Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.

Vol. 61,No. 10

Studies

on

the

Origin-Specific

DNA-Binding

Domain

of

Simian Virus 40

Large

T

Antigen

MICHAEL STRAUSS,tPEDRAM ARGANI, IAN J. MOHR, AND YAKOV GLUZMANt* ColdSpring HarborLaboratory, ColdSpring Harbor, Netw York 11724

Received 23 March1987/Accepted 29 June 1987

Theorigin-specific DNA-bindingdomain of simian virus 40largeTantigenwasanalyzed,and itsC-terminal boundarywasfound to beatorbeforeamino acid 259. This doesnotinclude the zincfingerstructuralmotif locatedatamino acids 302to320(J.M.Berg,Science232:485-486, 1986). Interestingly,N-terminalfragments of 266 and 272 amino acids andlargerdisplayeddramaticallyreducedorigin-binding activity.Inaddition, the specific DNA-binding propertiesof truncated proteins purified from both bacterial and mammalian sources

werecompared.Truncated T antigensfrom mammalian cells bound specificDNAfragmentsmoreefficiently

than did their bacterial counterparts. These results implicate posttranslational modification with a role in regulatingtheDNA-binding activityoflargeT antigen.

The multiple activities ofa single polypeptide chain are

often found sequestered within discrete domains, each of which is responsible for a particular function. The large T antigenencoded bySV40 providesanexcellent opportunity toexaminethisphenomenon. Thismultifunctionalproteinis involved in both lytic infection of permissive cells and malignanttransformation ofavarietyof cells(3,34);

further-more, extensivegenetic analysishas revealed that several of its functions operate independently of each other(4, 6, 10, 12, 13, 21, 22, 30).

Several of the many functions performed by large T antigen, namely initiation of viral DNA replication and autoregulation ofearly transcription (34), are mediated by specific DNA binding to two major sites, one of which

contains the viral origin of replication (5, 23, 31, 33). Previous mutational studies have implicated amino acids between residues 139 and 220 in specific DNA binding (13, 20); however, other genetic data suggest that amino acids beyond residue 300 areinvolved in specific bindingto viral DNAsequences. Whereasfragments containing 399 and 362

N-terminal aminoacids bindtotheviral origin ofreplication,

a smaller fragment containing 341 amino acids lacks this

activity (4). Furthermore, truncated T antigens containing theN-terminal 272 amino acids have been reported to lack specificDNA-binding activity (20, 29). These genetic studies

are complemented by results obtained from partial

proteo-lysis, sinceafragment spanning amino acids 131to371was

produced which retained the ability to bind to viral origin

sequences(27). This 240-amino-acid DNA-binding fragment islarge when compared withotherDNA-bindingdomainsof less than 100 aminoacids in proteinsofeitherprocaryoticor eucaryotic origin (1, 7, 9, 11, 15, 17, 19, 28). It therefore

seemsplausible that the region oflarge Tantigenresponsible forbinding totheviral origin would beofsimilar size. This prompted us toundertake a study aimed atfurther defining the limits of the T-antigen-specific DNA-binding domain,

*Corresponding author.

tPresent address: Zentralinstitut fur Molekularbiologie, 1115 Berlin-Buch, German Democratic Republic.

i Present address: Lederle Laboratories, American Cyanamid Co.,Pearl River, NY 10965.

with the ultimate goal of isolating and characterizing the minimal specific DNA-binding peptide domain.

Construction of BAL 31 deletion series. The ATG codon (nucleotide [nt] 5163) of a cDNA copy of simian virus 40 (SV40) large T antigen-coding sequences was converted to anNdeI site by oligonucleotide-directed point mutagenesis.

This cDNAwasisolatedas anNdeI-BamHIfragment, which

was directionally cloned into a T7 expression vector (32) between the NdeI and BamHI sites. This places the T-antigen cDNA under control of the T7gene10promoterand transcriptional terminator. This construct, pT7Tag,

pro-duces full-length T antigen in Escherichia coli (R. D.Gerard,

I. J. Mohr, M. Strauss, P. Argani, B. Stillman, and Y. Gluzman, manuscript in preparation) and served as the

starting materialforoursetofBAL 31 deletions.

Plasmid pT7Tag (Gerardetal., inpreparation)wascutat its unique PvuII site (SV40 nt 3506), and 5 ,ug of linear plasmid was treated with 1 U of BAL 31 (New England BioLabs)for2, 4, 8,or 12min.Thelongestincubation time

resulted inanaverage size decrease ofabout 600 basepairs. All four fractions were combined, phenol treated, and cut withBamHI toremovetheC-terminal partof thegeneatthe transcription terminator (32), and the endwasfilledwiththe

Klenowfragmentof DNApolymerase Iinthepresenceof all

four deoxynucleotides for 20 min at room temperature. End-filled DNA (0.5

Vg)

was incubated with 1 pLg of

nonphosphorylated Xba stop linker (see below for the

se-quence of this 14-mer; synthesized by Mark Zoller, Cold Spring Harbor Laboratory) and 10 U of T4 DNA ligase overnightat 14°C. CompetentE.coliDH5wastransformed,

and plasmids containingtheXba linkerwere usedto

trans-formtheBL21(DE3) expressionstrain(32). Extracts of 1-ml cultures(preparedbylysozymetreatment)werechecked for

origin-binding activity. Sequencing was done from the

BamHI site (adjacent to and downstream from the Xba linker) by the chemical degradation technique(14).

Constructionofrecombinant adenoviruses. The adenovirus vectorsused contained the coding regionof the truncated T antigens, starting from the HindIII site (nt 5171), covering theXba stop linker, and ending with aBantHI site derived

from pT7Tag. ThisBamnHI site wasfused tothe BclI site of the SV40 BclI-BamHI fragment, which contains the 3326

JOURNAL OFVIROLOGY, OCt. 1987, p.3326-3330 0022-538X/87/103326-05$02.00/0

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NOTES 3327

A A

Al; ( (;I.! - .111 .;6'S J'i. (S irl

t .I_

266r

+-''

cf-

3<-Z t ;Z:,a inc ta

B 272 266.1 259.3 708

__

E XE H 7

68--

55-

43-

25-FIG. 1. (A) Sequencesof truncated Tantigensin the vicinityof stopcodons. The top shows truncations createdbyfusion ofHindlll

sites atnt 4002 and 3476; the middle and bottom show truncations

generated byBAL 31treatment.Thefirstnucleotideisno.4043(1). CapitallettersrepresentSV40nucleotideoramino acid sequences. Lowercase letters represent nucleotidesequencesderived from the Xba stop linkeroradditional amino acids translatedfrom the linker

nucleotides.Thepresenceof additional amino acids translatedfrom the Xba linker sequence is reflected in the nomenclature of the

truncated proteins. Thus, 259.3 refers to atruncated protein

con-taining the N-terminal 259 amino acids from SV40 large Tantigen

and 3 additional amino acidsprovided byXbalinker sequences. The asterisks indicate carboxy termini of the proteins. (B) Purified T

antigens producedin E. coliand human HeLa cells. Sodiumdodecyl

sulfate-12.5%polyacrylamide gelofproteins (0.5to 1.5 ,ug) purified

from E. coli (E) or HeLa cells (H). The gel was stained with

Coomassie brilliant blue. The numbersonthe left indicate molecular size in kilodaltons.

polyadenylation signal. The HindIII-BamHI fragment was

expressed behind the major late promoter and one late mRNA leaderof adenovirus type5 after insertionintoearly region I ofa nondefective adenovirus vector (Y. Gluzman, manuscript in preparation).

DNA-binding reactions. Plasmid pOS1 contains the HindlIl (nt5171)-to-Ddel (nt 5228) fragment ofSV40DNA cloned between the HindlIl and BanII (nt 489) sites of

pAT153.The DdeI and BanII endswerefilled inby Klenow

polymerase before ligation. An equimolar mixture of three

origin-containing plasmids (pSVO +, which contains the

wild-type [wt] origin, consistingof sites I andII [31];

pOS1,

which contains site I

only;

and

pSVOdl3-site

II contains

SV40 nt 5209 to 128

[31])

was assembled and

digested

to

completion

with

TaqI.

This enzyme releases intact

SV40

origin-containing

sequences and generates several

plasmid-derived

fragments.

The mixture was then labeled with the

Klenow

fragment

of DNA

polymerase

I,

extracted with

phenol

and

chloroform,

and

precipitated

with ethanol.

Var-iousamounts ofT

antigen

wereincubated with 50 ngof this mixture in

origin-binding

buffer

(10

mM HEPES

[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.4],

100 mM

KCI

1 mM

MgCl2,

5%

glycerol,

50 ,ug of bovine serumalbuminper

ml)

for60 minat

0°C.

Purified

Pab416

(8)

(5 ,ug)

in

origin-binding

bufferwas

added,

andincubationwas

continuedfor an additional 20 minat

0°C.

A 10%

(vol/vol)

Protein

A-Sepharose

CL4B

(Pharmacia, Inc.) slurry

(100

RIl)

in NET buffer

(50

mM Tris

[pH

7.5],

150 mM

NaCl,

5 mM

EDTA,

0.05% Nonidet

P-40)

was

added,

and the reactions

were incubated on a rocker for 50 min at

4°C.

The beads

were

pelleted,

washed three times with NET

buffer,

sus-pended

in

1%

sodium

dodecyl

sulfate-25 mM

EDTA,

incu-batedat

65°C

for 15 min, and

electrophoresed

on

6%

native

polyacrylamide gels

in a Tris-borate-EDTA buffer system.

Gels were dried onto DE81 paper

(Whatman, Inc.)

and

autoradiographed

on Kodak XAR film.

Dephosphorylation

reactions.

Dephosphorylation

reactions

were as described in reference 18. T

antigen

in bufferF

(10

mM PIPES

[piperazine-N,N'-bis(2-ethanesulfonic acid); pH

7.0],

5 mM

NaCl,

0.1 mM

EDTA,

10%

glycerol,

1 mM

dithiothreitol,

0.5 mM

phenylmethylsulfonyl

fluoride

[26])

wasaddedtorection vessels

containing

30mMHEPES

(pH

8.0),

5 ,ugof bovine serum albumin per

ml,

and 1 mM

(final

concentration)

phenylmethylsulfonyl

fluoride.Calf intestinal

alkaline

phosphatase

(CIAP;

0.14 U per ,ugofT

antigen)

was

added,

and thereaction mixturewasincubated for12 minat

25°C.

One unit of

CIAP

activity

was definedas the

activity

which

hydrolyzed

1

,umol

of

p-nitrophenyl phosphate

in 1 minat

25°C.

To

begin

toaddress thelimits ofthe

DNA-binding

domain,

a cDNA copy of

large

T-antigen

coding

sequences was

cloned intoabacterial

expression

vectorunder control of the

phage

T7promoter

(32).

This cDNA clonewasthen usedto

generateaBAL31deletionseries from the

carboxy-terminal

endof the

protein-coding

sequences. A14-ntXba linker with stop codons in all three

reading

frames

(5'

CTAGTCT AGACTAG

3')

was used to

produce

truncated

proteins

containing

notmorethanfour additional aminoacidsattheir

carboxy

termini. The truncated

proteins

produced

in the T7

expression

system were

initially

assayed

for

origin-specific

DNA-binding

activity

in bacterialextracts.

Insertion ofthisstop linkerat theBclI site

(a.a.

682)

had

no effect on the

specific

DNA-binding activity

ofthe

trun-cated

protein

produced,

whereasinsertion of thesamelinker

into the PvuII site

(amino

acid

436)

resulted ina truncated

protein

which

displayed

slightly

reduced levels of

specific

DNA

binding (data

not

shown).

ThePvuII sitewastherefore

chosen as the

starting

point

for the deletion series. All

deletions between the PvuII and

HindIII

sites

(amino

acid

272)

produced

truncated

proteins

which either

completely

lacked or

possessed

significantly

reduced levels-of

origin-specific

DNA-binding activity

(data

not

shown).

Continued

screening

of smaller

proteins,

surprisingly,

revealed the presence of truncated T

antigens

which had

regained

the

ability

to bind DNA

specifically.

The C-terminal sequences of these

proteins

are

depicted

in

Fig.

1A.

In addition to

expressing

these truncated T

antigens

in E.

coli,

T-antigen

DNA sequences

coding

fortwoofthesetruncated

polypep-VOL.61, 1987

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[image:2.612.57.296.95.419.2]

3328 NOTES

PItOIG. 259.3

H E H

708

F1 t

a

2 3 3 2 3 2 3 2 3 2 '2i

B

5171 128

H3 Ddel 39I1 SphI

< < e--v

[image:3.612.62.299.72.363.2]

TI

FIG. 2. (A) Origin-specific DNA binding of truncated and full-size Tantigens. Various amounts of each purifiedTantigenwere

incubated withanequimolarmixtureofend-labeledfragments(lane M).After60minat0°C,Tantigenwasimmunoprecipitatedfrom the reactionand theboundDNAfragmentswereelectrophoresedon a

6% nativepolyacrylamide gel. Theamountsof Tantigenpresentin each set of reactions 1, 2, and 3 were 50, 150, and 450 ng,

respectively. The numbers at the top of the figure refer to the individualproteins purifiedfrom E.coli(E)orHeLacells(H). (B)

DNA templates containing SV40 origin sequences. These SV40 DNA fragments were present in equimolar amounts in all DNA-bindingexperiments. They containthewtSV40origin region(w.t.), siteI(), orsite 11(I),andtheirpositionsarenotedtotherightof theautoradiograms in Fig. 2A and 3. Thehorizontalarrows

repre-sentpentanucleotide-bindingsites for Tantigen(3), and the boxes delineatethe various sites(30). Theverticalarrows indicate cleav-age sites forrestriction endonucleases.

information as to the ability of the protein to

transiently

recognize specific sequences within the DNA fragment in

question. DNase footprinting studies have demonstrated

thatthetruncatedproteinsprotect thesameDNAsequences as full-length T antigen, although higher concentrations of

the truncated proteins are required to achieve similar de-grees ofprotection (W. Ryan and Y.Gluzman,

unpublished

data).

Equimolarquantities of DNAfragments containing site I

only (pOS-1), site II

(pSVOdl3),

and the entire wt

origin

(pSVO+) were mixed together to compare directly the

relative strength ofbindingtotheindividual

sites,

aswellas to the natural viral origin (Fig. 2B). Full-length T

antigen

from HeLa cells iscapable ofbindingtoallthree

fragments

in this mixture (Fig. 2A and 3). Itboundefficiently to both

the wt origin and the site I fragment at all concentrations examined;however, itboundtothefragment containing site

II atthe highestconcentration examined.

Interestingly,

the 272 and 266.1-amino-acid truncated

proteins

from E.

coli,

originally identified as non-DNA-binding proteins in our

crude-extract screening procedure, bound the wt

origin

fragmentwith low efficiency upon

purification.

Fusion

pro-teinsexpressed inE. coli,whichcontain theN-terminal272 amino acids of Tantigenalsobindto wtoriginsequences(D. Lane,

personal communication;

E.

Fanning, personal

com-munication). The smaller 259.3

protein produced

in E. coli standsin contrast totheaforementioned truncated

polypep-tides, sinceitefficiently bindstofragmentscontaining thewt

origin of

replication.

This resurgence of specific

DNA-binding activitysuggeststhatanobstructiontoDNA

binding

was removed between amino acids 266 and259. Examina-tion ofprotein-coding sequences between amino acids 266 and 259revealedthe presenceof fouradjacent glutamic acid residues. The presenceofthesenegatively charged residues proximaltotheDNA-binding domainmayaffectthebinding capability ofthefull-length

protein;

furthermore, these res-idues may have more pronounced deleterious effects when

they are present on the extreme carboxy terminus of the

Tog 259.31HW 259.3(E) 708(H)

CAP 4 - - -1

.~~

_

tides (266.1 and 259.3) were cloned into an adenovirus

expression vector and overproduced in mammalian cells (Gluzman, inpreparation). Proteins from both bacterial and mammalian sources were purified to homogeneity by im-munoaffinity chromatography on immobilized PAb419 (26;

Fig. 1B).

The DNA-binding properties of purified, truncated

pro-teinsfrommammalian andbacterialsourceswerecompared

withthoseofwtfull-lengthTantigen frommammalian cells. InthisDNA-bindingassay,theproteinwasincubated witha

mixtureoflabeledDNAfragments andimmunoprecipitated afterallowanceof time for protein-DNAinteraction. Specific DNAfragmentsboundtotheproteinwerethenvisualized by electrophoresis followed by autoradiography (16). The monoclonal antibody used in these studies, PAb 416 (8), recognizes adenaturation-resistant epitope locatednearthe

Nterminus of both truncatedandfull-length proteins. DNA binding, as measured by this assay, reflects formation of

stable, specific protein-DNA complexes but provides no

_

~~4o

-Wt.

- _ -I

2 3 2 3 2' 3 2 3 2 3 2 3

FIG. 3. Effect of dephosphorylation on origin-specific DNA

binding. Variousamounts of untreated purifiedTantigen(-) orT

antigen treated with CIAP (+) were incubated with an equimolar

mixture of end-labeled DNA fragments. DNA-binding reactions

wereperformedasdescribed in the legendtoFig.2. Theconditions

for CIAP treatment were as previously described (18). The high-molecular-weight bands appearing on the autoradiogram varied

between experiments and reflect nonspecific binding to protein A-Sepharose.

A

I I

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[image:3.612.321.557.472.641.2]

NOTES 3329 truncated T antigens. The fact that the purified truncated

proteins from E. coli bound only to fragments containing the wt origin but not to fragments bearing only site I or II at equivalent protein concentrations suggests that cooperativ-ity may exist between binding sites. It is also possible that higher protein concentrations or additional protein se-quences arerequired to recognize these sites individually. In

regard to the latter possibility, it is worth noting that

full-length Tantigen producedin E. coli fails to bind to site II only containing fragments at concentrations at which HeLa cell-produced T antigen binds efficiently (Gerard et

al., in preparation). Footprinting studies in progress will address this question further.

Significant quantitativeandqualitative differencesin

bind-ing were observed when proteins 259.3 and 266.1 purified

from HeLacells were used in this assay and compared with the bacterially produced proteins. Protein 266.1from HeLa cellsboundtofragments containingthe wtoriginatthreefold

reduced concentrations of protein and to fragments contain-ing site II at the highest protein concentrations. Protein 259.3

from HeLa cells bound DNA more efficiently than did

protein 266.1 and about as efficiently as did full-length T

antigen from HeLa cells. Protein 259.3 represents approxi-mately one-third of the length of full-size T antigen;

there-fore, identical-weight amounts ofeachprotein actually rep-resent athreefold molar excessofthe truncatedpolypeptide. Thus, in Fig. 2, lane 2 of259.3 and lane 3 offull-length T

antigen (708 amino acids) represent equivalent molar

quan-tities ofprotein inthe DNA-binding reactions. Full-length T

antigen first forms complexes with wt origin and site I

fragments, whereas site II fragments are bound at higher protein concentrations. However,the truncated mammalian proteins first form complexes with wt origin fragments and then begin to bind site IIfragments. It is possible that the

truncated proteins may be folded in a conformation which

enhances binding to site II fragments. Alternatively,

addi-tional amino acids may be required for certain truncated

proteinsto bind efficiently to site I. The latterpossibility is

unlikely, since truncatedT

antigens

shorter than 259 amino

acidsdisplay efficientbindingtositeI

fragments

(M.Strauss and D. McVey, personal

communication).

The fact that the truncated

proteins

from bacterial cells

exhibit differentDNA-binding propertiesfromtheir mamma-lian counterparts suggests that

posttranslational

modification provided by amammalian cell may influence

specific

DNA

binding. T

antigen

is

phosphorylated

at an amino-terminal cluster and a

carboxy-terminal

cluster

(24),

and

previous

studieshavedemonstrated that the

phosphorylation

stateof large T antigen affects its

ability

to

replicate

SV40

origin-containingDNA(18)andbindtoDNA sequencescontained

within the SV40 origin (18, 25, 27a). Upon

partial

dephos-phorylation

oflarge T

antigen

with

CIAP,

an up to 20-fold

increase in DNA

replication activity

in vitro was observed

undersome

conditions,

concomitant withafour- tofivefold

activation of

specific

DNA

binding

to site 11

(18).

It was

therefore ofinteresttoascertainthe effectofCIAPtreatment

on the

specific

DNA-binding

properties

of these truncated

proteins,which arealso

phosphorylated

inHeLacells

(data

not shown). When adenoviral vectors are used to

produce

full-length

T

antigen

in HeLa

cells,

truncated

proteins

con-taining

approximately

the first 130 amino acids are also

produced because of an incorrect

splicing

event. These

truncated

proteins

containthe amino-terminal

phosphoryla-tion cluster andarealso

phosphorylated. Furthermore,

these

same

proteins

are also substrates for

CIAP-mediated

dephosphorylation.

This demonstrates that truncated

pro-teins which possess

only

a

single phosphorylation

cluster

can be

partially dephosphorylated (approximately

80%) by

CIAP

(18).

We are

currently

examining

the

precise

sites

whichareaffected bythistreatment.

In agreement with

previously published data,

CIAP-treated T

antigen

displayed

a

slight

increase in

binding

to

fragments

containing

the wt

origin

and site I

only

and a

significant

increase in

binding

tosite II. The

binding

proper-ties of 259.3 from E. coli were not altered after CIAP

treatment

(full-length

T

antigen produced

in E. coliis

phos-phorylated

to a much lesser extent than HeLa

Tag;

R. D.

Gerard,

I.J.

Mohr,

M.

Strauss,

P.

Argani,

B.

Stillman,

and

Y.

Gluzman,

manuscript

in

preparation).

Similartreatment

of the truncated 259.3

protein produced

in HeLa cells resulted in

only

a

slight (if

any) increasein

binding

tobothwt

origin-

and site

Il-containing fragments.

Two

interpretations

ofthis resultare

possible.

In the

first, dephosphorylation

of the amino-terminal cluster has no role in

mediating

the

observed activation of DNA

binding,

while the

carboxy-terminal clusterof

full-length

T

antigen

is

entirely

responsi-blefor this

phenomenon. However,

asecond

interpretation

is

possible,

in which additional

protein

sequences,whichare

notconstituents of the minimal

DNA-binding

domainorthe

phosphorylation clusters,

are

required

to visualize the

acti-vation mediated

by

dephosphorylation.

Since both

phos-phorylation

clusters lieoutsidethe

region implicated

in DNA

binding,

removal of

phosphate

groups must then

indirectly

affect or

regulate

the

DNA-binding

activity

ofthe

polypep-tide.

The

DNA-binding

domain ofT

antigen, although

it can

function

independently,

appears to be a component of a

superstructure which modulates its

activity.

The actual

domainis less than 130 amino acids in size if the C-terminal

boundary

lies

proximal

to Pro 259 and the N-terminal

boundary

is taken to be

Lys-131 (29).

Once the domain is

released from the constraints

imposed by

the

larger

struc-ture, it is free to bind with greater

efficiency,

since the shorter 259.3

protein

binds DNA better than does its

longer

truncated counterparts. It is

interesting

that all ofthe

trun-cated

proteins

described here are

missing

the zinc

finger

structural motif

(Cys-X2-4-Cys-X2>15-His-X24-His)

at amino acids 302 to 320

(2).

Although

no functional role for this structure has been demonstratedfor

large

T

antigen,

it has

been

suggested

that thesestructuresmaybe involved in the

DNA-binding

activities manifested

by

several

eucaryotic

DNA-binding proteins (17;

see reference 2 for a

review).

Thus,

this

finger

structure cannotbea

major

determinantof

binding

specificity

for

large

T

antigen;

however,

it still may

be involved in

modulating

the

DNA-binding

activitiesofthis

polypeptide.

Posttranslational modification isan additional component

ofthis

regulatory

system which would

subject

it toan even

finer level of control. The fact that truncated

proteins

produced

in mammalian cells

display

increased levels of

DNA

binding

supports this notion. Protein

259.3, however,

does not

display

the characteristic activation of site II

binding following

dephosphorylation

exhibited

by

the

full-length

polypeptide.

This suggests that additional sequence

elements,

which are not constituents of the minimal

DNA-binding domain,

are

required

to visualize the activation in

site II

binding

brought

about

by

dephosphorylation.

These

dynamic

posttranslational

modifications,

which also lie

out-side the minimal

DNA-binding

domain,

are excellent

candi-dates foragentswhich would modulate the conformation of the superstructure and thus

change

the

accessibility

ofthe

DNA-binding

domain.

VOL. 61,1987

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http://jvi.asm.org/

3330 NOTES

We thank Bruce Stillman, Ed Harlow, John Anderson, and WinshipHerrfor critically readingthemanuscript,JoanCossettifor excellent technical assistance, and Marilyn Goodwin for help in preparing the manuscript.

This work wasfunded byPublic Health Service grant CA 13106 fromthe National Cancer InstitutetoColdSpring Harbor Labora-tory.

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2. Berg, J. M. 1986. Potential metal binding domains in nucleic acidbinding proteins. Science 232:485-487.

3. Botchan, M., T. Grodzicker, and P. Sharp. (ed.). 1986. DNA tumor viruses: control of gene expression and replication. Cancer cells, vol. 4. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

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5. DeLucia, A. L., B. A. Lewton, R. Tjian, and P. Tegtmeyer. 1983. Topography of simian virus 40 protein-DNA complexes: ar-rangementofpentanucleotide interaction sitesat theoriginof replication. J.Virol 46:143-150.

6. Gluzman, Y., J. Davison, M. Oren, and E. Winocour. 1977. Properties of permissive monkey cells transformed by UV-irradiated simian virus40.J. Virol. 22:256-266.

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