteins

(Mohr et al.. 1989).

3. Expression wYh a baculovirus system

A helper-independent baculovirus vector system has also been used for the high level expression of large T antigen genes in a eucaryotic ce11 environment (Lanford,

1988; Rice et al.. 1987). A recombinant baculovirus expressing the polyomavims large T antigen gene was constnicted by allelic replacement of wild type baculovirus polyhedrin gene sequences with the large T antigen coding region. A recombinant plasmid, pEV5 1LT, was constnicted by inserting an intronless constnict of the polyomavirus large

T gene into vector pEV5 1, a plasmid which facilitates the insertion of foreign genes into

70 the polyhed~region of the baculovirus Autographu cal~rnicamultiple nuclear polyhedrosis virus (AcMNPV) (Rice et al., 1987). Cotransfection of the resulting plasmid pEV5ILT with wild type AcMNPV DNA into a permissive insect ce11 Iine,

Spodoptera frugiperdu IPLB-SF21, resulted in allelic replacement of the wild type polyhednn gene of AcMNPV with the polyomaviw large T antigen gene under the control of the abundantly expressed polyhednn promoter (Rice et al., 1987).

Recombinant virus plaques were visually selected by their occlusion negative phenotype, resulting from polyhedrin replacement with the polyomavirus large T antigen gene. One widely used recombinant AcMNPV is named vEV5ILT (Rice et al., 1987). Expression of large T antigen can be carried out in either SF9 or High Five insect cells (Richardson,

1995). It was determined that the levels achieved with the baculovirus-based expression system are equal to or higher than the levels achieved with the adenovirus-based expression systems (Massie et al., 1986). A similar procedure was applied to SV40 large

T antigen; high levels of large T antigen synthesis were also observed (Lanford. 1988).

Both large T antipns have been proved to be hlly functional by a variety of studies.

4. Expression in yeast: Pichia pastoris expression system

As a eucaryote, Pichia pastoris has many of the advantages of higher eucaryotic expression systems such as protein processing, protein folding, and post-traaslational modification (for review, see Romanos et al., 1992), while being as easy to manipulate as

E. coli. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher

7 1 expression levels. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels (Buckholz and Gleeson, 1991 ; Cregg et al.,

1993; Romanos et al., 1992; Sreekrishna et al., 1988; Wegner, 1990). These features make Pichia very usefil as a protein expression system.

Pichia pasroris is a methylotrophic yeast, capable of metabolizing mêthanol as its sole carbon source. The fint step in the metabolism of methanol is the oxidation by the enzyme alcohol oxidase of methanol to formaldehyde using molecular oxygen. In addition to formaldehyde, this reaction generates hydrogen peroxide. To avoid hydrogen peroxide toxicity, methanol metabolism takes place within a specialized ceIl organelle, called the peroxisome, which sequesters toxic by-products away from the rest of the cell.

Alcohol oxidase has a poor affinity for O!, and Pichia pastoris compensates by generating large amounts of the enzyme. The promoter regulating the production of alcohol oxidase (AOXI) is used to drive heterolopus protein expression in Pichia (Cregg et al.. 1987). The AOXl gene cm be induced to give levels of up to 30% of total cellular protein by the addition of methanol (Couderc and Baratti, 1980).

There are two genes in Pichia pastoris that code for alcohoi oxidase: AOXI and

AOX.2. The AOXI gene is responsible for the vast rnajority of alcohol oxidase activity in the cell. While AOX2 is about 97% homologous to AOXI, growth with AOXZ on methanol is much slower than with AOXl (Cregg et ai., 1989; Koutz et al., 1989). This slow growth on methanol allows isolation of Mut' (Methanol utilization slow) strains

(aox 1) (see below). Expression of the AOXl gene is controlled at the level of transcription. In

methanol-grown cells, approximately 5% of the polyA' RNA is from the AOXI gene.

The regulation of the AUXI gene is a two step process: a repressiodderepression

mechanism plus an induction mechanism. Briefly, growth on glucose represses

transcription, even in the presence of the inducer methanol. For this reason, growth on

glycerol is recommended for optimal induction with methanol; howeever, growth on

glycerol (derepression) is not suficient to induce expression frorn the AOXI gene. The

inducer, methanol, is necessary for even detectable levels of AOXl expression (Ellis et

al., 1985; Koutz et al,. 1989; Tschopp et al., 1987)

Loss of the AOXl gene, and thus a loss of the alcohol oxidase activity. results in a

suain that is phenotypically Mut' (Methanol utilization slow). This results in a reduction

in the cells' ability to metabolize rnethanol. The cells, therefore, exhibit poor growth on

methanol medium. Mut' (Methanol utilimtion plus) refers to the wild type ability of strains to metabolize methanol as the sole carbon source. These two phenotypes are used

when evaluating Pichia trûnsformants for integration of the gene of interest.

Heterologous expression in Pichia pastoris can be either intracellular or secreted.

Secretion requires the presence of a signal sequence on the expressed protein to target it

to the secretory pathway. While several different secretion signal sequences have been

used successfully, including the native secretion signal present on some heterologous

proteins, success has been variable. The secretion signai sequence from the

Saccharomyces cerevisiae a factor peptide has been used with the most success (Cregg et

al., 1993; Scorer et al. 1993).

73 The major advantage of expressing heterologous proteins as secreted proteins is

that Pichia pastoris secretes very low levels of native proteins. That, combined with the

very low amount of protein in the minimal Pichia growth medium, means that the

secreted heterologous protein comprises the vast majority of the total protein in the

medium and serves as the first step in purification of the protein (Barr et al., 1992).

Howver, if there are recognized glycosylation sites (Asn-;Y-Ser/Thr) in the protein's

primary sequence. glycosylation may occur at these sites.

Based on the above knowledge, we attempted expression of polyomavinis large T antigen in Pichia pastoris. Our strategy was to insert an intronless large T antigen gene

into the yeast genome in such a way that the expression of the protein is under control of the AOX] gene promoter, and is therefore inducible in the presence of methanol. Since

multiple insertion events occur spontaneously, and usually give higher levels of gene expression (Cregg et al.. 1993), we screened the yeast transformanu and identified ce11

lines that contain multiple copies of the large T antigen gene and give relatively high

levels of expression. Characterization and purification of the yeast-derived large T antigen will be presented in Chapter 3. We Merdenved optimal conditions for binding of polyomavinis origin DNA by this large T antigen, and found that specific binding was strongly increased at low pH (6-7) (Chapter 4). This enhanced DNA binding at low pH enabled us to develop a novel gel mobility shifi assay for the detection of large T antigen in ce11 lysates (Chapter 5). By comparing the binding finhies of large T antigen to

DNA fragments containing one or more of the binding sites, we also demonstrated that large T antigen binds to sites 1/2, A, B, and C cooperatively (Chapter 4).

74 CHAPTER 2. MATERIALS AND METHODS

1. Cet1 cultures

1. Strains

Yeast Pickia pastoris suain GSll5 was purchased fiom invitrogen Inc. and used as the host strain for expression of polyomavirus large T antigen. It has a defect in the histidinol dehydrogenase gene (hi&) which prevents it fiom synthesizing histidine. Al1 expression plasmids carry the HIS4 gene which complements hi34 in the host, so transformants are selected for their ability to grow on histidine-deficient medium.

Hybridoma ce11 line F5 (Pallas et al., 1986) wu a gift from Carol Prives and

Marcel Bastin. and was used to produce monoclonal antibody against polyomavirus large

T antigen for irnmunoblotting and irnmunoaffinity chromotography. The F5 antibody is a

G, subclass immunoglobulin, and recognizes an 1 1-amino acid synthetic peptide, Leu-

Leu-His-Pro-Asp-Lys-Gly-G1y-Ser-His-Ala, corresponding to the putative F5 epitopr in polyomavirus large T antigen (arnino acid residues 40-50) (Wang and Prives, 1991). To prepare viral large T antigen, mouse 3T6 cells (ATCC) were infected with polyomavirus strain modori-AT3 (Bertin et al., 1993). Nuclear extracts of infected ceils were prepared according to a previously documented procedure (Sunstrom, 1991).

Insect ce11 line SB fiom Spodoptera hgiperda was used to generate stocks of a recombinant baculovirus vEV5 1LT that produces polyomavirus large T antigen (Rice et al., 1987). Both SB and vEV5 1LT were kindly provided by Marcel Bastin. Another

75 insect ce11 strain, High Five cells, a gift fiom Fernando Congote, was used to express polyornavirus large T antigen (Richardson, 1995).

Escherichia coli strain DH5aF' F'lenaX l hsdR I 7 (rim,') supE4.l thi-l recA1 gyrA (Na17 reZA l A(7ucZYA-argF)LI1 69 deo R (~80dlacA(ZucZ)MI S)] was used for cloning purposes and plasmid propagation. Competent cells were prepared as previously descnbed (Sambrook et al., 1989).

2. Media and growth coaditions

Pichia pastoris cells were grown on Yeast Peptone Dextrose (YPD)medium (1% yeast extract, 2% Bacto-peptone, and 2% dextrose) or Yeast Minimal Medium (1.34% yeast nitrogen base [DIFCO] without amino acids) as described (Sherman et al., 1986).

1% dextrose, 1% glycerol, or 0.5% methanol were added as carbon sources to minimal medium. and L-histidine was added to a final concentration of 0.004% when required.

Large T antigen-expressing yeast cells were cultured in a buffered glycernl-complex medium (BMGY)which contains 100 mM potassium phosphate, pH 6.0, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 4x10"% biotin, and 1% glycerol.

Induction of protein expression was done in a buffered rnethanol-complex medium

(BMMY) which is the same as BMGY but contains 0.5% methanol instead of 1% glycerol. Media were solidified by addition of 1.52% agar. Pichia pa.smris was grown at 28'-30°C. Doubling the of log phase Pichia pasturis in YPD is -2 hours. Ce11 density was measured by light scattering (One ODW=5x1 0' cells/ml). Hybridoma cells and 3T6 cells were grown at 37OC in Dulbecco Modified Eagle

Medium (DMEM) supplemented with 10% fetal bovine sem. The hybridoma cells were grown and split 1: 10 when ce11 density reached 4x1O6 cellslml; the cells were then incubated until the medium became acidic (yellow) and ce11 death occured (-5 days).

Monoclonal antibodies were harvested from hybridoma ceIl culture supernatant. Sf9 and

High Five cells were cultured ai 27OC in Grace's Insect Media without serurn supplement

(Invitrogen Inc.).

E. coli cells were propagated at 37OC in LB medium consisting of 1% Bacto- trypione, 0.5% yeast extract, 1% NaCl. Ampicillin, when required, was added at a concentration of200 pg/ml. For growth on solid media Bacto-agar was added at 1.5%.

3. Yeast and bacterisl transformation

Yeast cells were transfoned using the spheroplast method as described in the rnanud for Pichia pasloris expression (Invitrogen Inc.) (Cregg, et ai.. 1985; Himen et ai.,

1978). Bacterial tryisfonnations were performed as described (Hanahan. 1993).

4. Screening for yeast transformants expressing polyomavirus large T antigen

His' colonies were isolated aer spheroplast transformation, and were distinguished as Mut' or Mut' according to their growth on both Minimal Dernose medium (minimal medium supplemented with 1% dextrose) and Minimal Methanol medium (minimal medium supplemented with 0.5% methanol). Transformants were then screened for expression of polyomavims large T antigen by Western bloning (see below).

77 Cellular DNA of positive colonies was extracted (Sherman et al., 1986), and equd arnounts (-20 pg) of DNA from each ce11 line were sponed on nitrocellulose membranes afler denaturation by boiling. A "P-labeled and denatured DNA fragment containing the first 340 bp of the large T antigen gene was used to detect homologous sequence in the yeast DNAs by hybridization (Sherman et al., 1986). Radioactivity of each spot was quantitated by exposing the niuocellulose membrane to a storage phosphor screen that was analyzed in a Molecuiar Dynamics Phosphorimager. This quantitative procedure selected for multiple integration events, which usually give higher level expression of heterologous protein (Cregg, et al., 1985; Cregg, et al.. 1989).

II. Nucleic acids

1. DNA Extraction and enzymatic manipulations

Yeast genomic DNA was isolated by a procedure described by Sherman et ai.

(1986). Small scale preparations of plasmid DNA for analytical purposes were prepared from 10 ml overnight cultures of E. coli by the alkaline lysis procedure. Similarly, large scale stocks of plasmid DNA were purified fiom 1000 ml ovemight cultures of E. coli by centrifbgation in cesium chloride-ethidium bromide gradients (Sarnbrook et al., 1989).

Restriction endonuclease digestions of DNA, as well as phosphorylation and dephosphorylation of' DNA, were perfomed according to instructions fkom manufacturers (GIBCO/BRL, Pharmacia, Promega, or New Endand Biolabs). Isolation of desired DNA hgments from agarose gel slices was carried out ushg either the Gene

78 Clean (Bio 101 Inc.) or QIAquick (Qiagen) gel extraction kits or simply by centrifugation through a glus wool plug followed by phenol-chioroform (1 :1) extraction, chloroform extraction and ethanol precipitation. Ligation of isolated DNA fragments was carried out using 3 units of T4 DNA ligase (GIBCOIBRL) in the recommended buffer for a minimum of 12 hrs at 1SOC. Generation of blunt-ended DNA fragments was performed using the Klcnow fragment of E. coli DNA pol 1 (GIBCOBRL). Didtoxynucleotide chain termination sequencing of critical regions in plasmid constmcts was performed with a-["SI~ATP (Amersham) using the T7 Sequencing Kit (Pharmacia) according to the manufacturer's specifications for double-stranded DNA.

2. PCR amplification

Oligonucleotide prirners (Tabl were purchased fiom GSD Oligos, Toronto,

Canada. or GIBCOIBRL. Taq (GIBCO/BRL) or Ph (Stratagene) DNA polymerases were used according to the manufacturer's specifications. Typical 50 pl amplification reactions contained 35 pmoles of each primer. approxirnately 5-10 fmoles of template

DNA. and 0.2 mM each of (IATP, dGTP, dCTP, and dTTP (Pharmacia) in the buffer supplied by the manufacturer of the thermostable DNA polymerase. 30 cycles of 1.5 min at 9rloC, 1.5 min at SOC(sometimes lowered to 40°C in case of high AT content in the primer sequences), and 1.5 min at 72OC were followed by a final 5 min elongation step at

72'~. To internally label PCR products, the concentration of unlabeled dATP was reduced to 40 FM and 0.4 pM a-[32P]d~~P(3000 Ci/mrnol) (Amenham) was added to

# reactions.

79 To rnake end-labeled DNA for DNase 1 footprinting analysis, one of the two pnmers for each PCR was incubated with Y-[~'P]ATP and T4 polynucleotide kinase

(GIBCOBRL) before use in PCR. The primer labeling reaction was usually carried out in 20 pl volume and contained 35 pmoles of primer, 10 pmoles of y-["P]ATP (6000

Ci/mmol), and 15 units of polynucleotide kinase. The reactions were treated by phenol- chloroform (1 :1 ) extraction, and chloroform extraction before use in PCR.

PCR products were purified from 192% agarose gels prior to use as described above, unless the DNAs were biotinylated at one end. Biotinylated PCR products were extracted by chloroform to remove mineral oil before irnmobilization on streptavidin magnetic beads (Boehringer Mannhem) in binding buffer of 10 mM Tris-HCI (pH 7.9, 1 mM EDTA. 100 mM NaCl. Coupling occurs during incubation for 30 min on a tilted rotating wheel at room temperature. Concentrations of beads in a magnetic field were performed on a Magnetic Separation Stand (Promega) for 30-60 seconds. Beads containing bound DNA were washed twice with 10 mM Tris-HC1, 1 rnM EDTA. 1 M

NaCl and stored at 4OC in the washing buffer.

3. Plasrnid constructs

Polyomavirus strain AT3-Modori, generated by oligonucleotide-directed mutagenesis fiom strain AT3 (Bertin et al., 1993), contains four additional restriction endonuclease sites flanking large T antigen binding sites A, B and C (Fig. 16A). These sites were chosen to Uiwduce minimal changes to virai DNA; in particular, no

G(A/G)GGC consensus sequences were altered, and the distances between sites A, B, C

80 and 1/2 were unaltered. Plasmid pGEM-Modori contains AT3-Modori DNA cloned into the EcoR 1 site of plasmid pGEM-3Zf(-) (Promega). Two sets of mutants were generated as follows:

1) Deletion mutants. Plasmid pGEM-Modori was digested with Pst I and the 623- bp fragment containing the ocigin region of polyomavirus DNA (nt 5 179-53 1Y1-490) was cloned into the Pst I site in the polylinker of pGEM3Zf(-), resulting in parent plasmids pGEM-l/ZABC(+) or pGEM-l/ZABC(-), depending on the orientation of the insert. Plasmids containing individual binding sites or combinations of adjacent binding sites were derived from the parent plasmids by restriction cleavage, followed in sorne instances by blunt-ending, then religation. These deletion plasmids were constnicted by

Noelle-Ann Sunstrom and were named to describe the sets of binding sites they contain

(Fig. 16A) (Sunstrom, 1991).

2) Point mutants. Point mutations were introduced into the consensus

G(A/G)GGC binding sequences in each of binding sites A, B, or C within plasrnid pGEM-blodon by John Bertin, as previously described (Bertin et al., 1993). Mutants were named to describe which sites were mutated (Fig. 17A). For exarnple, mAmB specifies a mutant in which the two GAGGC sequences in site A and the two

G(A/G)GGC sequences in site B were mutated. Mutant A was named A1A2 in Bertin et al. (1993).

Using plasmids described above as templates, DNA fragments were made by

PCR. 1) Fragments containing the wild type ongin or deletion mutants were made from pGEM-1/2ABC and its derivatives by using Ml 3 universal primer and M13 reverse

81 Table 1. LW of deoryribonucleotide prirners

NAME SEQUENCE'

LT / 5' CCGGGGATCCTCGAGAAAAGAGA GATGGATAGAGTTCTGAGCAGA

LT 1 CfrlO I CTCCTCAGTTCCTCGCTC

LT 1 Apa 1 CCACCCTGTGTGTGTACA

TGTATTC

M 13 universal GTAAAACGACGGCCAGT

CAGGAAACAGCTATGAC

T7 AGTGAATTGTAATACGAC

Py 230 GTTCTAGCAGCCTTTCTTTG

Py (AT?):53-35

I 1 CTGCTTAATACTAAAAAAAACA 1 Biotin-Py j267 1 Biotin-GTGTGGmGCAAGAGGMG C CTGCAGTAA TACGACTCACTA TAGGGC- T7 GACCCCCCATAGTTGTCTGGGAA

'Al1 primers are written in the St+3' direction. Relevant restriction endonuclease sites are underlined. Bold letters indicate starting or terminating codon of large T antigen gene. lower case letters indicate inserted nucleotides. Italicized letters indicate T7 promoter. 'Source (letter) and position (nt numbers) of primer according to accession nurnber listed: LT (A3), large T antigen gene of A3 strain; pGEM, plasmid firom Promega; Py (AT3), AT3 strain of polyomavirus. nt numbers are written in the 5'+3' direction. primer (Table 1) (primer 1 and primer 2 in Fig. 16A). These fragments ranged in size from 736 bp (wild type) to 147 bp (site A alone). DNA products were intemally labeled by incorporating (~-"P)~A?Pduring PCR. ïhese DNAs were used in filter binding assays. 2) A set of 265-bp DNA fragments containhg the wild type origin or mutated binding sites were made from plasrnid pGEM-Modori and its derivatives by using primer

Py 220, and primer Py 5267 (Table 1) (primer 3 and primer 1 in Fig. 17A). DNAs were either intemally labeled as descnbed above, or end-labeled at nt 5267 (methods described above). These DNAs were used for filter binding assays. for gel mobility shifi assays, and for DNase 1 footprinting assays. Al1 PCR-generated DNA fragments were purified and were quantitated by measurement of radioactivity.

III. Protein analysis

1. Western blotting and silver staining

Western bloning analysis was based on a procedure described by Towbin et al.

( 1979). Either polyclonal antibody 3e 1 (Sunstrom. 199 1) or monoclonal antibody F5 were used for the assays. Silver staining protocol was adapted fiom previously described procedures (De Moreno et al., 1985; Momssey, 198 1).

2. ImrnunoaffMy column preparation

The F5 hybridoma ce11 culture supematant was harvested and centrifuged at 3000 rpm for 10 min at room temperature. 500 ml of the supematant was then passed through

83 a 10 ml Protein A Sepharose CL-4B (Pharmacia) column at 4'~. The column was

washed with 100 ml 50 mM Tris-HCI (pH 7.0), 50 mM NaCI, 0.3% NaN, at OC, and the

F5 antibody retained by the column was eluted at 4'~with 20 ml of a 50 mM glycine (pH

2S), 150 mM NaCl solution. The purified antibody was concentrated either by dialysis

against PEG2000 at 4'~or by centrifugation in a 15 ml concentrator with a 30 kD

molecular weight cut-off (Amicon). Conjugations of the antibody to CNBr-activated

Sepharose 48 (Pharmacia), or Afigel-10 (Bio-Rad) were performed by recommended

protocols from manufacturers of the beads. Briefly, 20 mg of concentrated F5 antibody

@re-dialyzed against coupling buffer: 100 rnM NaHCO, [pH 8.31, 0.5 M NaCI for

Sepharose 4B; 100 mM NaHCO, [pH 7.51 for Afigel-1 O) was incubated with either 1.5 g

of Sepharose 48 (swollen and washed with 1 mM HCI before incubation) or 10 ml of

Affigel-1 O beads (washed with distilled water) at J°C ovemight (Sepharose 48) or for 4

hn (Affigel-IO). For Sepharose 4B, the mixture was filtered with a sintered glas filter, the gel was washed with coupling buffer, and soaked in 0.1 M Tris-HCI (pH 8.0) to block the remaining active groups on the beads. For Afigel-10, 1 M ethanolamine (pH 8.0)

was added to the mixture (0.1 ml per ml of gel) to block the remaining active groups.

Both antibody-conjugated beads were washed extensively with 10 mM Tris-HCl (pH

7.9, 140 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA before use.

3. Expression and purification of polyomavirus large T antigen

Yeast Pichia posroris transfomants E-3 (His'Muf) or E-51 (His'Mut3 (Chapter

3) were used to express polyomavirus large T antigen. Typically, a 5-ml YPD culture of

84 E-51 cells was inoculated into 500 ml BMGY medium; the cells were grown at 28°-300C in a shaking incubator (250-300 rpm) until the ce11 density reached an OD,=2-4

(approximately 12- 15 hrs) and the cells were in log-phase growth. Cells were harvested by cenuifuging at 1500 rpm for 10 min at room temperature. The ce11 pellet was resuspended in 1000 ml of BMMY medium to induce expression. When strain E-3 was usrd. culture was startrd in 1000 ml BMGY media and subsequently transfmed to 200 ml BMMY media, because E-3 cells grow slowly in BMMY medium. 5 ml methanol was added per liter BMMY culture (final concentration of 0.5% rnethanol) every 12 hrs to maintain induction. After incubation for 60 hrs, cells were harvested and centrifuged at 2000 rpm for 10 min. Ce11 pellets were washed twice with breaking buffer (50 mM

NaH,PO, [pH 7.4],200mM NaCI, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol. and 5% glycerol). Cells were resuspended in 120 ml breaking buffer and were broken by votexing with equal volume of glass beads (0.5 mm diameter). Ce11 lysates were separated from the glass beads by centrifugation at 500 rpm at J°C for 2 min, and further clarified by centrifugation at 10,000 rpm at 4OC for 20 min.

Al1 of the purification procedures were performed at 4OC. 23 ml of yeast ce11 lysate was diluted 1 :2 in loading buffer (10 mM Tris-HCI [pH 7.51, 140 mM NaCI, 1 mM dithiothreitol, 1 mM EDTA. 0.1% Triton X-100) and incubated with 10 ml of F5 monoclonal antibody-conjugated Sepharose/Afigel- 1O beads for 3 hr. Beads were collected by centrifugation at 500 rprn for 5 min, and then wûshed in conical tubes sequentially with 50 mi loading buffer, 50 ml loading baer without Triton X-100

(twice), and 20 ml of 50 mM Tris-HC1 (pH 6.8). AAerwards, the beads were loaded into

85 two 10-ml plastic columns, and each column was Merwashed with 10 ml of 50 mM

Tris-HCl (pH 8.0), 100 rnM NaCl, 1 mM EDTA, 1 rnM dithiothreitol, 20% glycerol.

Large T antigen was eluted f'rom washed beads with 20 mVcolumn of 50 mM Tris-NaOH

(pH Il), 500 rnM NaCI, 1 mM EDTA, 1 rnM dithiothreitol, and 20% glycerol. The eluate was immediately neutralized by adding 10 ml of 1 M Tris-HC1 @H 6.8), and dialyzed against 10 mM potassium phosphate ((pH 7.0), 50 mM NaCI, 1 nM EDTA, 1 mM dithiothreitol (DTT),20% glyceml. Large T antigen was concentrated From 50 ml to about 5- 10 ml by centrifugation in 15-ml concentrators with 30 kD molecular weight cut- off (Amicon), and was the predominant protein species when analyzed by gel electrophoresis afler purification. The quantity of large T antigen was determined both by cornparison to protein standards visualized on silver-stained gels and by colorimetnc anaiysis. which is based on the color change of Coomassie brilliant blue G-250 dye in response to various concentrations of protein. Large T antisen concentrations were measured by cornparison to a standard cwe set up by using bovine serum albumin.

Approximately 1 mg of purified large T mtigen was obtained from 500 ml BMGY culture of E-51, or 1000 ml BMGY culture of E-3. Purified large T antigen was aored at

-70°C in the dialysis buffer descnbed above.

When large T antigen was purified fiom baculovinis-infected cells (Rice et al.,

1987), recombinant baculovirus vEV5lLT stock was generated in Sf9 cells. Large T antigen was expressed in High Five cells (Richardson, 1995) according to the

Baculovirus Expression Manual fiom Invitrogen Inc. and published methods (Rice et al.,

1987; Richardson, 1995). The High Five cells were infected with an multiplicity of

86 infection (MOI) of 5. After incubation for 48 hrs, cells were scraped fiom plates and centrifuged at 1O00 rpm for 10 min at 4OC. Ce11 pellets were resuspended in lysis buffer

(10 mM Na,HPO,, 2 mM KH2P0,, [pH 7.41, 140 rnM NaCl, 3 mM KCI, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100) in a concentration of 1o6 cells per

100 pl buffer, and incubated on ice for 45 min (vortexing at 10 min intervals to assist

Iysis). Cellular debris was pelleted by centrifugation at 1000 rpm for 10 min at -toc,and supernatant was used for purification of large T antigen by an imrnunoafinity procedure as described above.

IV. Assays for protein-DNA interactions

1. Filter binding assay

A procedure modified from previously published methods (Borowiec and

Hunvitz 1988; Hinkle and Chamberlin, 1972; Lorimer et al.. 1991) for binding of DNA to nitrocellulose filten was utilized. Purified large T antipen was incubated with "P-

Iabeled DNA fmgrnent at 37OC in 60 pl binding solution containing 50 miM NaCI. 7 mM

MgCl,, 83 pg bovine serum albumin per ml, 1 mM dithiothreitol, 1 mM phenylrnethylsulfonyl fluoride, 10 ng/ml aprotinin, and 17 pg sheared salmon sperm

DNA per ml. Buffers were 50 rnM sodium acetate (pH 5.0-5.6), 50-100 mhf potassium phosphate (pH 6.0-7.6), or 50-100 mM Tris-HCI (pH 7.0-8.5). ATP (Boehringr

Mannheim or Phannacia), when used, was dissolved in distilled water and pH was adjusted with K,HPO, or NaOH. After incubation for 20-25 min, the niunires were

87 filtered by capillary flow through 13 rnrndiameter nitrocellulose filters (Schleicher &

Schuell BA-85, 0.45 mm pore size) that had been boiled in 0.8% sodium dodecyl sulfate, washed in water, and presoaked in washing buffer (5 mM MgCl,, 100 mM NaCl, and SU mM sodium acetate, potassium phosphate, or Tris-HCl adjusted to the pH corresponding to those used for binding reactions). The filters were then washed with 2 ml of washing buffer, dried, and bound radioactivity was measured by liquid scintillation counting.

Bound DNA was expressed as the percent of input radioactivity remaining bound to filters.

2. DNase 1 footprinting analysis

Binding reactions were carried out in the same buffers used for the filter binding assays. In a volume of 70 pl, 0.4-0.6 pg of large T antigen and 20 fmol of labeled origin

DNA were incubated for 20 min at 37'C at pH 7.0-7.4 (Tris-HC1 buffer) or pH 6.0

(potassium phosphate buffer). 1 mM ATP was included in the reaction when indicated.

The reaction was then added to 5 pl of 5 mM CaC1,. 10 mM MgCl,, 10 pgml sheared salmon sperm DNA. After 1 min at room temperature, 0.02-0.05 unit of DNase I

(GIBCO/BRL) was added. Digestion was allowed to proceed for 1 min at room temperature and was terminated by adding 70 pl of 100 rnM EDTA. 2 M ammonium acetate, 0.2% sodium dodecyl sulfate, and 100 pg/ml calf thymus DNA. DNA was extracted with phenoVchloroform and subjected to electrophoresis on 12% polyacrylamide-8M urea gels, which were dried and exposed to X-ray film or to storage phosphor screens that were amiyzed in a Molecdar Dynamics Phosphorimager.

88 In case of solid phase DNase 1 footprinting (Sandaltzopoulos and Becker, 1994;

Sandaltzopoulos et al., 1995), DNAs were purified and immobilized on magnetic beads as descnbed in the "PCR amplification" section. Large T antigen binding reactions were carried out as descnbed above. DNase 1 digestion was terminated by addition of an equal volume of 4 M NaCl, 100 mM EDTA. While still attached to the magnetic beads, the nicked DNA fragments were purified by washing once with 2 M NaC1,20 mM EDTA, and once with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The beads were mixed with 5-8 pl of loading buffer (80% formamide, 10 mM EDTA, 0.05% xylene cyanol, 0.05% bromophenol blue). Samples were denatured for 10 min at 8j°C and loaded on a 12% denaturing polyacrylamide gel. Beads did not intrrfere with the electrophoresis. so their removal before loading was not necessary.

3. Gel electrophoregis mobility shift assay

"P-labeled DNA was incubated with large T antigen at 37'~for 20 min in 60 pl binding solution containing 50 mM potassium phosphate (pH 6.0 or 7.6), 7.5% glycerol, and the other components described above. Where indicated, glutarddehyde was added at a concentration of 0.1% and incubation was continued for 5 more min. Samples were directly loaded ont0 5% polyacrylarnide gels and electrophoresis was carried out in 50 mM potassium phosphate buffer (pH 6.0 or 7.6), 1 mM EDTA. for 1.5-3 hrs at 100 volts.

Gels were dried and exposed to X-ray film.

4. DNA immunoprecipitation assay

89 This DNA binding assay was essentially done as described by McKay (198 1) and

Sunstrom et al. (1 99 1). Bnefly, large T antigen preparations were incubated in a 1 ml volume containing 20 mM phosphate buffer (pH 7.0), 2 mM dithiothreitol, 0.01% bovine serum alburnin, 0.1 M NaCl, 1 mM EDTA, 0.05% Nonidet P-40, 0.008% aprotinin, 1 rnM phenylmethylsulfonyl fluonde and 3% dimethyl sulfoxide. To this mixture was added 15 ng of "P-labeled Hhf I digest of pABC 1?+ and 1 pg of unlabeled calf thymus

DNA. After one hou incubation at 4'C, the large T antigen-DNA complexes were immunoprecipitated by incubation at 4OC for 30 min with 2 pl of polyclonal antibody

3eI, followed by 50 pl of a 50% solution of formalin-fixed Staphylococcus aureus cells

(Calbiochem). The immune complexes were then washed twice with 1 ml of 10 rnM

Tris-HCI (pH 8.0). 150 mM NaCl, 0.5% Nonidet P40. Finally, the DNA was released from the immune complex with 1% SDS, 10 mM EDTA. deproteinized with phenol and chlorofonn. and ethanol precipitated. DNA was analyzed by electrophoresis in a 2% agarose gel. Before autoradiography, agarose gels were irnrnersed in ethano1 for 30 min and dned under vacuum with heating. Gels were exposed to X-ray film.

5. Glycerol gradient centrifugation

A. Protein-DNA complexes: 5 ng of ~-~'~-labeled265-bp DNA Fragment was incubated in the presence or absence of large T antigen in 120 pl of 100 mM potassium phosphate buffer (pH 6, pH 7, or pH 7.6), 50 rnM NaCl, 7 rnM MgCl,, 10 pg/ml bovine serum albumin, 1 mM dithiothreitol, 2 rnM phenylmethylsulfonyl fluoride, 10 nghl aprotinin, and 1 pg sheared calf thymus DNA for 20 min at 37OC. Reactions were then

90 layered ont0 3.8 ml 840% (dw) glycerol gradients containing 100 mM potassium phosphate buffer (pH 6, pH 7, or pH 7.6), 50 mM NaCI, 1 mM dithiothreitol, and 10 ng/mi aprotinin. Gradients were centrifuged in a Beckrnan SW-60 rotor at 30,000-35,000 rpm for 30-60 min at 20°C, and 20 195yl fractions were collected nom the top. The bottom of the centrifuge tube was washed with 195 pl 2% SDS, which was treated as fraction 2 1. Radioactivity in each Fraction was measured with a scintillation counter.

B. Large T antigen aloae: 0.5 pg large T antigen was incubated in the buffers described above; gradients were centrifùged for 30 min at 30.000 rpm. Large T antigen was detected by spotting aliquots of each fraction onto a nitrocelluIose membrane and incubation with polyclonal antibody 3el (Sunstrorn et al., 1991), followed by alkaline phosphatase-conjugated anti-rat antibody (Sigma). Immuno blotting results were quantitated by scanning densitometry and cornparison with results obtained by blotting dilutions of purified Iarge T antigen.

6. Duplex DNA fragment unwinding assay

Unwinding reactions (20 pl) contained 10 mM Tris-HCI @H7.7), 30 mM potassium phosphate (pH 7.7), 7 mM MgCl,, 5 mM ATP, 10 ng/ml aprotinin, 10% glycerol, 1 mM dithiothreitol, 0.8 pg bovine serurn albunirn, 1 ng of labeled polyomavirus origin DNA, 0.8 pg E. cofi single-stranded DNA binding protein

(Pharmacia) and 200 ng purified polyomavirus large T antigen. Mixtures were incubated at 37OC for 50 min. Reactions were terminated by addition of 1 pl 10% SDS, 1 pl 0.5 M

EDTA, and 10 pg proteinase K. Incubation was continued at 37'~for 30 min followed

91 by 55'C for 10 min. Samples were directly loaded ont0 a 6% nondenaturing polyacrylarnide gel and subjected to electrophoresis. CHAPTER 3

Production of Active Polyomavirus Large T Antigen in Yeast Pichia pastork

Yu-Cai Peng and Nicholas H. Acheson

Department of Micro biology and Immunology, McGill Universiy, 3 77.5 University Street, Montreal, Quebec, Canada H3A 2B.l

This chapter is derived fiom a manuscript published in Vims Research, May 1 997,49:4 1-47 ABSTRACT

The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector PHIL-D2.

Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOXI) promoter. Large T antiyn was purifièd by immunoaffnity chromatography. We showed that yeast-derived large T antigen bound specifically to a

DNA fragment that contains the polyornavirus replication origin, protected the four known major binding sites in the origin against DNase 1 digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent marner. This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for ifi vitro studies. INTRODUCTION

Polyornavinis large T antigen is a 780-785 amino acid, multifunctional nuclear phosphoprotein which interacts with cellular regulatory proteins involved in ce11 growth and division, and directs initiation of viral DNA replication (Cowie and Kamen, 1984;

Murakami et al., 1986; Pipas, 1992). Large T antigan binds to polyomavirus DNA at four distinct sites near the replication origin, denoted 112, A, B, and C, by recognizing specific target sequences consisting of the pentanucleotide 5'-G(A/G)GGC-3' (Bondeson et al.,

1995; Cowie and Karnen, 1986) and hrther unwinds the double-stranded DNA (Wang and Prives, 199 1). Binding at site 1/2 is followed by a conformational change that leads to formation of hexamers of large T antigen; the DNA is subsequently unwound and replication of viral DNA is carried out by cellular proteins (Bhattacharyya et al.. 1995).

Binding at sites A, B, and C stimulates DNA replication in vivo (Prives et al.. 1987;

Weichselbraun et al., 1989), probably by concentrating the protein at the replication origin. Recent studies showed that RNA polymerase 11 molecules are blocked just upstream of site A on polyomavirus DNA when large T antigen is bound to this site in infected cells (Bertin et al., 1992; Brabant and Acheson, 1995). Further in vitro studies of

the mechanisms by which large T antigen regulates transcription require large amounts of purified, active large T antigen.

Helper-independent adenovinis (Mansour et al.. 1985; Massie et al., 1986) and baculovinis (Rice et al., 1987) expression systems have been developed which produce 5-

10 fold more large T antigen than do polyomavinis-infecte cells. These recombinant

95 large T antigens are fùnctional, but culturing mammalian cells or insect cells is costiy and timeîonsuming.

The methy 10 trophic yeast Pichia pastoris has been developed as an efficient system for high level production of foreign proteins (Clare et al., 1991; Cregg et al.,

1987; Faber et al., 1995; Sreekrishna et al., 1989). A major advantage of this organism is the ease with which it cm be grown to high cell density in an inexpensive, defined medium. This expression system uses the promoter from the methanol-induced alcohol oxidase gene, AOXl (Koutz et al., 1989). A nurnber of proteins have been expressed, either vicl secretion (Barr et al., 1992) or intracellularly (Clare et al., 1991; Cregg et al.,

1987; Sreekrishna et al., 1989). to exceptionally high levels in Pichiu pastoris. We now report that Pichia prrsforis is capable of providing reasonable quantities of biologically active large T antigen for in vitro studies.

RESULTS

Integration of polyomavinis large T antigen gene into yeast Piclth pastoris genome and expression of the large T intigen

We first attempted to express large T antigen extracellularly by using the S. cerevisiae a-mating type factor sequence as a secretory signal (Brake et al., 1984). An introniess constnict of the large T antigen gene was inserted into vector pPIC9

(Invitrogen Inc.) in such a way that the a-factor cleavage site preceded the initiation

96 codon of large T antigen (Fig. 7A). Recombinant plasmid pPIC9-PyLT was transfected into Pichia pastoris strain GSM(his4) spheroplasts (Cregg et al., 1985). Integration of the large T antigen gene into the yeast genome may be realized via homologous recombination between the transforming DNA (AOXI gene sequences and the His4 gene sequences, Fig. 7C) and regions of homology within the genome (Cregg et al., 1985;

Cregg et ai., 1989). Because there is no ye& origgin of replication in the expression vector, His- transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome.

Colonies able to grow in the absence of histidine were cultured in the presence of methanol and screened by Westem blotting for the ability to secrete large T antigen into the culture medium. No such transformants were found; transformed cells expressed a protein slightly Iarger than large T antigen that was retained in the cells (data not shown), suggesting that the signal sequence was not properly recognized and cleaved.

An invacellular expression cassette lacking the signal sequence was then constructed by transfemng an Xho I fragment containing the large T antigen gcne from pPIC9-PyLT to the vector pHIL-D2 (Invitrogen Inc.) (Fig. 7). Transformants were induced with methanol and ce11 lysates were analyzed by Western blotting using either monoclonal antibody LT 1 (Oncogene Science) or polyclonal antibody 3e 1 (Sunstrom et al., 199 1) against polyomavinis large T antigen. A 100 kDa protein was detected in about one half of 200 transformants. Quantitative dot-blotting analysis (Clare et al., 1991;

Romanos et ai., 199 1) was used to determine the copy number of large T antigen genes in transformants, since high level expression often results fiom multiple tandem integrations

97 Fig. 7. Construction and sequence of plasmid pHIL-D2-FyLT. A) Cloning strategy. The polyomavinis large T antigen coding region was isolated hmplasrnid pADBM5- PYLT (Massie et al., 1986; Sunstrom et al., 1991). Unique Xho l restriction sites were introduced at both ends of the coding region by PCR; short mows show positions of primers. A three-way ligation between PCR-generated end fragments and a long central fragment was used to reduce the chance of introducing mutations into the gene via PCR. Recombinant plasrnid pPIC9-PyLT (not shown) was constructed by introducing the resulting ,Kho I fragment into plasmid pPIC9. The ?(ho I fragment was isolated from this plasmid, blunt-ended, and introduced into EcoR I-cleaved, blunt-ended plasmid pHIL-D2. Appropriate orientation of the large T antigen gene in recombinant plasmids \vas determined and PCR-synthesized linker regions were sequenced. B) Sequence at the ends of the insert. This sequence results from ligation of the blunt-ended EcoR I and Xho I sites, and from introduction by PCR of a (untranslated) signal cleavage site (Glu-Lys- Arg-Glu) upstrearn of the large T antigen ATG. The ATG initiation codon and TGA termination codon of large T antigen are shown in bold letters. C) Structure of expression plasrnid pH1L-D2-PyLT. The intronless coding region of polyomavirus large T antigen was inserted between the 5' AM1 promoter and 5' AOXl transcription termination sequence (TT). The integration of large T antigen gene into yeast chromosome is facilitated by homologous recombination between the AOXl and HIS4 gene sequences and their counterparts in the yeast genome. The expression of large T antigen in yeast cells is driven by the AOXI promoter and is inducible by methanol. 5' AOXl: An -1000 bp fiagrnent containhg the AOXl promoter, also targets plasrnid integration to the AOXI locus; 3'AOXl (TT): Native transcription termination and polyadenylation signai from AOXl gene (-260 bp); HLW Pichia wild-type gene coding for histidinol dehydrogenase (-2.4 kb) and used to complement Pichia his4 strains; 3' AMI: Sequences fiom the AOXI gene that are mer3' to the TT sequences (-650 bp), targets plasmid integration at the AOXl gene; ~mp~:Ampicillin resistance gene; fl ongin: Bacteriophage fl ongin of replication (458 bp). Xho 1 CfrlO 1 Api I Xho I: h - 3-way 1 igation Xbo l 1 Xho l

Inscn into vcctor pPIC 9, transfomi E.coli Dtl-5a. re- digest the amplilied plasmid with Xho 1. isolate PyLT gcnc fragment, blunt ends Digest with EcoR 1. blunt ends

Mix, ligak. tmsforrn lE.coli DH-Sa

Remaining 'A' of the Polyoma T-Ag native AOX I 'ATG' I 5' AOXl

HIS 4 (Clare et al., 1991). We identified transformants E-3 and E-51, both of which contain multiple copies of the large T antigen gene. E-3 lacks an intact AOXl gene and grows slowly in methanol media (His'Muf), while E-51 has an intact AOXl gene and grows well in methanol media (His'Mut'). The expression of large T antigen in both E-3 and E-

51 was detectable by 18 hr fier induction with 1% methmol, and reached maximum levels at about 60 hr. We chose transformant E-3 to cany out analysis and purification of yeast-derived large T antigen in this report.

Specific DNA-bindiag and purification of the yeast-derived hrge T antigen

A modified McKay irnmunoprecipitation assay (McKay, 198 1) was used to determine whether yeast-derived large T antigen has specific DNA binding properties similar to those of large T antigen derived from marnmalian or insect cells. An E-3 ceIl lysate was incubated with "P-labeled fragments of pABC12+ (Sunstrom, 1991), a plasmid which contains a copy of the intergenic region of the polyomavirus genome

inserted into the Pst I site of plasmid pGEM3Zf (0) (Promega). Specific irnrnunoprecipitation of the 762-bp Hinf I fragment containing the intergenic region was observed by autoradiography of agarose gels (Fig. 8), while nothing was precipitated by the yeast lysate transfonned by vector PHIL-Dî alone (data not shown). This result demonstrates that yeast-derived polyomavirus large T antigen has DNA-binding properties similar to those of authentic large T antigen (Lorimer et al., 1991).

Large T antigen was purifid by binding to monoclonal antibody Fj (Pallas et al.,

1986) covaiently cross-linked to Sepharose (Phamacia) or Affi~gel-1 O (Bio-Rad) beads. Fig. 8. Specific binding of yeast-derived polyomavirus large T aatigen to the intergenic region of polyomavirus DNA. Yeast transformant E-3 was grown in

BMGY medium (Clare et al., 1991; Cregg et al., 1987) at 30" in a shaking incubator

(250 rpm) until the culture reached an OD,=2-4 (1 OD, unit = 5x10' cells/rnl). Cells were harvested, resuspended in BMMY medium containing 0.5% methanol (Clare et al.,

1991; Cregg et al., 1987) using 115 of the original culture volume, and incubation was continued for 60 hr. Cells were pelleted and broken by vortexing with acid-washed glas beads in breaking buffer (50 mM Nd2PO4 [pH7.4], 1 mM phenylrnethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol), and the ce11 lysate \vas clarified by centrifugation (10,000 rpm, 20 min, 4OC). Hinf I fragments of plasmid pABC 12+ (Sunstrom, 1991) were end-labeled by incubation with the Klenow fragment of DNA polymerase 1 and [~~'PI~ATP.50 pl of yeast ce11 lysate (representing about

5x10' cells) or 50 pl of a nuclear extract from 0.8~10'polyomavims-infected mouse 3T6 cells (Sunstrom. 1991) were incubated in a total volume of 1 ml with 15 ng of labeled

DNA fragments at 4OC for 1 hr in binding buffer (20 mM potassium phosphate [pH 7.01,

0.01% bovine senim albwnin, 1 mM EDTA. 1 mM dithiothreitol, 0.05% NP-40, 3% dimethyl sulfoxide, 100 mM NaCl, 1 pg/ml sheared salmon sperm DNA, 1 mM phenylmethylsulfonyl fluoride, 0.008% aprotinin), followed by incubation with 2 ~1 polyclonal antisem for 30 min (MacKay, 1981). Fixed Staphylococcus aureus cells were used to precipitate antibody-bound DNA. DNA was solubilized and analysed by electrophoresis in an agarose gel (Sunstrom et al., 1991). Lane 1 represents 4% of input

DNA; lanes 2 and 3 contain DNA fragments bound by the E-3 ce11 lysate and the polyomavinis-infected 3T6 ce11 extract, respectively. The numbers to the left of the autoradiograph show lengths, in nt, of the Hinf I fragments of the plasmid pABC 12+.

Fig. 9. Analysis of immunoaffmity-purified large T antigen by Western blotting and silver staining. An E-3 ce11 lysate was diluted 1 :2 in loading buffer (10 mM Tris-HCl

[pH 7.51, 140 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.1% Triton X-100) and incubated with F5 (Pallas et al., 1986) monoclonal antibody-conjugated Sepharose beads for 3 hr at 4OC. Beads were then washed sequentiaily with loading buffer, loading buffer ivithout Triton ,Y-100 (nvice), 50 rnM Tris-HCI (pH 6.5), and finally widi 50 mM Tris-

HC1 (pH &O), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol. An oliquot of the beads (lane 1) was reserved and analysed by electrophoresis on an 8% polyacrylamide gel in Tris-glycine buffer (Ausubel et al., 1988) at 50 volts for 3 hr.

Large T antigen was eluted from washed beads with 20 mM Tris-HCl (pH 8S), 500 mM

NaCl, 1 mM dithiothreitol, 1 mM EDTA, 20% glycerol, containing 80 ~gmlof peptide

Leu-Leu-His-Pro-Asp-L ys-Gly-Gly-Ser-His-Al (Wang et al., 199 1 ) (lane 2); or with 50 mM Tris-NaOH (pH 1 l), 500 mM NaCI, 1 mM EDTA. 1 mM dithiothreitol. 20% glycerol (lanes 3 and 4), from identical aliquots of beads. Lanes 1-3: proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and subjecied to Western blotting analysis (Towbin et al., 1979) using polyclonal antibody 3e 1 (Sunstrom. 199 1 ).

Lane 4: proteins (andysed on an identical gel run simuitaneously) were directly visualized by silver staining (De Moreno et al., 1985). HC, heavy chah of mouse IgG conjugated on Sepharose beads.

Elution with a peptide conesponding to amino acids 40-50 of large T antigen (Wang and

Prives, 199 1) (lane 2 in Fig. 9) was less efficient than elution with a pH 11 buffer (Dixon et al., 1985) (lane 3 and lane 4); however, large T antigen eluted by both methods had equivalent DNA-binding activity (not shown). Elution with 50% ethylene glycol was also less efficient than with pH 11 buf5er (not shown). Al1 assays descnbed below were carried out with large T antigen eluted with pH 1 1 buffer. Eluates were concentrated with a 15-ml Centriplus concentrator (30 kD cut-off, Amicon). Final concentration of large T antigen was estimated to be 10-30 ngml by colorimetric analysis or by cornparison with

Coomassie brilliant blue-stained standards.

We performed DNase 1 footprinting assays with purilied large T antigen, using a labeled DNA fragment covering the polyomavirus intergenic region (Fig. 10). Sites 112,

Al B, and C were protected against DNase 1 digestion as previously shown (Lorimer et al. 991). The arnount of large T antigen required to protect its binding sites against

DNase treatment (0.6 pg for 6 ng DNA) is very similar to the values previously reported using baculovirus-produced large T antigen (0.75 yg for 7 ng DNA; Lorimer et al.. 1991).

Purified large T antigen unwinds duplex DNA fragment

We also perfomed a DNA unwinding assay with purified large T antigen. A labeled double-stranded DNA fragment containing the polyomavirus replication ongin was incubated with large T antigen in the presence of E. coli single-stranded DNA- binding protein (Goetz et al., 1988; Wang and Prives, 1991). The deproteinized DNA was then analyzed by electrophoresis in a polyacrylamide gel. Incubation with large T Fig. 10. DNase 1 footprinting reveals protection of large T antigen binding sites by yeast-derived large T antigen. 6 ng of a 6 19-nt DNA fragment including polyomavirus nt 5267-5312/1-490, generated by PCR and labeled at the 5' end at nt 5267 with "P, was incubated for 30 min at 37OC with O or 0.6 pg of immunopurified large T antigen in 100 pl of 50 mM Tris-HC1 (pH 7.9, 7 mM MgCl,, 100 mM NaCl, 4 mi! ATP, 1 mM dithiothreitol, 10 ng/ml aprotinin, 2 mCi phènylmcthylsulfonyl fluoride, 3 pg bovine serum albumin and 1 pg sheared salmon sperm DNA. The solution was added to 100 pl of 5 mM CaCII, 10 mM MgCl?, 10 pg/ml sheared salmon sperm DNA and incubated for

1 min at roorn temperature. 0.034 unit of DNase I was added and after 1 min at room temperature, the digestion was terminated by adding 200 pl of 200 mM NaCl, 50 mM

EDTA, 1% sodium dodecyl sulphate, 100 @ml tRNA. DNA was extracted with phenoVchloroform and precipitated with ethanol. The pellets were washed with 70% ethanol. dried, dissolved in 80% deionized focmamide, 10 mM EDTA. and trace arnounts of bromophenol blue and xylene cyanol. and subjected to electrophoresis on a 12% polyacry lamide-8 h.1 urea gel. The gel was dried and autoradiographed. M. molecula. weight markers.

Fig. 11. Yeast-derived large T antigen unwinds duplex DNA fragment. 1 ng of a

265-nt DNA fragment including polyomavims nt 5267-53 1211-220, generated by PCR and labeled at the 5' end at nt 5267 with "P, was incubated for 50 min at 37'~ with 0.8 pg

E. coli single-stranded DNA binding protein (Pharmacia), and O (lane 1) or 200 ng (Iane 2 and 3) irnmunopurified large T antigen in 20 pl of 10 mM Tris-HCI (pH 7.7), 30 rnM potassium phosphate (pH 7.7), 7 miLI MgCl,, 10 @ml aprotinin, 10% glycerol, 1 mi dithiothreitol, 0.8 pg bovine serurn albumin. Incubation was in the presence (lane 1,3) or absence (lane 2) of 5 mM ATP. Reactions were terminated by addition of 1 pl 10% SDS,

1 pl 0.5 M EDTA, and 10 pg proteinase K. Incubation was continued at 37'C for 30 min followed by 5j°C for 10 min. Sarnples were directly loaded ont0 a 6% nondenaturing polyacrylamide gel and subjected to eiectrophoresis; DNA bands were visualized by autondiography. Denat.: DNA was heat-denatured before loading on the same gel, but the lane was cut and juxtapositioned next to the other lanes. = ++ LT Ag +-+ ATP antigen in the presence, but not in the absence, of ATP led to unwinding of a portion of the input DNA and its migration as single-stranded DNA (Fig. 11). Thus polyomavims large T antigen purified fiom Pichia pastoris cm specifically bind to DNA containing target G(A/G)GGC pentanucleotides, can protect its binding sites on DNA from DNase 1 digestion, and cm unwind double-stranded DNA, three biological activities show by iarge T antigen made in mammalian or insect ceils.

DISCUSSION

Pichin pastoris expression systems have been show to produce high levels (0.1 -

10 g/liter) of a variety of different foreign proteins (Barr et al., 1992; Clare et al., 1991 ;

Cregg et al., 1987; Faber et al.. 1995; Koutt et al., 1989; Sreekrishna et al., 1989). The level of expression of polyomavinis large T antigen that we attained is far lower than those reported for other proteins. Generally, 0.5-1 mg large T antigen was purified from

1 liter of yeast culture. This is comparable to yields reported fiom bacuIovirus-infected insect cells (cl mg per liter) (Rice et al., 1987; Lorimer et al., 1991), and is some 50-fold higher than we obtained from polyomavims-infected 3T6 cells (not shown). About one haif of the expressed large T antigen was iost in the pellet &er ce11 lysis and about 25% of large T antigen bound to antibody-conjugated beads could not be eluted even with the pH 11 buffer (data not shown). Given that large T antigen is a multifunctional regdatory protein, hi& level expression of it may be toxic to the cells (Romanos et ai., 1992). The

105 propagation of Pichia pastoris is easy and much less expensive than the propagation and infection of insect ce11 cultures. It may be possible to scale up expression and make large amounts of purified protein suitable for structural studies. CHAPTER 4

Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Yu-Cai Peng and Nicholas H. Acheson

Department of ~Cficrobiologyand Imrniinology, McGill Universi& 3775 University Street, Monfreol, Quebec. Canada

This chapter is derived fiom a manuscript published in Journal of Viroiogy, September l998,72:7330-7340 CONNECTING TEXT

In the preceding chapter, we described an simple and inexpensive method for production and purification of polyomavirus large T antigen. We also showed that the

Pichia-derived large T antigen binds to viral origin DNA and protects the four binding sites 12, A, B, and C from DNasr 1 digestion. Since specific DNA binding by large T antign is affected by many factors, as shown by previous studies, we asked what would be the optimal conditions for our large T antigen to bind to origin-containing DNA. In this chapter, we describe the characterization of the effect of pH and ATP on large T antigen DNA binding using three different DNA-binding assays (filter binding, DNase 1 footprinting, and gel retardation). We then proceed to carry out a systematic study of binding to origin mutants lacking one or more binding sites, to determine whether binding to one site influences binding to neighbonng or distant sites. Polyomavims large T antigen binds to multiple 5'-G(A/G)GGC-3'pentanucieotide sequences in sites 1/2, A, B and C within and adjacent to the origin of viral DNA replication on the polyomavinis genome. We asked whether binding of large T antigen to one of these sites cm influence binding io other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4-7.8, ofien used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase 1 footprinting.

Results showed that large T antigen binds cooperatively to its four binding sites in viral

DNA. suggesting that large T antigen bound to one of these sites stabilizes large T antigen bound to oiher sites via protein-protein contacts. Sites A, B and C may therefore augment DNA replication by facilitating binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 112, and ATP specifically enhanced protection against DNase I digestion in the central 10- 12 bp of site 112, where hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-

DNA complex, and Mermore that ATP stimulates their assembly into hexamers at site

112 by a "handover" mechanism mediated by these protein-protein contacts. INTRODUCTION

Polyomavirus large T antigen initiates DNA unwinding and replication via eiaborate interactions with the virai replication origin (Bhattacharyya et ai., 1995).

Specific DNA binding by this 785-arnino acid protein is mediated by a domain that lies between amino acids 282 and 398, as defined by deletion mutants (Sunstrom et al., 1991).

Large T antigen binds to a target consensus pentanucleotide sequence, 5'-G(G1A)GGC-

3', which is present in multiple copies in the replication origin region between the early transcription start site and the transcriptional enhancer (Bondeson et al., 1995; Cowie and

Kamen, 1984, 1986; Dilworth et al., 1984; Pomerantz et al., 1983). Immunoprecipitation and DNase 1 protection assays showed that four distinct sites on polyomavirus DNA, denoted 1/2, A, B, and C, are bound by large T antigen in vitro (Cowie and Kamen. 1984;

Dilworth et al.. 1984; Pomerantz et al., 1983). Site 1/2, whict is situated within the core origin of DNA replication (Hendrickson et al., 1987; Katinka and Yaniv, 1983; Luthman et al., 1982: Muller et al., 1983; Prives et al., 1987), contains four closely-spaced consensus pentanucleotide sequences arranged symmetrically as two partly overlapping pairs on opposite DNA strands (Cowie and Karnen, 1986; Dailey and Basilico, 1985;

Pomerantz and Hassell, 1984; Schelier and Prives, 1985). Sites A, B, and C are located between the core replication origin and the eariy transcnption unit. These sites contain respectively two, two, and four target pentanucieotide sequences in polyomaWus strain

A3 and its derivatives (Bertin et al., 1993; Dailey and Basilico, 1985; Prives et al., 1987;

Triezenberg and Folk, 1984). Adjacent pentanucleotides are spaced approximately one

I IO tum of the DNA helix apart in each of these three sites, implying that large T antigen molecules bound to adjacent pentanucleotides are aligned on one side of the helix.

Mutagenesis and methylation interference experiments showed that binding of large T antigen to adjacent pentanucleotides within a given site is cooperative, as removal of one pentanucleotide sequence fiom a site contairing three sequences reduced binding afT~nity by a factor of 10 (Colvie and Kamen, 1956). Large T antigen of the closely related simian virus 40 shares extensive sequence homology with its polyornavirus couterpart, and also recognizes G(A1G)GGC pentanucleotide sequences on DNA (Pipas, 1992;

Pomerantz and Hassell, 1984).

Large T antigen molecules can oligomenze; most preparations contain varying amounts of monomers, dimers, trimers, tetramen. and hexamers in solution (Bradley et al.. 1982; Dean et al., 1992; Gidoni et al.. 1982; Wang and Prives. 1991a). Incubation with ATP stimulates hexarner formation (Dean et al., 1993; Wang and Prives, 1991a), presumably by inducing a conformational change in large T antigen. In the presence of

ATP, two hexamers of simian virus 40 large T antigen assemble on viral DNA at site II in the simian virus 40 replication origin (Dean et al., 1987c; Dean et al., 1992; Mastrangelo et al., 1989; Parsons and Tegtmeyer, 1992; Wessel et ai., 1992b); each hexarner is ceniered on one of the pairs of closely-spaced G(A/G)GGC sequences in site II (Parsons and Tegtmeyer, 1992). It has been posnilated, but not show directly, that hexamers of polyomavirus large T antigen also assemble at the analogous site 112 on polyomavinis

DNA. Hexamers are circular structures that enclose the DNA like a wheel about an axle

(San Martin et al., 1997; Wessel et al., 1992b). Large T antigen hexamers unwuid DNA

111 in the replication origin, leading to the initiation of bidirectiond DNA replication by cellular DNA polymerase a:primase, which is bmught to the origin by interaction with large T antigen (Moses and Prives, 1994; Murakami and Hurwitz, 1993b).

What is the role of polyomavinis large T antigen binding sites A. B and C in viral

DNA replication? Although these sites are not absolutely required to direct large T antipn-mèdiated DNA replication in vivo (Bertin et al., 1993; Muller et al., 1983;

Weichselbraun et al., 1989), their presence augments DNA replication in transfected plasmids (Weichselbraun et al., 1989) and is required for optimal virus replication in permissive mouse cells (Bertin et al., 1993). Large T antigen bound more strongly to sites A, B and C than to site 1/2 in the absence of ATP (Cowie and Kamen. 1984;

Dilworth et al., 1984; Pomerantz et al., 1983). but ATP strongly increased the afinity of

large T antigen for DNAs containing site 1/2 (Lorimer et al.. 1991), probably by stimulating formation of hexamers.

We decided to re-examine the binding of large T antigen to its multiple sites in the replication origin region of polyomavirus DNA. by use of point and deletion mutants in target DNA hgments. In the course of setting up DNA binding assays, we also studied the effects of pH and of ATP on binding. We found that specific binding to DNA is strong and stable at pH 7 and below, but is weaker and reversible above pH 7.4; that ATP

stabilizes binding of large T antigen to DNAs that contain site 112; and that in the presence of ATP, large T antigen preferentially protects the central 10-12 nt of site 1/2 against DNase 1 digestion. Using a variety of DNA binding conditions, we found that

large T antigen binds cooperatively to its multiple sites in the replication origin. These

112 observations suggest a mode1 in which the assembly of hexamers of large T antigen at the replication origin is facilitated by "handover" of reversibly-bound large T antigen molecules from sites A, B and C to site 112.

RESULTS

Specific origin-binding activity of polyomavirus large T aatigen is enhanced at pH 7 and below

We wanted to quantitate binding of purified polyomavirus large T antigen to

DNA fragments containing the multiple G(A/G)GGC consensus pentanucleotide sequences present in the region of the polyomavims replication origin. Binding of protein-DNA complexes to nitrocellulose filters is a simple. rapid and easily quantifiable method of measunng protein-DNA binding (Borowiec and Hurwitz 1988b; Deb and

Tegtmeyer. 1987; Hinkle and Chamberlin, 1972; Lorimer et al., 1991). We simplified the binding solution to include only NaCl, MgCl,, a reducing agent and a buffer? in addition to protease inhibitors and nonspecific cornpetitor DNA (see Materials and Methods). To characterize our DNA binding assay, we exarnined the influence of different components in the binding reaction. nie pH of the solution was varied between 5.0 and 8.5 by use of different buffers, and filter binding of a 736-bp "P-labeled polyomavinis origin DNA fragment containing the four large T antigen binding sites 112, A, B, and C (Bertin et al.,

1993; Cowie and Kamen, 1984) was determined in the presence or absence of large T

113 antigen (Fig. 12A). Optimal binding was observed between pH 5.6 and 7.0, with a maximum at pH 6.0, and binding activity fell off sharply at pH 7.6 or higher.

Previous DNA binding assays of polyomavirus or simian virus 40 large T antigen were c'mied out at various pH values between 7 and 8 (Borowiec and Hurwitz, 1988b;

Deb and Tegtmeyer, 1987; Lorimer et al., 1991; Mastrangelo et ai., 1989; Pomerantz and hassell, 1984; Pomerantz et al.. 1983; Scheller and Prives, 1985; Vogt et al., 1986).

Although differences in binding to nonspecific (Dom et al., 1982; Montenarh and

Henning, 1982; Oren et al., 1980) or specific DNAs (Deb adTegtmeyer, 1987) were noted, no systematic study of the variation of DNA binding with pH has been published.

Since polyomavirus large T antigen purified from Pichia pastoris had not been previously characterized. we asked whether increased DNA binding below pH 7 was peculiar to this source. We therefore purified polyomavirus large T antigen made in insect cells by a recombinant baculovirus (Rice et al., 1987) which has been used extensively in other studies (Bhattacharyya et al., 1995; Lorimer et al.. 1991; Maton et al., 1995: Wang and Prives 199 la, 1991 b), and measured its DNA binding activity as a function of pH; the results (not shown) were similar to those shown in Figure 12A To deterrnine whether increased binding at low pH is specific for DNA containing large T antigen binding sites, we performed filter binding assays with DNAs either containing or lacking G(A/G)GGC consensus sequences. The results showed that binding to nonspecific DNA increased as the pH was lowered, but remained less than 2% of input

DNA at pH 7.0, and less than 10% of input DNA at pH 6.0, under conditions where 70 to

90 % of specific DNA was bound (Fig. 12B). Furthemore, we canied out DNase 1

114 Fig. 12. Effect of pH on DNA-binding activity of polyornavinis large T antigen. A)

Binding to nitrocellulose filten of 3 ng "P-labeled 736-bp DNA fragment containing the wild type polyornavinis origin region was carried out in 50 mM baen of different pH

(pH 5.0-5.6: sodium acetate; pH 6.0-7.6: potassium phosphate; pH 8.0-8.5: Tris-HCI) after incubation in the presence (black bars) or absence (gray bars) of 100 ng large T

ÿntigrn. B) Binding of large T antigsn to nonspecific DNA remains iimited at low pH.

100 ng large T antigen was incubated with the 736-bp DNA fragment (black bars) or with

DNA lacking binding sites (gray bars), and retention of radioactive DNA on filters was determined. C) The same DNA fiagrnent was incubated with increasing arnounts of large T antigen in potassium phosphate buffer at pH 6.0 (triangles) or pH 7.5 (squares).

Radioactivity bound to filters in the absence of large T antigen was subtracted from results to give corrected specific binding values. O no LT Ag LTAg O nonspecific DNA B Ispecific DNA - footpnnting assays at different pH values, and found that binding of large T antigen to origin DNA at pH values between 6.0 and 7.6 gave discrete footprints (see below) similar to those previously reported (Cowie and Kamen, 1984; Lorimer et al., 199 1 ; Marton et al., 1995), although much higher concentrations of large T antigen were needed at pH 7.4 and above than at lower pH values.

Figure 12C shows that as little as 5 ng of large T antigen per 60 pl reaction was sufficient to bind to a fraction (12%) of a Iabeled polyomavirus origin DNA fragment when binding was carried out at pH 6.0, and binding was maximal at about 100 ng of large T antigen per reaction. In contrast, binding at pH 7.5 required substantially higher concentrations of large T antigen. The difference in binding finity at pH 6.0 compared with that at pH 7.5 was estimated, from the difference in the initial dopes of the two curves, to be 10- to 20-fold. Clearly, the effect of pH on DNA binding by polyomavirus large T antigen is important, and must be taken into account when carrying out binding studies,

Large T antigen-DNA complexes are stable nt pH6-7, but are unstable at pH 7.6

In previous reports, DNA binding by polyomavirus or simian virus 40 large T antigen could be detected by gel mobility shifi assays only after fixation of protein-DNA complexes with glutaraldehyde (Dean et al., 1987~;McVey et al., 1996; Murakami and

Hurwitz, 1993b). The enhanced binding that we found at Iow pH encouraged us to try

DNA band retardation with unfixed complexes using a pH 6.0 bufTer during electrophoresis. A 265-bp, 32~-labeledDNA fiagrnent contauiing al1 four binding sites

116 was incubated with large T antigen in binding solutions containing potassium phosphate buffer at pH 6.0 or 7.6, and reactions were loaded onto a 5% polyacrylamide gel and subjected to electrophoresis in pH 6 buffer (Fig. 13A). Men the binding reaction was carried out at pH 6 (lane 2), dl of the radioactive DNA migrated as a band very close to the position of the loading well. Pnor fixation of DNA-protein complexes by glutaraidehyde (lane 3) had no effect on the migration of these complexes. When the binding reaction was carried out at pH 7.6 (lane 5), surprisingly, the migration of al1 of the DNA was also retarded. However, pnor fixation of these complexes with glutaraldehyde resulted in a very small proportion of the input DNA being present in the retarded band (lane 6).

We explain these results as foilows: complexes formed at pH 6 between target

DNA and polyomavinis large T antigen are stable for the several houn during which they were subjected to electrophoresis at pH 6, resulting in a retarded DNA band. Complexes form less readily at pH 7.6, but when the pH 7.6 binding reaction was loaded onto the pH

6 gel (lane 5), the pH of the reaction dropped before electrophoresis began, alloning stable DNA-protein complexes Co form at the lowered pH present in the loading well. On the other hand, glutaraldehyde fixation of large T antigen that had remained unbound (or not stably bound) to DNA at pH 7.6 inactivated its ability to bind to DNA. leading io reduced binding after the reaction was loaded ont0 the pH 6 gel (lane 6).

We aiso prepared binding reactions at either pH 6 or pH 7.6 and subsequently loaded the reactions ont0 a 5% polyacrylamide gel run in pH 7.6 buffer (Fig. 138).

When binding was carried out at pH 6, some DNA remained in the retarded band, but

117 Fig. 13. Stability of DNA-protein complexes subjected to eleetrophoresis at pH 6.0 or pH 7.6. A) 1 ng of a "P-labeled 265-bp DNA fragment containing the origin region was incubated without large T antigen (lanes 1 and 4) or in the presence of 100 ng large T antigen (lanes 2, 3, 5, and 6) at 37OC for 20 min, in potassium phosphate buffer at either pH 6.0 (lanes 1-3) or pH 7.6 (Ianes 4-6). Glutaraldehyde (0.1 %) was added to reactions 3 and 6. and incubation was continued for 5 more minutes. Al1 reactions were analyzed on a 5% polyacrylamide gel made and run in potassium phosphate buffer, pH 6.0. B) 1 ng of the same DNA fragment was incubated without large T antigen (lanes 1 and 4) or with

50 ng (lanes 2 and 5) or 100 ng (lanes 3 and 6) large T antigen. at pH 6.0 (lanes 1-3) or pH 7.6 (Ianes 4-6). Electrophoresis was carried out in potassium phosphate bufTer, pH

7.6. pH6.0 pH7.6

O 0.1 0.1 O 0.1 0.1 LT(pg)

œ 9 + g + Glu. pH6.0 pH7.6

O 50 100 O 50 100 LT (ng) most of the DNA-protein complexes dissociated during electrophoresis, leading to a smear of radioactive DNA between the position of fkee and bound DNA (lanes 2 and 3).

Very Iittle retarded DNA was seen when binding was camied out at pH 7.6 (lanes 5 and

6). These results show that much of the large T antigen initially bound to DNA at pH 6 dissociated when the complexes were exposed to pH 7.6. nierefore, we conclude that large T antigen-DNA complexes are stable at pH 6 but are unstable at pH 7.6. dissociating upon extended incubation at that pH in the absence of free large T antigen.

The increased stability of large T antigen-DNA complexes at low pH may explain the more efficient binding of this protein to DNA at these pH values.

ATP does not affect DNA binding at pH 7 or below, but stabilizes a fraction of large

T antigen-origin DNA complexes at high pH

AIT was shown to increase the affinity of polyomavims large T antigen for DNA fragments within the viral replication origin, when binding was done at pH 7.8 (Lorimer et al.. 1991). Because we found that the affinity of large T antigen for origin DNA was significantly greater below pH 7 than at higher pH values. we decided to test the effect of

ATP on DNA binding as a function of pH, using the nitrocellulose filter binding assay.

Binding to ongin DNA was stimulated two-fold by 5 mM ATP in Tris-HCI b&er at pH

7.8 under our binding conditions (Fig. 14A), in agreement with previous results (Lorimer et al., 1991); however, we could detect no stimulation of binding by ATP in Tns-HC1 buffer at pH 7.0 or in potassium phosphate bufTer at pH 6.0 (Fig. 14A). In other experiments (results not shown), the use of different concentrations of large T antigen

119 Fig. 14. ATP stimulates DNA binding at pH 7.8 but not at pH 7 or below. A) 1 ng of a "P-labeled 265-bp origin-containing DNA fragment was incubated with 100 ng large

T antigen in 100 mM potassium phosphate buffer (pH 6.0) or Tris-HC1 bufTer (pH 7.0 or

7.8), in the absence (gray bars) or presence (black bars) of 5 mM ATP. Bound DNA was detected by the nitrocellulose filter binding assay. B) 8 ng of a 5'-end-labeled 6 Wbp

DNA fragment was incubated at pH 6.0 without large T antigen. or with 100 ng or 200 ng of large T antigen in the absence or the presence of 4 mM ATP. After DNase 1 digestion, products were analyzed by electrophoresis and autoradiography. Protected regions corresponding to binding sites ln, A. B, and C are shown by bnckets.

- = 0.1 0.1 0.2 0.2 LT (pg) -+++ATP (1 0-100 ng per reaction) or ATP (0.1-5 mM) did not result in stimulation of binding at pH

7 or below. In addition, a DNase 1 footpnnting assay revealed no significant effect of

ATP on either the level of DNA binding or the extent of the protected regions on target

DNA at pH 6 (Fig. 148).

Since we had shown that large T antigen-DNA complexes were unstable at pH 7.6 but stable at pH 6.0. we asked whether ATP acted by stabilizing protein-DNA complexes at high pH values. We assembled complexes at pH 7.0 and subsequently diluted samples

16-fold into pH 7.0 or pH 7.8 buffer containing a 100-fold excess of unlabeled target

DNA, in the presence or absence of 5 mM ATP. Aliquots were either filtered immediately (2 min or less) upon dilution, or were incubated at O°C for various times before filtration, to measure the arnount of DNA remaining bound by large T antigen.

Figure I5A (upper cwe) shows that large T antigen-DNA complexes formed at pH 7 were relatively stable afier dilution into pH 7 buffer; only 4% of these complexes dissociated within 2 min of dilution, and 29% dissociated during a subsequent 2-hour incubation in pH 7 buffer at oOC.

Dilution of complexes into pH 7.8 buffer in the absence of ATP (bottom curve) led to npid dissociation of one-half of the complexes formed at pH 7, consistent with the results of the gel mobility shift assays described above. The remaining complexes remained intact, but subsequently dissociated with a haif-life of approximately 3 hom.

When dilution into pH 7.8 bufFer was carried out in the presence of ATP, a fraction of the large T antigen-DNA complexes was protected fiom rapid dissociation. In this experiment (Fig. 1SA), 50% of the complexes formed at pH 7 dissociated after dilution to

121 pH 7.8 in the absence of ATP, while only 28% of the complexes dissociated in the presence of ATP. Therefore, about No-fifis (22/50) of the complexes that dissociated rapidly upon dilution to pH 7.8 were pmtected against dissociation by ATP. This population of ATP-protected complexes, however, subsequently dissociated more rapidly than the remaining complexes, and had dmost completely disappeared after 2 hours at dC (haif-life of 1 hou).

Stobilization of complexes by ATP depends on the presence of site 112

ATP stimulates formation of hexamers of both polyomavirus and simian virus 40 large T antigens in the absence of DNA (Dean et al., 1992; Wang and Prives, 1991a).

The stimulatory effect of ATP on binding of simian virus 40 large T antigen to target

DNAs has been ascribed to hexamer formation on DNA (Borowiec and Hunvitz. 1988b:

Deb and Tegtmeyer, 1987; Lorimer et al., 1991). Hexamer formation is specific to binding site II on SV40 DNA (Joo et al., 1997; Parsons et al., 1991), which is analogous to site li? on polyomavirus DNA (DeParnphilis, 1993; Hassel1 and Brinton, 1996). We therefore pstulated that the fraction of protein-DNA complexes protected from dissociation by ATP consists of hexamers formed on the DNA upon dilution of complexes fiorn low to high pH in the presence of ATP. If this were the case, complexes made with a target DNA lacking site 112 should not be protected by ATP from dissociation upon dilution to high pH, as hexamers would not be expected to fom on such DNA. The results of such an expenment are show in Figs. 15B and 15C. Protein-

DNA cornpiexes containhg sites C, B, A and 112, fonned at pH 6 (Fig. 15B), were very

1Z? Fig. 15. Kinetics of dissociation of protein-DNA complexes upon dilution from low to

high pH in presence or absence of ATP. A) 1 ng/60 pl of a "P-labeled 265-bp DNA

fragment containing sites 112, A, B, and C was incubated at 370C for 25 minutes with 100 ng large T antigen in 100 mM Tris-HC1 buffer (pH 7.0). 60 pl aliquots were then diluted into 1 ml ice-cold Tris-HCl buffer, pH 7.0 or 7.8, containing al1 other components of the binding solution (see Materials and Methods) as well as a 100-fold excess of unlabeled

265-bp DNA fragment, in the presence or absence of 5 mM ATP, as noted. Mixtures were filtered after incubation at O'C for the indicated times and filters were washed with the dilution buffer. Results are expressed as percent of protein-DNA complexes originally present in undiluted 60 pl aliquots of the pH 7.0 binding reactions. B) A similar expenment was canied out except that the initial binding was carried out in 100 mM potassium phosphate buffer, pH 6.0, and dilution was in potassium phosphate buffer at pH 6.0. or Tris-HC1 buffer at pH 7.0 or 7.8. C) A "P-labeled 260-bp DNA fia-ment containing binding sites A, B and C but lacking site 112 was incubated with large T antipn at pH 6.0 as in B, then diluted with pH 6.0 or 7.8 buffer as shown. Error bars show maximum difference in radioactivity retained on filters using duplicate sarnples.

Error bars are omitted fiorn the overlapping curves in B for the sake of clarity, but are similar to those shown on the lower curves. D) DNase 1 Footprinting analysis performed at pH 7.4 on ongin DNAs containing ail four sites (lefi panel) or lacking site 1/2 (right panel). ATP increased protection of site 1/2 (lefi panel), but had no effect on protection of sites A, B, and C in the absence of site 1/2 (right panel). œ

rn œ

œ 4pH 7.0, no ATP +pH 7.8,s mM ATP

rn +pH 7.8, no ATP

1 I 1 I 1 1 O 30 60 90 120 Tirne (min) -0- pH 6.0, no ATP +pH S.O,S mM ATP +pH 7.0, no AT? +pH 7.0,s mM ATP +pH 7.8, no AT? +pH 7.8,s mM ATP

60 Time (min) +pH 6, no AT? +pH 7.8, no ATP +pH 7.8,s rnM ATP

60 Time (min) LTAg + + ATP - -+ 1E 'Y- w-& I

-. ] ail

rlL +- 1 stable when diluted into pH 6 buffer (top curves), and nearly as stable when diluted into pH 7.0 buffer (middle cwves). ATP had little effect on these complexes when present during dilution to pH 6 or 7. However, ATP protected a hction of the complexes fiom rapid dissociation upon dilution in a pH 7.8 buffer (bottom curves), in agreement with the results shown in Fig. 15A. In a parallei experiment carried out with a target DNA containing only sites A, B and C (Fig. 15C). more than one-half of large T antigen-DNA complexes dissociated upon dilution to pH 7.8, but ATP did not protect these complexes from dissociation (Fig. 15C). Similar results were found using other DNAs containing various combinations of binding sites A, B and C but lacking site 112 (not shown).

DNase I footprinting experiments cmied out at pH 7.4 aiso showed that ATP stimulated binding to site 112 on a DNA fragment cont&ning al1 four sites (Fig. 15D, lefi panel), but had little effect on binding to a DNA fragment lacking site 1/2 (Fig. IjD, right panel).

These results suggest that the stabilizing effect of ATP on large T antigen-DNA complexes upon dilution to pH 7.8 is due to the formation of hexamers of large T antigen at site 1/2 on a fraction of the target DNA molecules.

Binding of large T antigen to origin DNA fragments containing one or more adjacent binding sites

Having defined optimal conditions for DNA binding assays, we proceeded to ask w hether poly ornavirus large T antigen binds independenti y or cooperativel y to its muliiple target sites in the polyomavim origin region. In a f~ set of experiments, we prepared target DNAs from which one or more of sites 1/2, A, B and C were deleted (Fig.

134 Fig. 16. Binding of large T antigen to DNAs contaioing combinations of adjacent binding sites. A) The Pst I fiagrnent of polyomavims AT3-Modori DNA that contains the replication origin and large T antigen binding sites 112, A, B and C is shown in the upper panel as part of plasmid pGEM-1/2ABC(+). Arrowheads show the G(A/G)GGC consensus sequences within each site. Numbers indicate the positions of the restriction sites show. Primers used for PCR aniplification oî fragments within pGEM plasmids are shown at each end. In the lower panel, horizontal lines show sequences retained in deletion mutants made by cutting and religating plasmids between different restriction sites in the Pst 1 fragment or the pGEM polylinker (pGEM and polylinker sequences are not show). For sake of clarity, al1 deletion mutants are show in the same orientation; however. mutants BC and B were denved from plRABC(-) and therefore primers 1 and

2 are inverted for those plasmids. B) 6 fmoles of labeled DNA fragments containing different combinations of adjacent binding sites (noted at the right) were incubated with various arnounts of large T antigen (1 ng = 10 fmoles) in potassium phosphate buffer, pH

6, and filter binding assays were carried out as described in Materials and Methods. primer 1 t,,, 4 4 4 4 >D++ primer 2 -7' 1 I 1 I I g7- rl12ABC -ABC -BC 16A), and measured binding as a function of large T antigen concentration using the filter binding assay at pH 6.0, in the absence of ATP. Under these conditions, binding to the wild type ongin region reached a maximal value of 85% (Fig. 16B); half-maximal binding was achieved with 50 ng large T antigen per 60 pl reaction. Binding to DNAs containing only site A or site B was barely detectable; each of these sites contains only

~VOG(AIG)GGC consensus bindhg sequences, and previous studies using such sites also showed inefficient binding (Bondeson et al., 1995; Cowie and Karnen, 1984; Wright et al., 1984). However, fragment AB, containing both sites A and B, had an afinity for large T antigen about ten-fold higher than A or B separately. Fragment C, which contains four G(A/G)GGC consensus sequences, showed about the same affinity for large T antigen as Fragment AB. Addition of site B, or both A and B. to site C increased binding by factors of about 2 (BC) or 4 (ABC), respectively, as determined by the concentration of large T antigen required for haif-maximal binding of wild type DNA. Thus two inherently weak binding sites (A and B) strengthened binding of large T antigen to a moderately strong binding site (C) when positioned adjacent to diat site. Furthemore, fragment 112A bound about as well as fragments AB or C, but cornbining 112A and BC increased binding by a factor of about 2.5 (cornparison of half-maximal binding of

1/2ABC vs. that of BC).

These results show that large T antigen binds cooperatively to its multiple binding sites in the region of the replication origin. Binding of large T antigen is some 50-fold stronger to DNA containing al1 four sites tban to DNA containing only site A or B, and 5- fold stronger than to DNA containing only site C. We also measured DNA binding at pH

126 7.0 in Tris-HCl buffer for most of the deletion mutants described in Fig. 16A (data not shown). Although overall binding was lower, the relative binding aninities to the different DNAs were the same as shown in Fig. 16B.

Binding of large T antigen to DNA targets containing mutated binding sites

One drawback of the use of deletion mutants as shown in Fig. 16 is that the DEiA fragments used have different sizes (ranging from 147 to 736 bp) and contain different flanking sequences; these could affect binding affinities. We tested binding to PCR- generated DNA fragments of different sizes, and detected no major differences (data not shown). However, the availability of DNAs containing point mutations in the

G(A/G)GGC consensus sequences within sites A, B and C (Bertin et al., 1993) allowed us to determine binding strengths of a set of DNA targets identical in size and sequence except for a smail number of nucleotide substitutions (Fig. 17A). This set of DNAs also allowed determination of binding strengths of different combinations of sites than were possible with the collection of deletion mutants; al1 of these DNA fragments contain an intact site 113.

Mutation of either site A, B, or C (mA, rnB, or mC) reduced binding of large T antigen to the origin DNA fragment by only 1.3- to 1.6-fold (Fig. 17B), as measured by the ratios of large T antign concentration at half-maximal binding. It is of interest that mA and mC had nearly identicai binding strengths; only two G(A/G)GGC sequences are mutated in mA but four are mutated in mC, and the results in Fie. 17B showed that site C alone binds much more strongly than site A. This suggests that the proximity of sites

127 Fig. 17. Binding of large T antigen to DNAs containing mutated binding sites. A)

The region containing binding sites 112, A, B and C is shown in the upper panel. Below are show mutants generated by introducing point mutations into consensus pentanucleotide sequences in each of sites A, B or C, or combinations of these sites

(Bertin et al., 1993). Mutants are named for the mutated sites they contain. Arrowheads show the G(AeG)GGCsequences that remain intact in each plasmid. PCR amplification of the regions between nt 220 and 5167 for each mutant was carried out by use of primers 3 and 4. B) 6 fmoles of labeled DNA fragments containing mutations in

G(A/G)GGC consensus sequences in sites A. B and C were incubated with various arnounts of large T antigen in Tris-HC1 buffer, pH 7.0, and filter binding assays were carried out. primer 3 primer 4 - bDDD 4 4 4 4 bb4* - affects the overall binding strength of the DNA fragment. Fragment mA contains a gap of some 70 nt between sites 112 and B, while the three sites (B, A and 1/2) in mC are separated by only 25-30 nt each.

The double mutant mAme bound significantly less strongly than either mA or mB. Fragment mAmB required a 2-fold higher concentration of large T antigen for half- maximal binding than did wiid type DNA; however, at a concentration of 100 ng large T antigen per 60 pl, 5-fold more wild type DNA bound than mAmB DNA. Thus, at low concentrations of large T antigen, the differences in binding strengths between wild type

DNA and mAmB, rnAmC, or mBmC were greater than at higher concentrations of large

T antigen. Fragment mAmB also bound less strongly than did mC, although both mutants are missing four G(A/G)GGC sequences, and DNA fragments containing both sites A and B or site C alone had very sirnilar binding strengths (Fig. 17B). This difference may be due to a 100-nt gap between site C and site 1R in mutant mAmB,that may render cooperative interactions less efficient.

DNAs containing oniy site A or site B in addition to site 1/2 (mBmC and mAmC) bound about 7-fold less well than DNA containing both sites A and B (mC). Thus, mutation of a weak binding site (B or A) strongly affected binding of large T antigen to the remaining sites on the target DNA. Finaily, a DNA target containing only site 112

(mArnBmC) barely showed any binding to large T antigen at the highest concentration used. This experiment was carried out at pH 7.0 in Tris-HCI bufTer, in the absence of

ATP; sirnilar results were obtained at pH 6, except that higher overall levels of binding were detected. DNase 1 footptinting of mutant DNAs in the preseace of large T antigen

Filter binding and gel retardation assays do not provide information on the

occupancy of each of the binding sites on the various DNAs we used. To understand

more precisely the nature of the cooperative interactions between large T antigen and its

binding sites, we carried out DNase i footprinting experirnents (Fig. 18) using the mutant

DNAs described in Fig. 17A. This experiment was carried out at pH 7 using 400 ng large

T antigen per binding reaction, but essentially identical results were obtained at pH 7.4

using a higher concentration, and at pH 6 using a lower concentration of large T antigen.

Fig. 18A shows the footpnnt of three single mutants (mA, rnB and mC) compared to that of the wild type origin. As expected, there was no protection against DNase 1 digestion in the mutated region of each DNA, but al1 other sites were protected to approxirnately the siune degree as in the wild type DNA. This agrees with the results in Fig. 17B. which showed that single mutations in these sites reduced overall binding afinity for large T antigen by only a small arnount.

Fig. 18B shows results with double and triple mutants. It is instructive to examine protection of site 112 in these four mutants and in cornparison to wild-type DNA. There was little binding of large T antigen to site 112 in DNA of the triple mutant mAmBmC, as could be expected from examination of Fig. 17B. Protection of site 1/2 was strongly increased by the presence of site C, some 100 nt distant from site 1/2, in DNA of mutant mAmB. Mutants mAmC and mBmC showed only partial protection of site IR, and fkthermore showed little protection of site B (in mAmC) or site A (in mBmC);

130 Fig. 18. DNase I footprinting of DNAs coataining mutated binding sites. 20 fmoles of 265-bp end-labeled DNA fragments containing binding sites mutated as shown at top were incubated in the absence (-) or presence (+) of 400 ng large T antigen in Tris-HCl buffer, pH 7, and then incubated with DNase 1, as described in Materials and Methods.

DNAs were analyzed on 12% polyacrylamide-8M urea gels. Regions protected against

DNase I digestion are indicated by brachts. A) Footpnnt of diree single mutants (do mB and mC) compared to that of the wild type origin. B) Footprint of double (mAmB, mAmC, and mBmC) and triple (mArnBmC) mutants.

even though these sites were intact in the target DNA. If we compare these pattems to the patterns for single mutants rnA, rnB, and mC, it is clear that binding of large T

antigen to site B requires the presence of either site C or site A, and binding to site A requires the presence of either site B or site C, in addition to site 1/2, which is present on al1 of these DNAs. These results Merdocument the cooperative nature of the binding of large T antigen to polyorna~imsorigin DNA. In particular, they rmphasize that optimal binding to site 1/2 requires the presence of either site C or sites A and B.

ATP specifically enbances protection igainst DNase 1 digestion of the central 10-12 base pairs of site 112

We then tested the effect of ATP on footprint patterns at pH 7.4 ushg the different mutant DNAs show in Fig. 17A. In our initial expenrnents. we observed what appeared to be increased protection against DNase 1 digestion by 4 rnM ATP throughout site 1/2. and decreased protection in sites A, B, and C. However, we found that 4 mM

ATP reduced overall DNase I activity (perhaps by sequestering free Mg"), leading to increased levels of uncleaved DNA and decreased levels of cleaved DNA. particularfy in regions close to the labeled end of the DNA (smaller DNA fragments). We therefore reduced the ATP concentration from 4 to 1 mM, and titered amounts of DNase in each reaction so that similar levels of cleavage took place in the presence and absence of ATP, by making sure that the amount of uncleaved, full-length DNA was similar in each digested reaction. Under these conditions ATP had little or no effect outside of site IR on wild type DNA or any of the mutant DNAs. Sample results with mutants mAmB and

132 rnAmBmC are show in Fig. 19A, and a phosphorimager tracing of a gel lane using mutant mC is shown in Fig. 19B. Precise alignrnent of the footprint pattern with the

DNA sequence was done by digesting end-labeled DNA with different restriction endonucleases and comparing the migration of those DNAs with the footpnnt pattern of the sarne DNA run in the same gel (results not shown); Hp II cuts between nt 9 and 10 on the boitom (labeled) strand, and Sph I cuts between nt 32 and 53 on the same suand.

Therefore determination of the sites where ATP altered large T antigen protection of

DNA was accurate to within one or two nucleotides in this region.

Figs. 19A and 19B show that ATP had distinct effects in different parts of site 1/2.

At the early (lefi) side of site 112 a major DNase-sensitive site at nt 37 was not protected by large T antigen at this concentration in the absence of ATP. Binding in the presence of ATP led to protection at this site, but little or no enhancement of protection in the adjacent region of site 112, between nt 35 and 22 (compare thick and thin continuou lines in Fig. 19B). This region contains one of the four GAGGC sequences in site 1/2.

However, ATP strongly enhanced protection of the central region between nt 21 and nt

11, shown by the heavy bracket to the nght of Fig. 19A and the dark shading of the sequence in Fig. 19B. Furthemore, protection of the region fiom nt 10 and beyond

(lighter shading) was also enhanced, but to a lesser extent than in the central region. The central region contains two overlapping GGGGC consensus binding sequences, and represents approximately one tum of the DNA helix. Similar results were obtahed when the expenment was canied out at pH 7, but ATP had no detectable effects on footprint patterns at pH 6.0 (Fig. 14).

133 Fig. 19. ATP enhances protection of the central portion of site 10 ngainst DNase 1

digestion. A) 20 fmoles of 265-bp labeled DNA fragments containing binding sites

mutated as show at top were incubated with (+) or without (0) 600 ng of large T antigen

in Tris-HCl binding buffer, pH 7.4, in the presence or absence of 1 mM ATP. DNase 1

footprinting was then carried out. A DNase-sensitive band that corresponds to nt 37 in

polyomavirus DNA, between sites A and 112, is noted by a star. The domain whose

protection is enhanced in presence of ATP is noted by a thick black bracket on the right

side. B) Phosphorimager protile of relative band intensities in the region denoted by the

two-headed anow in (A), from a DNase I footprint using mutant DNA mC. The DNA

sequence in this region is shown above the density profile; nucleotide numbers refer to

the polyomavirus stmin A3 genome. G(A/G)GGC sequences on each DNA strand are designated by arrows. The dark box indicates the region whose protection is strongly

enhanced in the presence of ATP; the lighter box indicates a region whose protection is

weaidy enhanced in the presence of ATP. The star denotes the same band marked by a

star in (A). - + + - + +LT - +== + ATP I I CCAGACCATCCGGAGGC- GGC CT C T C CGTCTCGTACCCCTCCG CCC CA C A C DISCUSSION

The results presented in this paper show that polyomavirus large T antigen binds in a cooperative fashion to its multiple target sites within and adjacent to the origin of

DNA replication. Binding of large T antigen to sites A, B and C facilitates binding to site

12, in the core replication ongin, where hexamers presurnabiy fom to initiate unwinding and DNA replication. The presence of these auxiliary sites (Bertin et al., 1993;

DeParnphilis, 1993; Weichselbraun et al., 1989) near the origin may therefore allow initiation of DNA replication at substantially lower concentrations of large T antigen than would be possible in their absence.

No previous studies have anempted to quantitate binding of polyomavirus large T antigen to origin DNA in which individual binding sites were deleted or mutated.

Binding to DNA Fragments containing isolated sites A, B or C, or some combinations of these sites, was first show by immunoprecipitation of large T antigen-DNA complexes

(Cowie and Kamen. 1984, 1986; Pomerantz et al., 1983). Site A in polyomavirus main

A2 contains three GAGGC pentanucleotide sequences (in strain A3 and derivatives used here there are two pentanucleotides [Bertin et al., 1993; Dailey and Basilico, 1985; Prives et al.. 1987; Triezenberg and Folk, 19841); inactivation of any one of these three sequences by methylation at a single G residue reduced binding by large T antigen by a factor of about 10 (Cowie and Kamen, 1986). This led Cowie and Karnen (1986) to propose that large T antigen molecules bind cooperatively to the adjacent G(A/G)GGC sequences, approximately one helical turn apart, within individual sites (A, B or C).

13 5 These results, as well as DNase footprinting studies (Cowie and Karnen, 1984; Lonmer et al.. 1991; Peng and Acheson, 1997), also suggested that each large T antigen molectde occupies approximately one helical tum when bound to target DNA, and that mutual interaction of these closely-packed large T antigen rnolecules helps to stabilize their binding to DNA. The cooperative binding described in this paper involves large T antiyn molecules bound to sites that lie between 20 and 100 nt apart, and in which

G(A/G)GGC sequences are not always on the same DNA suand. Our results (Figs. 16-

18) suggest that large T antigen bound to any of sites A, B, C and 1/2 cm interact with large T antigen bound to al1 of the other sites.

We propose a model (Fig. 20) to account for these results. This model allows interactions between DNA-bound large T antigen molecules by virtue of folding of the

DNA in this 170-nt region into a compact protein-DNA complex. Interactions between sites C and 1/2. A and B, A and 1/2, and B and 112 are suggested by contacts between spheres representing large T antigen rnolecules bound to each set of sites. Interactions between site A or B and site C can be imagined by folding the top and bottom parts of the complex vertically above the plane of the page. DNase 1 footprinting (Cowie and Kamen,

1984; Lorimer et al.. 199 1 ; Peng and Acheson, 1997; and this paper) shows that there are unprotected regions between each of the four sites, show as DNA not covered by protein in the model. In addition, there is increased DNase 1 sensitivity at positions located between sites At B, and C, upon binding of large T antigen (Fig. 18 and Bhanacharyya et al., 1995; Mastrangelo et al., 1989), consistent with the bending or distortion of the DNA helix that would be necessary to bring the various large T antign molecules into contact

136 Fig. 20. Mode1 for cooperative binding of large T antigen to ongin DNA. Monornen of large T antigen (spheres) are show bound to the multiple G(PJG)GGC sequences within sites C, B, A and 112 on origin DNA in such a way that they cm interact cooperatively with each other. The structure containing sites A and B could fold upwards to enable contact with site C as well as site 112. with each other. However, we do not clah to know the exact path of the DNA through this multiprotein complex, nor the stability of such a complex in the cell.

We observed a strong pHodependence of binding by large T antigen to specific

DNA, fiom a maximum at pH 6 to nearly no detectable binding at pH 8.5. Previous M vitro binding studies were carried out at pH values between 7 and 8 (Borowiec and

Hunvitz, 1988b; Deb and Tegtmeyer, 1987; Lorimer et al., 199 1 ; Mastrangelo et al.,

1989; Pomerantz et al., 1983; Scheller and Prives, 1985; Vogt et al., 1986); however, no quantitative data on the pH dependence of specific DNA binding by either polyomavirus or simian virus 40 large T antigen have been reported. Binding over the range of pH values tested (6.0 to 7.8) was specific for G(NG)GGC sequences on DNA, and the pattern of DNase 1-protected regions on origin DNA was similar (in the absence of ATP) across this pH range. Moreover, cooperative binding to the multiple binding sites on origin DNA was observed at al1 pH values tested. Stronger binding at low pH cm be explained by increased stability of large T antigen-DNA complexes at low pH values. shown by resistance to dissociation upon dilution or during gel electrophoresis. Increased binding strength and increased stability of protein-DNA complexes at low pH dlows more sensitive detection of large T antigen by nitrocellulose filter-bindinp or gel retardation assays than was previously available. Purification of large T antigen by binding to DNA at low pH and release at high pH might also be possible. The pH effect should also be considered when interpreting any studies on biochemical activities of large

T antigen mediated by DNA binding, since the decrease in binding strength as pH rises is panicularly steep in the range between pH 7.4 and 8.0, wd in many binding and

138 repiication assays (Borowiec and Hurwitz, 1988b; Deb and Tegtmeyer 1987; Lonmer et al., 1991; Mastrangelo et al., 1989; Scheller and Prives, 1985; Vogt et al., 1986).

What might be the biologicd significance of the variation in binding as a function of pH? Most mammalian cells have an intracellular pH of 7.1-7.3, although values ranginp from 6.8 to 7.5 have been measured (Demaurex et al., 1996; Roos and Boron,

198 1; Strazzabosco and Boyer, 1996). Invacellular pH is regulated by a NaiH' antiport and by HCO3- exchange (Grinstein et al., 1989; Roos and Boron, 198 1; Strazzabosco and

Boyer, 1996). Regulation of a number of cellular processes, including ce11 spreading and attachment, DNA replication, and cellular proliferation, has been correlated with changes in intracellular pH or in the activity of the Na'/H' antiport (Deitmer and Rose, 1996;

Gnnstein et al., 1989; Strazzabosco and Boyer, 1996). At pH 7.2. the binding &ity of large T antigen for ongin DNA is intermediate between its maximum. at pH 6.0, and its minimum. at pH 8 and above (Fig. 12A). It is possible that polyomavirus DNA replication may be responsive to changes in intracellular pH as a result of the pH- dependence of the affmity of large T antigen for its DNA target. In particular, lower intracellular pH may favor binding to origin DNA and therefore accumulation of suficient large T antigen molecules near the replication origin, and higher pH rnay favor mobilization of these bound protein molecules and the formation of hexamea by a

"handover" mechanism (see below).

Previous reports showed that An, in the presence of Mg2', stimdated binding of both polyomaWus and simian virus 40 large T antigens to their respective origh DNAs when binding reactions were carried out at 370~in pH 7.5-7.8 bufTers (Borowiec and

Hurwitz, 1988b; Dean et al., 1WC; Deb and Tegtmeyer, 1987; Lorimer et al., 199 1). We

confirmcd that ATP stimulates binding by polyomavinis large T antigen to an origin

DNA fiagment at pH 7.8, when measured by the nitrocellulose filter binding assay, but

found no effect of ATP on binding at pH 7 or below. We fbrther found that a fraction of

the protein-DNA complexes formed at pH 6 or 7 was protected against dissociation upon dilution to pH 7.8 when ATP was present. This protective effect was seen when a DNA

fragment containing the entire origin region was used, but was not seen with fragments lacking site 1/2. ATP is required for generation of hexameric foms of sirnian virus 40 and polyomavirus large T antigen, either in solution or at the replication origin

(Mastrangclo et al., 1989; Muller et al.. 1983; Wang and Prives, 1991a); hexamers of simian virus 40 large T antigen have been show to fom at site II (Dean et al., 1992;

Wessel et al., 1992b), homologous to polyomavinis site 1/2 (DeParnphiiis, 1993). It is likely that the effect of ATP on DNA binding results from its ability to stimulate hexamer formation at site IR. Hexamers may not fom on DNA below pH 7, either because of a changed protein conformation at low pH, or because large T antigen molecules are t00 tightly bound to G(NG)GGC pentanucleotide tarpt sequences on DNA and therefore cannot be released to form hexamers. Hexamer formation upon shifting to higher pH in the presence of ATP could stabilize protein-DNA complexes. Hexamers probably bind to

DNA by topologicaily enclosing the DNA double helix within the central hole formed by the circular hexamer (San Martin et al., 1997; Wessel et al., 1992b), rather than by recognizing and interacting with specific nucleotides. The ATP-stabilized complexes

140 generated upon dilution to hi& pH (Figs. 15A and 15B) decayed relatively rapidly, with a half-life of about 1 hr. This could be due to instability of hexamers, but hexamea of simian virus 40 large T antigen were shown to be stable for seveml hours in vitro (Dean et al., 1992; Wessel et ai., l992b). Altematively, hexamers may simply fdl off the ends of the linear DNA fragments to which they are bound in our in vitro assay.

-41 pH 7.4, -4TP specifically enhanced protection of the central 10-13 bp of site

1/2 when less than saturating concentrations of large T antigen were used (Fig. 19). A previous study of the eEect of ATP on the DNase I footprint pattern of wild-type polyomavirus origin DNA (Lorimer et al., 199 1) showed increased protection of a region including sites A and 1/2. but did not detect specific enhancement in this part of site 112.

However, those experiments were carried out under different conditions (higher concentration of large T antigen, pH 7.8), and the opposite DNA strand was labeled, making it dificult to detect closely-spaced DNase-sensitive bands in site 1/2.

Studies of the structure of polyomavirus origin DNA bound by large T antigen in the presence of AMPPNP, a non-hydrolysable analogue of ATP (Bhattacharyya et al.,

1995), detected KMn0,-sensitive nucleotides at positions 10 and II. and 20 to 22 (Fig.

5). These sites are located at the borders of the 10- 12 bp region in the center of site 1R whose protection against DNase 1 digestion is enhanced in the presence of ATP. These

KMn0,-sensitive sites may reveal distortion of the DNA helix at the edges of bound protein molecules. Taken together, our data and those of Bhattacharyya et al. (1995) suggest that ATP, or AMPPNP, induces the formation of a complex of large T antigen that covers the central part of site 1/2 over a single tum of the DNA helix. In contrast,

14 1 simian virus 40 large T antigen protects a larger region of the homologous site II against

DNase 1 digestion (Borowiec and Hurwitz, 1988b; Parsons and Tegtmeyer, 1992), and there are no KMnû,-sensitive sites generated at the equivalent positions in site II

(Bhattacharyya et al., 1995). Sirnian virus 40 large T antigen has been shown to form double hexarners at site II, via a cooperative process directed by the two halves of site II

(Parsons and Tegtmeyer, 1992). A single hexmer of polyorna~inislarge T antigen may fonn at the center of site 112. This is consistent with the different structure of these sites: in simian virus 40 site II, the central GAGGC pentanucleotides are separated by 1 nt, perhaps allowing formation of a hexamer on each half of site II; however, in polyomavirus site 1/2, the central GGGGC pentanucleotides overlap by 2 nt (the 3'- terminal GC on each strand). Furthemore, simian virus 40 DNA replication begins at approximately the same site on each DNA strand (Hay et al., 1984), while polyomavirus begins replication near nt 30 on the early strand, but some 16 nt beyond (nt 46) on the late strand (Hendrickson et al., 1987). Therefore, it is possible that a single hexamer forms at site 112 and begins unwinding and replication in the early direction, followed by formation of a second hexamer once replication has begun. This would also account for the existence of unidirectional rolling-circle replication known to take place in polyomavirus (Bjursell, 1978). Such unidirectional replication could result fkom the progression of a single replication fork if the second hexamer were not assembled.

The cooperative binding of large T antigen to its multiple target sites on origin

DNA, coupled with the reversibility of binding at inmcelluiar pH values, suggest a mode1 for the pathway of assembly of hexamers of large T antigen leading to the

142 initiation of DNA replication at the ongin (Fig. 21). This mode1 proposes that large T antigen monomers bind cooperatively to DNA via interactions with the G(A/G)GGC pentanucleotides in al1 four sites, forming a complex similar to that show in Fig. 21.

Such cooperative binding would tend to concentrate large T antigen to one or a few DNA molecules, avoiding nonfunctionai binding of a smail nurnber of monomers arnong several DNA molecules. This could be important to the eficiency of DNA replication in the beginning of the replication cycle, when low amounts of large T antigen are present.

Large T antigen molecules bound to DNA then assemble into hexamers at site 1/2 in the presence of ATP, which presumably induces a conformational change in large T antigen favoring hexarner formation (Lorimer et al., 199 1 ; San Martin et al., 1997; Wessel et al.,

199%). Hexamer assembly would be Favored by the proximity of large T antigen molecules and their mutual interaction in the protein-DNA complex; they can therefore be "handed over" from sites A, B, and C, to which they are reversibly bound, to site 1/2, where hexamer formation occurs. When large T antigen is present at high concentrations, it could alternatively assemble directly from solution onto hexamers growing at site 1/2.

A single hexamer is shown in Fig. 21, reflecting our observations on enhanced DNase I protection at the center of site 1/2 by ATP. Whether a second hexamer can subsequently form adjacent to this hexamer, as with sirnian virus 40 (Parsons and Tegtmeyer, 1992;

Weisshart and Fanning 1996; Wessel et al., 1992b), or foms ody after the displacement of this hexamer during unwinding and initiation of DNA replication, is not known. Fig. 21. "Handover" mode1 for assembly of hexamers nt replication origin.

Monomers of large T antigen (spheres) bind cooperatively to the four binding sites in origin DNA to form a cornplex as shown in Fig. 20. In the presence of ATP, bound monomers in contact with each other dissociate from the G(A/G)GGC sequences to which they are bound, and rearrange to form a hexamer at the center of site 1/2, which can unwind DNA and allow initiation of DNA replication. A second hexamer may subsequently form by a similar rearrangement. cooperative binding 1 CHAPTER 5

Enhanced Binding to Origin DNA at Low pH Enables Easy Detection of Polyomavirus Large T Antigen by Gel Mobility Shift Assay of Unfued Complexes

Yu-Cai Peng and Nicholas H. Acheson

Deparrment of hficrobiologv and Imm unology. McGil l L'niversity, 3 77) Universip Street, hfontreal, Quebec, Canada H3A 2B-l

This chapter is derived fiom a manuscript published in Journal of Virological hfethods, March 1999,711: 13- 19 CONNECTING TEXT

In the preceding chapter, we showed that large T antigen forms stable complexes with target DNA at pH 6, but the complexes dissociate when the pH is shifted to 7.8.

Stable binding at pH 6 enables analysis of large T antigen-DNA complexes by an easy gel mobility shift assay. Such an assay was not available previousiy and should be useful for studies of large T antigen. In this chapter, we further characterize of the gel retardation method, demonstrating that it can be applied to cnide ce11 lysates containing large T antigen from different sources. We also show that large T antigen-DNA complexes fomed at low pH are massive, probably because large T antigen self- associates at low pH. This explains why bound DNA hardly entered the gels as shown in

Fig. 13 of Chapter 4. ABSTRACT

Enhanced, stable binding by polyomavirus large T antigen to the viral DNA

replication origin at pH 6 allowed the development of a gel mobility shift assay for the detection of large T antigen. Such assays were not possible at pH 7.6 without previous

fixation, due to instability of the complexes. We demonstrated that the gel mobility shifi assay at pH 6 is very sensitive, allowing the detection of as little as 5 ng large T antigen, and is highly specific for DNA containing G(A/G)GGC target sequences. We used this method to detect large T antigen in crude ce11 lysates from transformed yeast ce11 lines or nuclear extracts from infected insect cells. Large T antigen-DNA complexes remained at or near the loading well in 5% acrylamide or 1.5% agarose gels. indicating that these complexes are very large. Giycerol gradient analysis showed that protein-DNA complexes formed at pH 6 were massive, and that large T antigen also formed large complexes when incubated at low pH in the absence of DNA. These results show that pH has a major effect on binding of large T antigen to its multiple target sites in the viral origin of DNA replication, presumably by affecting protein-protein interactions that are important for the stability of large T antigen-DNA complexes. INTRODUCTION

Polyomavinis large T antigen is a multihnctiond protein which binds to specific

DNA sequences (Bondeson et al., 1995; Cowie and Karnen, 1984, 1986; Pomerantz and

Hassell, 1984), has enzymatic activities (helicase, ATPase) (Gaudray et al., 1980; Seki et

al., 1990; Wang and Prives, 1991b), interacts with several cellular proteins (DNA

polymerase a:primase, replication protein A, retinoblastorna protein Rb, p107)

(Desjardins et al., 1997; Moses and Prives, 1994; Murakami and Hunvitz, 1993a; Pilon et al., 1996), and regulates viral DNA replication as well as host ce11 growth and division

(for review, see Pipas, 1992). Large T antigen has also been implicated in the replation

of transcription of viral genes (Cogen, 1978; DaiIey and Basilico, 1985; Farmene and

Folk, 1984; Kern et al., 1986), and cm block passage of RNA polymerase II molecules

when bound to viral DNA (Bertin et al., 1992, 1993: Brabant and Acheson, 1995).

Polyomavirus and the closely-related sirnian virus 40 have been used as models to study eucaryotic DNA replication, and their large T antigens. key proteins for viral DNA

replication and cell transformation, are widely used in oncology, ce11 biology, and molecular biology.

Large T antigen binds cooperativeiy to four distinct sites on viral DNA designated

A, B, C, and 1/2 by recognizing a consensus pentanucleotide, 5'-G(NG)GGCJI, which is present in multiple copies nearby the viral ongin of replication (Bhattacharyya et al.,

1995; Bondeson et al., 1995; Cowie and Kamen, 1984, 1986; Peng and Acheson, 1997,

1998; Pomerantz and Hassell, 1984). DNA-bound large T antigen is believed to form

148 hexamers at site 1/2 where it subsequentiy unwinds origin DNA and initiates viral DNA replication (Lorimer et al., 1991 ; Wang and Prives, 199 1a). We have suggested that sites

A, B, and C play a role in accurnulating large T antigen molecules nearby the replication origin and "handing over" these protein molecules to site 1/2 duhg formation of hexamers (Peng and Acheson, 1998).

We previously showed that binding of polyomavirus large T antigen to its target

DNA is strongly dependent on pH (Peng and Acheson, 1998); binding is optimal at pH 6, and declines sharply above pH 7.4. Large T antigen-DNA complexes formed at pH 6-7 were stable at those pH values, but a fraction of complexes dissociated rapidly when diluted in buffers at pH 7.6 or higher, indicating that increased binding at low pH is due at least in part to increased stability of protein-DNA complexes (Bondeson et al., 1998;

Peng and Acheson, 1998). In previous reports, binding to DNA by polyomavirus or sirnian virus 40 large T antigen could be detected by gel mobility shift assays ody after fixation of protein-DNA complexes with glutaraldehyde (Dean et al.. 1987c; Lorimer et al., 1991 ; McVey et al.. 1996; Murakami and Hurwi~1993b). The enhuiced binding that we found at low pH encouraged us to try DNA band retardation with unfixed complexes using a pH 6 buffer during electrophoresis. Our results showed that this assay is a useful method for the detection of large T antigen-DNA complexes (Peng and

Acheson, 1998). The purpose of this study is to characterize the features of this method. RESULTS

Gel mobility shift assay of large T antigen-DNA complexes at pH 6: sensitivity and

specificity

Figure 22 illustrates the sensitivity of the gel mobility shift assay. Different

amounts of immunopurified large T antigen derived from a recombinant Pichia pastoris

strain (Peng and Acheson, 1997) were incubated with a 265-bp, 32~-labeledDNA

fragment containing al1 four binding sites in binding solution containing potassium

phosphate buffet at pH 6. Binding reactions were loaded onto a 5% polyacrylamide gel

and subjected to electrophoresis in pH 6 potassium phosphate buffer. Free DNA was

well separated fiom retarded DNA, which migrated as a band very close to the position of

the loading well. Figure 22 shows that as the concentration of large T antigen in the

binding reactions was incteased (lane 2-a), increasing arnounts of the retarded DNA band

were detected. As linle as 5 ng large T antigen in a 60-pl binding reaction mixture was suflicient ro shift the migration of a portion of the labeled DNA. and the migration of virtually al1 of the DNA was retarded in the presence of 100 ng large T antigen.

We further carried out a gel mobility shift assay with a mixture of specific and nonspecific DNA fragments in the same binding reaction (Fig. 23). The results show that

large T antigen cari sz!ectively bind to a 762-nt DNA containing G(A/G)GGCtarget sites in a mixture of other DNAs and retard its migration without affecthg the migration of nonspecific DNAs. Since in this experiment the specific DNA fragment represented only a small proportion of the labeled DNA present, the selectivity of the gel mobility shifi assay for specific DNA is high.

150 Fig. 22. Gel mobüity shift of large T antigen-DNA complexes subjected to

electrophoresis nt pH 6.0. Polyomavims large T antigen was purified from yeast

Pichia pastoris strain E-3 as described previously (Peng and Acheson, 1997, 1998). A

265-bp DNA fragment including polyomavirus nt 5267-5312/1-220 was made by PCR with the primers 5'-GTTCTAGCAGCCTTTCTTTG-3'(polyomavims nt 220-201) and 5'-

GTGTGGXnGCAAGAGGAAG-3' (polyomavims nt 5267-5287). Plasmid pABC 1/2+ (Sunstrom et al., 1991; Peng and Acheson, 1998) was used as template and

DNA was intemally labeled by incorporating (a-3'P)d~TPduring PCR. One nanogm of labeled DNA was incubated with different amounts of large T antigen (shown above figure) at 37'C for 20 min in 60 pl binding solution containing 100 mM potassium phosphate (pH 6). 50 rnM NaCl, 7 mM MgCl?, 1 mM dithiothreitol. 10 ng aprotinidml,

7. j% glycerol, 42 pg bovine serum aIbumin/ml. and 1.7 pg sheared calf thymus DNA/ml.

Samples were directly loaded onto a 5% polyacrylamide gel and electrophoresis was carried out in 50 mM potassium phosphate buffer (pH 6). 1 mM EDTA, for 2 hrs at 100 volts. The gel was dried and exposed to X-ray film.

Fig. 23. Gel mobility shift assay is specific for DNA containing binding sites for large T antigen. A Hinf 1 digest of plasmid pABClR+ was 3'-end-labeled with (a-

3'~)d~TPusing the Klenow fragment of DNA poiymerase, to provide DNA fragments containing or lacking specific binding sites. The labeled DNA fragments were incubated at pH 6 in the absence (lane 1) or presence (lane 2) of 100 ng purified large T antigen in the same binding reactions as described in Fig. 22. Electrophoresis was canied out at pH

6.0. Sizes of DNA fragments are shown at leh; the 762 bp fragment contains the polyomavirus origin region.

Since the polyomavirus large T antigen we used was purified from yeast Pichia pastoris, we asked whether DNA retardation at pH 6 was peculiar to this source. We

therefore purified polyomavirus large T antigen made in insect cells by a recombinant

baculovirus (Rice et al., 1987) which has been used extensively in other studies

(Bhattacharyya et al., 1995; Bondeson et al., 1995; Li et al., 1997; Moses and Prives,

1994; Rose and Schdfhausen, 1995), and performed a gel mobility shifi assay under the

same conditions as descnbed above. The result (Fig. 24, lane 1) was similar to results

shown in Fig. 22 for large T antigen derived from Pichia pastoris.

Application of gel mobility shift assay to crude lysates of cells expressing large T

antigen

Having defined the sensitivity and specificity of this method, we tested unpurified

Iarge T antigen from different sources. As show in Figure 24, 5 pl of nuclear extract

from large T antigen-expressing insect cells (lane 2), or 5 pl of ce11 lysates fiorn large T

antigen-expressing yeast ce11 lines E-3 (lane 3) and E-5 1 (lane 4), formed complexes with

labeled DNA that could be detected by our assay. However, a cell lysate from yeast

strain G-l 1, lacking a recombinant large T antigen gene, did not give rise to a distinct

retarded band at the position of the loading well (lane 5). Cnide ce11 lysates, which are expected to contain various non-specific DNA-binding proteins, gave rise to a smear of

labeled DNA throughout the gel, indicating the presence of many different nonspecific

protein-DNA complexes, but these are easily distinguished from complexes retarded

nearby the loading well. This method can therefore be used for detection of large T

153 Fig. 24. Use of gel mobility shift assays with crude lysates of cells expressing large T

antigen. Yeast ce11 lysates fiom transformants E-3, E-51 and G-11 were prepared as

described in Peng and Acheson (1997). Strains E-3 and E-51 were transformed with the

large T antigen gene, and showed different levels of large T antigen expression; suain G-

1 1 was transformed with vector DNA and did not express large T antigen. A baculovirus

expression system (Rice et al., 1987) was nlso used as an alternative source of large T

antigen. Recombinant baculovirus vEV5 I LT stock was generated in Spodoptera firgiperda (SB)cells. Large T antigen was expressed in High Five cells according to

published rnethods (Rice et al., 1987). Large T antigen was purified as described (Peng

and Acheson, 1997) From a nuclear extract of infected High Five cells. One nanogram of

"P-labeled 265-bp ongin DNA fragment was incubated with 50 ng purified baculovirus

large T antigen (lane l), 5 pl vEV5 1LT-infected High Five ce11 nuclrar extract (lane 2), 5

pl yeast ce11 lysates from E-51 (lane 3), E-3 (lane J), or G-1 1 (lane 5), as descnbed in Fig.

22. Electrophoresis was carried out at pH 6.0, and the gel was dned and analyzed with a

Molecular Dynmics PhosphorIrnager. 1. purified LT (Baculovirus-derived) 2. nuclear extract from Baculovirus-LT infected High Five cells 3. ceIl lysate from yeast strain E-51 (LF) 4. ce11 lysate from yeast strain E-3 (LV) 5. cell lysate from yeast strain G-11 (LT-) antigen expression in crude lysates of different ce11 types, avoiding costly and tinte- consuming assays based on use of specific antibodies.

Glycerol gradient aoalysis of large T antigea-DNA complexes

The retarded large T antigen-DNA complexes barely entered 5% polyacrylarnide gels (or l.j% agarose gels, data not shown), suggesting that they might be very large.

We therefore canied out experiments to determine the size of large T antigen-DNA complexes formed in buffers of different pH. Complexes were forrned by incubation of radioactive DNA with large T antigen in buffers at pH 6, 7, or 7.6. Binding reactions with or without large T antigen were sedimented on glycerol gradients made in the sarne buffers. The distribution of unbound DNA or large T antigen-DNA complexes was determined by measuring radioactivity in gradient fractions (Fig. 25). Thyroglobulin, a protein with a molecular weight of 669 kD, was sedimented in a parallel gradient as a size standard. Complexes formed at pH 7.6 sedimented slightly faster than unbound DNA, as shown by a shoulder in the regions of fractions 4-6 (only a fraction of the input DNA was bound under these conditions). Complexes formed at pH 7.0 sedimented more heterogeneously throughout the gradient, and some of these complexes were recovered from the pellet. Complexes formed at pH 6.0 also sedimented heterogeneously throughout the gradient, but a large fraction of these complexes were recovered fiom the pellet. In this case, very little DNA remained unbound, as little radioactivity sedimented in the first few fractions of the gradient. These results suggest that large T antigen forms very large complexes with target DNA at pH values of 7 or below, and that the size of the

155 Fig. 25. Glycerol gradient analysis of large T antigen-DNA complexes formed at pH 6, 7, or 7.6. Five nanograms (13,500 cpm) of a '*P-labeled 265-bp DNA fragment were incubated at 37'C for 20 min in the presence or absence of 500 ng large T antigen in

120 pl binding buffer at pH 6.0, 7.0 or 7.6. Reactions were loaded ont0 3.8 ml 840%

(w/w) glycerol gradients made in buffers of the sarne pH values, but without bovine serum aibumin. Gradients were centrifugcd in a Beckman SW-60 rotor at 35,000 rpm for

60 min at 20°c,and 20 195-pl fractions were collected from the top. The bottom of the centrifuge tube was washed with 195 pl 2% sodium dodecyl sulfate, which was treated as fraction 21. Radioactivity in each fraction was measured by scintillation counting.

Thyroglobulin (M. Wt. 669 kD) sedimented in fractions 3-5 in a similar gradient. Cpm in fractions 1-3 (top of gradient) for DNA alone, DNA plus large T antigen at pH 7.6, and

DNA plus large T antigen at pH 7.0 are not show to allow better dispiay of remaining fractions. Pellet (fraction 21) for DNA plus large T antigen at pH 6.0 contained 4244 cpm. -o- DNA alone +pH 7.6, DNA+LT -t. pH 7.0, DNA+LT +pH 6.0, DNA+LT

O 5 10 15 20 Fraction complexes increases as the pH decreases. Most of the complexes fonned at pH 7 or below sedimented substantially faster than thryoglobulin, which sedimented in fractions

3-5 in gradients like those shown in Fig. 25; we estimate that the larger complexes may contain hundreds of large T antigen molecules.

We further asked whether large T antigen also cm self-associate at low pH in the absence of DNA, by incubating large T antigen alone in binding reactions at pH 6, 7, or

7.6 and centrifuging the protein in glycerol gradients of the corresponding pH. Large T antigen was detected by immunoblotting aliquots of each fraction. The results (not shown) indicated that large T antigen by itself foms large complexes upon incubation in buffers below pH 7, but not at pH 7.6. Both polyomavirus and simian virus 40 large T antigens have been found to form oligomers (Runzier et al., 1987; Wang and Prives,

1991a); however, formation of large complexes of protein has not been described previously. The mechanism by which low pH facilitates this self-association is unknown.

Influence of binding sites on gel retardation

Lady. we asked whether the formation of these large complexes depended on the presence of the multiple binding sites in the origin region. DNA fragments made from a series of origin mutants, which carry point mutations in the severai consensus pentanucleotide sequences in each of sites A, B, or C, or in combinations of these sites

(Bertin et al., 1993; Peng and Acheson, 1998), were used to perform the gel mobility shift assay. The results (Fig. 26) showed that origin DNA fragments with one or more of sites

A, B, and C mutated were dl retarded, when bound to large T antigen, to a position

157 nearby the loading well, implying that the size of the complexes does not depend on the number of binding sites on DNA. However, DNA with fewer binding sites bound large T antigen less eficiently, as show previously by other methods (Peng and Acheson, 1998). Fig. 26. Binding of large T antigen to DNAs containing mutated binding sites.

Mutants generated by introducing point mutations into consensus pentanucleotide sequences in each of sites A, B, or C, or combinations of these sites were described previously (Bertin et al., 1993; Peng and Acheson, 1998). Mutants are named for the mutated sites they contain; for example, mAmB DNA has mutations in the four pentanucleotides present in sites A and B. PCR amplification of die regions between nt

220 and 5267 for each mutant was cked out as in Fig. 22. Six femtomoles of labeled

DNA fragments were incubated at pH 6.0 with 100 ng large T antigen, and binding reactions were loaded ont0 a 5% polyacrylamide gel nui in pH 6.0 phosphate buffer. Bound DNA

Free DNA DISCUSSION

The gel mobility shift assay we describe here is simple, sensitive, and inexpensive. The binding buffer contains only NaCl, MgCl,, a reducing agent and a pH 6 potassium phosphate buf%er,in addition to protease inhibitors and nonspecific competitor

DNA. For elertrophorrsis, we used a bbuffer of 50 mM potassium phosphate, 1 mh.I

EDTA. Target DNAs can be made by PCR and labeled either at 5' or 3' ends or intemally. Previously-described gel mobility shifi assays with large T antigen need glutaraldehyde fixation and are much less sensitive. At least 100 ng purified large T antigen was required to retard the migration of a detectable arnount of DNA (Dean et al.,

1987c; Lorimer et al., 1991; Murakami and Hurwitz, 1993b; McVey et al., 1996), whereas our assay could easiiy detect less than 5 ng of large T antigen. The simplicity of this assay and the ease with which quantitative results can be obtained make it ideal for detection of large T antigen in ce11 extncts, or for quantitative studies on the effects on specific DNA binding of mutations in DNA targets or in the amino acid sequence of large

T antigen. It should also be possible to use this gel mobility shifi assay for selection of sequences in complex cellular genomes that bind with high aflïnity to large T antigen. CHAPTER 6. SUMMARY AND CONCLUSIONS

Many fundamental questions in eucaryotic molecular biology have been examined by using large T antigens. Large T antigen is the major regdatory protein in the polyomavirus life cycle. Although not as well charactenzed as the corresponding large T antigen rncoded by SVJO, it is nonetheirss an interesting modei protein for studies of the control of eucaryotic transcription, DNA replication and ce11 groivth. In this thesis, we have utilized yeast-derived polyomavirus large T antigen to understand how this protein intencts with the viral origin of DNA replication. Previous studies showed that binding of large T antigen to the viral origin is mediated by a number of G(A/G)GGC pentanucleotide sequences, which are conserved in both SV30 and polyomavirus replication origins and are recognized by both large T antigens (Cowie and Kamen. 1984;

Pomerantz et al.. 1983; Pomerantz and Hassell. 1984; DeLucia et ai.. 1983; Jones et al.,

1984). Four distinct sites in the polyomavirus origin region. sites A, B, C. and 1/2. each containing two or more pentanucleotides. had been shown to be protected by large T antigen against DNase 1 digestion (Cowie and Karnen, 1984; Dilworth et al., 1984). Our studies showed that binding of large T antigen to the four sites is cooperative, and that specific origin DNA binding by large T is strongly afYected by pH. In the discussion that follows, 1 will recapitulate the conclusions drawn in Chapten 3, 4, and 5 with occasional reference to relevant studies and to possible fuhüe experiments that may address unanswered questions. 1. Expression of large T antigen in Piclriapastoris

In Chapter 3, we described a yeast Pichia pastoris system which was used for production of active polyomavirus large T antigen. The methylotrophic yeast Pichio pastoris has been developed as production systern for recombinant proteins. The favorable and advantageous characteristics of this species have resulted in an increasing number of biotechnolopical applications. As a consequence, Pichia pastoris is rapidiy becoming the system of choice for heterologous eucaryotic gene expression in yeast (for review, see Gellissen and Hollenberg, 1997; Hollenberg and Gellissen, 1997; Sudbery,

1996). Large T antigen expressed in Pichia pastoris migrates in polyacrylarnide gels with an apparent molecular weight of 100 kD, as does authentic large T antigen produced in mouse cells infected by polyomavinis (Fig. 9). The protein cm bind to viral origin

DNA specifically and protect the four distinct binding sites, as well as does authentic large T antigen (Figs. 8 and 10). Moreover, yeast-derived large T antigen can unwind double-stranded DNA molecules containing viral ongin sequences (Fig. 1 1). Because

DNA unwinding is carried out by large T antigen hexarners (Wessel et ai., 1992a; Wessel et al.. 1992b) and requires an intact zinc finger structure, ATPase fiction, and domain for ATP binding (Loeber et al., 199 1; Lorimer et al., 1993; Rose and Schaffhausen, 1995;

Wang and Prives, 1991b; Wun-Kim and Simmons, 1990), we conclude that the yeast- denved large T antigen is biologically functional. The yeast transfomants we obtained are genetically stable. The relatively high level expression (-1 mg protein per liter of culture) and the ease of culturing yeast cells make this system cheaper and easier than the baculovirus expression system, which is widely used currently for production of both

162 SV40 and polyomavirus large T antigens for in vitro studies (Lanford, 1988; Rice et al.,

1987).

However, ou overall expression level is fa lower than that reported for other proteins using the Pichia pastoris system (Cregg et al., 1987; Sreekrishna et al., 1989;

Clare et al., 1991; Faber et al., 1995; Koutz et al., 1989; Barr et al., L 992). It is possible that expression of large T antigen in the yeast cell exerts some negative effecrs on ce11 growth or protein expression itself. Previous studies showed that SV40 large T antigen expressed in Saccharomyces cerevisiae affects the ce11 cycle (Nacht et al., 1995). Yeast cells expressing SV40 large T antigen showed morphological alterations as well as growth inhibition. In vivo cross-linking experiments further showed that large T antigen coimmunoprecipitated p34--' from extracts of large T antigen-expressing yeast cells and altered the kinase function of p34CD"'(Nacht et al., 1 995).

To puri@ the protein, we used an immunoaffinity protocol with monoclonal antibody F5 that recognizes the HPDKGG box in polyomavirus large T antigen (Pallas et al., 1986; Pipas, 1992). This method is adapted fiom one used to purify large T antigen expressed in insect cells (Dixon et al., 1985; Wang and Prives. 1Wl), and is probably the most efficient one presently available. However, it is expensive to produce monoclonal antibodies, and scaling up the purification procedure by a factor of 10 or IO0 would therefore be difficult. We inserted a sequence coding for a polyhistidine tag at the N- terminus of large T antigen into the viral genome, in an attempt to make a protein that can be purified by aflïnity chromatography on a nickel resin. However, this insertion rendered the virus nonviable (data not shown). Presumably, the modified large T antigen

163 is not properly folded, or the insertion causes other lethal changes in the virus. To render

protein purification easier and cheaper, it might be worth trying expression in Pichia pastoris of recombinant large T antigen with a polyhistidine tag, inserted at the N-or C- terminus or even intemally.

Attempts to crystallize large T antigen have so far been unsuccessful, probably

because of hrterogeneity resulting from nonuniforni post-translationai modifications

(Cole, 1996). We do not know how our large T antigen is modified. Post-translational modifications are consewed between yeast and higher eucaryotes (Faber et al., 1995;

Romanos et al., 1991). The phosphoproteins fos and c-rnyc are correctly phosphorylated

in Saccharomyces cerevisiae (Miyamoto et al., 1985; Sarnbucetti et al., 1986). Possibly,

Pichia-derived large T antigen is more homogeneous than large T antigen preparations

from mammalian cells. It will be of interest to generate suficient arnounts of yeast-

denved large T antigen to try crystallization.

11. pH effect on origin DNA binding by large T antigen

To characterize the DNA-binding activity of purified polyomavirus large T antigen preparations, we examined the influence of different components in the binding

reaction. The results presented in Chapter 4 show that specific DNA binding by

polyomavirus large T antigen is optimal at pH 6 to 7 and is 10- to 20-fold lower at the

more commonly-used pH 7.6. We stumbled upon this discovery via an emin

experimental design. We originally added unbufTered ATP solutions to DNA binding assays and found strongly increased binding as a fiuiction of ATconcentration;

164 however, unbuffered ATP solutions also reduced the pH of the binding reaction.

Elimination of ATP and the use of buffers at different pH values revealed that specific

DNA binding was optimal at pH 6.

In our studies, specific DNA binding by polyomavims large T antigen as a function of pH was shown by: 1) low levels of binding to DNAs lacking G(A/G)GGC binding sites, using a filter binding assay (Fig. 12); 2) specific gel retardation of an origin

DNA fragment in the presence of a number of other nonspecific DNA fragments (Fig.

23); and 3) generation of the same DNase I footprint by binding of large T antigen to labeled origin DNA at pH 6 (Fig. 14B) as that seen at pH 7.6-7.8 (Cowie and Kamen,

1984, 1986; Lorimer et al., 1991). Increased binding is independent of the buffer used to

Vary pH. since Tris-HCI, sodium acetate, and potassium phosphate buffers were used in different experiments with similar results.

How does reduced pH enhance specific DNA binding by large T antigen?

Analysis of large T antigen-ongin DNA complexes showed that a fraction of complexes fomed at pH 6 dissociate rapidly upon dilution into a pH 7.8 buffer. but dilution into low pH buffer led to only very slow dissociation (Fig. 1S), indicating that the protein-DNA complexes are stable at low pH but unstable at pH 7.8. This observation was codinned by studies using surface plasmon tesonance to record interactions of large T antigen with binding site C (Bondeson et al., 1998). Therefore, the effect of pH on DNA binding is likely to result at least in part fiom the increased stability of large T antigen-DNA complexes at low pH values.

Increased stability of protein-DNA complexes could result fiom increased affinity

165 of large T antigen for individual G(A/G)GGC consensus sequences on target DNA.

Specific DNA binding by SV40 large T antigen has been shown to be mediated by a limited nurnber of amino acids (in regions A and B2) as descnbed in Chapter 1 (Simmons et al., 1990a); these residues are highly conserved between SV40 and polyomavirus large

T antigens (Fig. 4) (Simmons et al., 1990b). NMR studies of the SV40 DNA binding domain indicate that a involving a histidine residue (His203) is important for binding to the G(A/G)GGC motif (Luo et al., 1996). Since this region, including the histidine residue (His356), is conserved in polyomavirus large T antigen, it is possible that the decreased stability of polyomavirus large T antigen-DNA complexes at high pH is caused by titration of imidazole groups in this histidine residue (Bondeson et al., 1998).

Altemativeiy, stimulation of binding could result from an effect of pH on the interactions between large T antigen molecules in such a way as to facilitate specific

DNA binding by affecting protein-protein interactions. Indeed, glycerol centrifugation analysis indicated that large T antigen, either on DNA (Fig. 25) or by itself (data not shown), foms large complexes upon incubation in buffers below pH 7.0. but not at pH

7.6. In addition, gel mobility shifi assays showed that the DNA-large T antigen complexes were too big to migrate into the polyacrylamide gel (Figs. 13,22,23,24). The mechanism by which low pH facilitates this self-association is unknown. Since complexes formed between baculoviw-expressed large T antigen and viral origin DNA were also retarded on the top of the gel in a gel mobility shift assay (Fig. 24, lane l), it is not likely that this oligornerization is unique to yeast-derived large T antigen.

Oligomerization of DNA-binding proteins is common. It was found that

166 cooperative non-specific DNA binding by lambda cl repressor can only be carried out by octamers, suggesting that the state of oligomerization of the repressor molecule modulates its affinity for DNA sequences (Pray et al., 1998). The self-association and cooperativity of the CIrepressor is rnediated by a C-terminal domain, while an N-terminal domain mediates DNA binding (Pray et al., 1998). Studies with mutant super-repressor proteins of the ûp repressor systern of E. coli damonstrated that increased operator binding by the super-repressor resulted from protein oligomenzation on DNA; and the oligomerized protein molecules have an aitered conformation (Reedstrom et al., 1996).

Purified eucaryotic transcription terminator factor TTF-1 was dso show to form aggregates by glycerol gadient sedimentation analysis (Sander et al., 1996). Self- associated TTF-1 has a lower DNA binding activity unless the N-terminal 185 amino acids, which mediate the protein oligomenzation, are removed (Sander and Grummt.

1997; Sander et al., 1996).

Our studies indicated that oligomenzed large T antigen molecules at pH 6 have an increased specific DNA binding activity. It is possible that, at low pH, some arnino acid side chains become positively-charged, therefore enabling interaction with negatively- charged amino acid side chains. Altematively, hydrophobic domains may become exposed at low pH, resulting in mutual interactions between large T antigen molecules.

Low pH may also decrease the net charge of large T antigen molecules, preventing charge repulsion between molecules. The caiculated isoelectric point of polyomavirus A3 large

T antigen (without modifications) is 6.35; at this pH, the net charge of the protein should be zero. SV40 large T antigen has been shown to form aggregates at pH 7.5, in the

167 Figure 27. Oligomerization and DNA binding of polyomavirus large T antigen.

Large T antigen monomers can interact with each other to fonn oligomea. The

interactions stabilize binding of large T antigens to target DNA, resulting in cooperative binding to multiple sites in the viral origin of DNA replication. ATP stimulates hexamer

formation of large T antigen, either in solution (upper right) or on DNA (lower right).

Low pH increasrs oiigomerization of large T antigens (iower lefi). Oligomers of large T antigen have multiple adjacent DNA binding domains, and can therefore bind more strongly to DNA than rnonomers. Hexarners formed in solution do not bind to DNA well because the DNA binding domains are not exposed to the pentanucleotide sequences on

DNA (Dean et al., 1992). This figure was made by Dr. Nicholas H. Acheson. ATP L

Hexamei Monomers LDNA Low pH 1 "*f

Oligomen

f Hexamer on DNA presence of magnesium ions; 5 mM MgCl, causes more aggregation than 2 mM MgCl,,

in the absence of other salts (Wessel et al., 1992a). Mg" could act as a bridge to bring

adjacent large T antigen molecules together. In our studies, 7 mM Mg", in addition to

50-1 00 mM potassium phosphate or Tris-HCI buffers and 50-100 mM NaCI, was present

in al1 binding reactions. We do not know if ~g'-plays a role in large T antigen oiigomeriwtion.

Large T antigen molecules oligomerized at low pH could bind efficiently to target

DNAs because multiple DNA binding domains would be pre-aligned to bind to multiple

adjacent target pentanucleotides (see Fig. 27). DNA binding by monomen in solution, at

higher pH, would be less efficient because individuai monomers would have to align, one

by one, on the target DNA to form a stable cornplex. It will be of interesr to identify the

domains on polyomavirus large T antigen which mediate this pH-dependrnt protein

oligomeriwtion, and to determine if cooperative binding also depends on these domains.

III. Applications of the pH effect on large T antigen DNA binding

Previous gel mobility shift assays of either polyornavirus or SV40 large T antigen

required prior fixation of protein-DNA complexes (Dean et ai., 1987; Lorimer et al.,

1991; McVey et al., 1996; Murakami and Hurwitz, 1993b). Our findings show that large

T antigen binds with low affinty at pH 7.6, and upon electrophoresis at this pH, the

complexes dissociate. The use of pH 6 for binding and electrophorisis under the

conditions we described allows band retardation that is highly specific for target DNAs.

The simplicity of this assay and the ease with which quantitative results can be obtained

169 make it ideal for studies on the effects on specific DNA binding of mutations in the binding sites on DNA targets or in the amino acid sequence of large T antigen.

Application of this method to crude lysates of cells expressing large T antigen avoids costly assays based on use of specific antibodies for detection of protein expression

(Chapter 5).

Li rnay be possible to puri& large T antigen by binding to immobilized DNA at low pH followed by release fiom the DNA at high pH. A number of DNA binding proteins, including various RNA and DNA polymerases, hormone recepton, and repressors, have been purified by nonspecific DNA-cellulose and DNA-agarose af5nity chromatography (Arndt-Jovin et al., 1975; Kadonaga and Tjian, 1986; Rosenfeld and

Kelly, 1986). SV40 large T antigen was purified by chromatography with DNA-cellulose

(Oren et al.. 1980), while a variation of the DNA-cellulose method was used to enrich for polyomaims large T antigen (Gaudny et ai.. 198 1). In those studies. large T antigen was separated from other cellular proteins by differential affinities to DNA at different salt concentrations. We prepared a DNA-cellulose column by conjugating a 3823 bp linear

DNA containing the polyornavirus origin region to fibrous carboxymethyl cellulose

(Whatman) (Poni2ak and Dean, 1978; Potuzak and Wintersberger, 1976). Binciing of large T antigen at pH 6 to this column was very efficient. However, Tris-HCI buffers of pH 8, 9, and 11 (containing 7 rnM Mg2+)failed to elute the protein as eficiently as expected, although about 30% of the bound protein came off the column at pH 11 (data not shown). Other factors such as ionic strength and temperature may play roles in the dissociation of large T dgen fiom immobilized DNA. Since magnesium has been

1 70 show to cause aggregation of SV40 large T antigen on nonspecific DNA (Wessel et al.,

1992a), elimination of magnesium fiom the elution buffer might help the elution of large

T antigen. Further experiments are required to characterize this method.

IV. ATP effcct on origin DNA binding by large T antigen

As noted in Chapter 1, previous reports showed that ATP, in the presence of Mg',

strongly stimulated binding of both polyomavirus and SV40 large T antigens to their

respective origin DNAs when binding reactions were carried out at 37OC at pH 7.5-7.8

(Borowiec and Hurwitz, 1988; Dean et ai., 1987; Deb and Tegtmeyer, 1987; Lorimer et

al., 199 1 ). ATP is required for generation of hexarneric forms of SV40 and polyomavinis

large T antigen. either in solution or at the replication origin (Mastrangelo et al., 1989;

Reynisdottir et al.. 1993; Wang and Prives, 199 la); these forms enable initiation of DNA replication at the origin (Fanning, 1992). Our results show that ATP had no effect on the

interaction of polyomavirus large T antigen with a polyomavirus DNA fiapent containing al1 four binding sites (112. A, B. C), when the binding reaction was carried out at or below pH 7 in Tris or potassium phosphate buffer, 7 mM Mg", at 37'~(Fig. 14).

ATP was also show to have a diminished effect on binding by SV40 large T antigeen to origin sequences in a pH 7 buffer at O°C compared with a pH 7.5 buffer at 37'C (Deb and

Tegmeyer, 1987). These results suggest that binding by large T antigen to DNA containing G(G/A)GGC target pentanucleotides is independent of ATP at pH values between 6 and 7, where large T antigen tends to form oligomers. However, at pH 7.5-7.8 and 37"C, binding to target DNAs is less efficient because large T antigen is less stably

171 bound. ATP is proposed to alter the conformation of bound large T antigen to form

stably bound complexes at the ongin region (Dean et al., 1987~;Mastrangelo et al., 1985;

Dean et al., 1992; Mastrangelo et al., 1989; Panons et al., 1991). nie conformational

change effected by ATP to increase binding at high pH may well be the same change

induced by ATP that stimulates hexamer formation, and increased binding may simply

result from the formation of large T antigen hexamers on a proportion of the bound DNA

target molecules. Observations that ATP stimulates large T antigen binding to DNA

contiiining site 1/2, but not to DNAs lacking site 1/2 (Fig. 15D) indicate that the ATP

effect is focused on a DNA domain where pentanucleotides are amnged to facilitate

hexarner formation.

Our observations also suggested that experiments on the effects of factors on

DNA binding by polyomavirus large T antigen should take into account the sharp

increase in specific DNA binding activity as the pH decreases from 8.0 to 7.0; if binding

assays are done in that pH range, it is possible that added components may change

binding afinity by means of subtle changes in pH. Such studies should preferably be carried out near the optimai pH for binding (for example, pH 7.0), where small changes in pH will not strongly affect binding; or strong buffer systems should be used to maintain the desired pH.

V. Cooperative binding of large T antigen to viral origin of DNA replication

Using a series of point mutants and deletion mutants in the viral regdatory region, we demonstrated that binding of large T antigen to sites C, B, A, and 1/2 is cooperative

173 (Chapter 4). Binding of protein-DNA complexes to nitrocellulose filters, gel retardation assays, and DNase I footprinting assays showed that binding of large T antigen to one site stimulated binding to other sites 20 to 100 nt distant, and that binding to inherently weak sites is strengthened if two or more such sites are present on the same DNA molecule.

These findings suggest that large T antigen molecules bound to DNA interact with each other to mutually stabilize their binding. We proposed a mode1 in which large T antigen molecules bound to sites C, B. and A are handed over to site 112, where hexamers are formed, via these protein-protein interactions (Fig. 20). Cooperative binding to adjacent binding sites is a common feature among viral origin-binding proteins. For example, the

Epstein-Barr virus nuclear antigen 1 (EBNA 1) assembles cooperatively on the viral latent origin of replication (Summers et al., 1996); the origin binding proteins El and E2 of papillomavirus also bind cooperatively to the viral ongin of replication. which contains binding sites for both proteins, forming an El-EZ-ori complex which is essential for initiation of DNA replication (Berg and Stenlund, 1997; Le Moal et al., 1994; Sedman et al., 1997). In addition, experiments measuring the binding of SV40 large T antigen to viral origin DNA sequences suggested that the binding of the protein to site 1 influences its binding to sites II and III (Myers et al., 198 la). Using cloned origin DNAs containhg al1 three sites, or only sites II and III in DNase 1 protection experiments, these authors demonstrated that SV40 large T antigen bound to its sites in a sequential and cooperative manner. In the absence of site 1, the binding of large T antigen to sites II and III was markedly decreased. Therefore, site 1 appeared to facilitate the binding of SV40 large T antigen to sites II and III. Parsons et al. (1991) reported that SV40 large T antigen

173 assembles into a double hexamer complex at the core origin (site II) in a cooperative fashion. Binding to the early half of the origin stimulates binding to the late half of the origin (Parsons et al., 199 1).

Cooperative bixiding of proteins to DNA is often mediated by specific protein- protein interactions. In previous DNA binding studies, Sunstrom (1991) showed that

Full-iength polyomavirus large T antigen bound more sirongly to DNA fragments containing al1 binding sites (1/2, A, B, and C) than to fragments missing one site (1/2AB or ABC), in agreement with our results. However, an amino-terminal deletion mutant of large T antigen, NI 75 (lacking the N-terminal 175 arnino acids), did not show increased binding to I/ZABC compared with 1QAB or ABC. This observation, carried out with cmde extracts using an immunoprecipitation assay, indicates ihat the integrity of the arnino terminus may be required for cooperative binding to the multiple sites. Lack of cooperative binding of the mutant NI75 may be caused by the absence of a contact sire needed for protein-protein interaction with 0th large T antigen monomers, thereby preventing the formation of oligomers. Future experirnents should test cooperative binding and oligomerization of purified large T antigens with different deletions in the N- terminal region.

In figure 21 of Chapter 4, we suggested a pathway for binding of large T antigen to the origin region and hexamer formation at site 1R. The cooperative binding of large

T antigen to its multiple target sites, coupled with the reversibility of binding at intracellular pH values (pH 7.1 to 7.3), suggests a role for sites A, B and C in polyomavirus DNA replication. Sites A, B and C may act as auxiliary elements for viral

174 DNA replication, a role compatible with the attraction of large T antigen to the origin via revenible binding following by transfer to site I/2 on polyomavirus DNA. It has been show that replication could be enhanced by a factor of 10 when binding site B or binding site C was also present on a plasmid containing sites 112 and A (Weichselbraun et al., 19 89). S ite-directed mutagenesis showed that mutation of consensus pentanucleorides in sites A, B, and C gave risr to srnalier plaques. aithough these mutant viruses are still viable (Bertin et al., 1993). Both in vivo studies indicate that sites A, B, and C increase the efficiency of viral DNA replication.

VI. Formation of hexamers of large T antigen at site 112.

As introduced in Chapter 1, formation of hexamers of laree T antigen on site 1/2 is essential for initiation of viral DNA replication. Hexamers diston the double-stranded

DNA in the core origin and act as a helicase in DNA replication. In the case of SV40, double hexamers are fonned at site II before initiation of bidirectional DNA replication

(Borowiec and Hurwitz. 1988b; Dean et al., 1987~;Masuange10 et al., 1989). However, there is no direct evidence showing large T antigen hexamer Formation at the polyomavirus DNA replication origin. Our DNase 1 footprinting analysis indicated that, in the presence of ATP, large T antigen preferentially protects the central two overlapping pentanucleotides of site 112 when the protein concentration is at an unsanirating level

(Fig. 19). Our observations, combined with structural differences between the polyomavinis and SV40 core ongins (Fig. 3), as well as Kh(ln0, modification resdts

(Fig. 5) fiom Bhattachayya et al. (1995), suggest a single hexamer mode1 which is

175 presenied in Chapter 4 (Figs. 20 and 21). According to our model, large T antigen hexamer formation in polyomavinis core origin is sequentiai. One hexamer is formed first in the center of site 112; this hexamer distorts the core origin DNA and then moves upstrearn to unwind DNA, leading to initiation of DNA replication on the early strand.

Tnis would be followed by assembly of a second hexamer at site 1/2 and this hexamer would initiatc replication on the late strand.

The single hexamer model can explain why polyomavirus DNA replication begins near nt 30 on the early strand but some 16 nt beyond (nt 46) on the late strand (Fig. 3A)

(Hendrickson et al., 1987). If the two hexamers are not formed simultaneously, DNA replication could initiate at different locations. In the case that a second hexamer fails to form, viral DNA replication would be unidirectional. Such unidirectional rolling-circle replication was indeed noted in previous studies (Bjurseil, 1978). However, some questions about this rnodel remain. If a double-stranded conformation is required for the correct formation of the second hexamer, what is the mechanism for the central palindrome to rernain double-stranded while the PuPy strand bias region and NT-rich region are unwound or untwisted? Double hexamers form at site II of the SV40 core origin, in which the four copies of pentanucleotides are separated by 1 nt; adjacent pentanucleotides are separated by 2 nt and the central two pentanucleotides overlap by 2 nt in site 1/2 of the polyomavinis core ongin. If the inability of polyomavinis large T antigen to form double hexamers is caused by structural differences on target DNA, might it be possible to assemble double hexamen by changîng spacing beîween the pentanucleotides in site 112? In vitro studies on formation of large T antigen hexamers on

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