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Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.


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CopyrightC)1991,American Society for Microbiology

Specific Mutation of



Site within the ATP-Binding

Region of Simian Virus 40 Large T Antigen


Department of Biological Chemistry and Molecular


andDepartment of Pathology,2HarvardMedical School andDana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115

Received 8January 1991/Accepted 13 May 1991

Inan attempt to distinguish simian virus40 (SV40)large T antigen (T) binding toATP from hydrolysis, specific mutationsweremade intheATP-bindingsiteofTaccordingtoourmodelfor the site (M. K. Bradley, T. F.Smith, R. H. Lathrop,D. M.Livingston, and T.A. Webster, Proc.Natl.Acad.Sci. USA 84:4026-4030, 1987). Two acidic residues predictedtomakecontactwith themagnesium phosphatewerechangedtoalanines. The mutated Tgene was completely defective for viral DNA synthesis and for virion production, and itwas

dominant defective for viral DNA replication. The defective T gene encodeda stable product (2905T) that oncogenically transformedmousecelllines. 2905T, immunoprecipitated from transformed-cellextracts,bound SV40 origin DNA specifically and, surprisingly, itwas activeasanATPase. A recombinant baculoviruswas

constructedfor the production and purification of themutantprotein fordetailed biochemical analyses. 2905T had only 10% of the ATPase and helicase of wild-typeT.The Kmof 2905T forATP in ATPaseassays wasthe

same astheKmof wild-type T. ATP activatedthe ATPase activityof wild-type T,butnotof 2905T. As tested by gel bandshiftassay,2905T boundtoSV40 originDNAandtoindividual sites I andIIwithaffinities similar to that of the wild type. However, ATP did notmodulate the DNA-binding activity ofmutantT tosite II. Therefore, this mutation in the ATP-binding site inT resultedindefects in the interactionbetween the protein andATP thatappearedtobe responsible for the determination oftheactivestateofTforDNA bindingversus


Nearly all of the viral peptide large T antigen (T) is required for the replication of simian virus 40 (SV40) DNA. T is the only virally encoded protein necessary, with all

otherproteins for replication provided by the host cell (23, 37). Thereareseveral biochemical activities associated with T and itsreplication activity, including binding specificallyto the SV40 DNAorigin of replication (23), hydrolysis of ATP (62), andaDNAhelicaseactivity that melts double-stranded

substrate (64, 74). If single-stranded DNA-binding proteins

are added, T can unwind double-stranded linear templates

(18, 25, 27, 30, 55). Evidence that these activities are

associated with viral DNAreplicationcomesfrom mutations

in the T gene that inactivate either the enzyme or

origin-specific DNA-binding activities resulting in inactivation of DNAreplication (2, 15, 17, 36, 39, 41, 42, 46, 57, 61, 66, 67, 73). Data from immunological studies have shown that T-specific monoclonal antibodies that inhibit any of these

activities also inhibit replication in vitro (lla, 63, 73). By investigating how T combines these activities for the repli-cation of viral DNA, we are likely to gain insight into the mechanismsinvolved in initiating viral DNA synthesis.

The dataonT indicate that thereareatleasttwoseparate domains of the protein, includingoneforbinding DNA and oneforbinding ATP (23). Both the specific andnonspecific DNA-binding activities have been mappedtobetween amino acids 131 and 260 ofT, asdeterminedby makingtruncations


tPresent address: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge,MA02149.

tPresent address: DepartmentofNeurosurgeryandDepartment ofMicrobiology& Immunology, Albert Einstein Collegeof Medi-cineand MontefioreMedicalCenter, 111 East 210thStreet, Bronx, NY 10467.

and point mutations in the gene (1, 51, 61). A

carboxy-terminal region of T between residues 413 and 528 includes at least three different contacts with ATP, as shown by

binding studies using ATP and ATP analogs (6, 10, 16). Binding of the analogs to T blocked the ATPase activity, indicating that theATP-bindingsitewasthecatalytic site. T

proteins containing mutations within the same

carboxy-terminalregion have been showntobe defective for ATPase activity as well (15, 17, 39, 41). As expected, the DNA

helicase and unwinding activities required that both the N-andC-terminal regions remain intact (18, 25, 30, 46, 74, 76). These experimental data established the biochemical do-mains but provided little information about how T coordi-nated theseactivities forinitiating the replicationprocess.

One possibility raised by recent experiments was that T

regulated its initiation of SV40 DNA replication by the bindingaswellashydrolysis of ATP.Ithas been shownthat bindingtothe SV40originwasinfluencedbythepresenceof ATP without hydrolysis, but the exact effect of ATP has been debated.Investigators havereportedATP inhibition of DNAbinding (71),ATP stimulation of DNAbinding (4, 22), and ATP neutralization of inhibition by magnesium ions (Mg2+) (56). More recent data analyzing the SV40 origin DNA after incubation with T plus ATP showed that the DNA was significantly distorted. An openingoruntwisting

ofthe DNAwas induced inregions adjacentto the specific binding sites within the minimal origin (5, 20, 50). These different experimental techniques have revealed the

com-plexity of the interaction ofT, ATP, and DNA. First, we

needtocharacterize morecarefullythe effect ofATPonT. Then,wemighthavesomeidea how the interactionof T with ATP affects the physical changes in DNA that lead to replication.

Precedentforaregulatoryrole for ATP in the initiationof 4973

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provided by

biochemical and





systems. For






has been shown to be


for the coordination of initiation of


in Escherichia coli


DnaA, for





replication (oriC),

and for








oriC. An


"prepriming" complex

is then

formed with the

replication proteins DnaA, DnaB,

and DnaC

at oriC(58, 59). Similarto DnaA, SV40Tbindsto


DNA sequencesand then opensan

adjacent region

toform the


fork as described above.


T has the


to form a


with the DNA



by binding polymerase

ot(26, 63). Unlike






and is


tobe involved


in additional steps of DNA


We know from bio-chemical studies that the

DNA-binding activity

of the

pro-teinis affectedby ATP asoutlined


and the

ATPase-active form of T, a part of the helicase


can be induced by binding nucleotide (6).


theactivities of both DnaA andTin



apparently regulated


ATP. Genetic analyses of the


site in the






In order to

analyze genetically




hydrolysis activity




moreinformation aboutthelocation ofthe



in the


Amodel was


to aid in


the location and structure ofthe

ATP-binding region



This model


that amino acids




would form a central element within a five-strand

P-sheet extending

fromamino acids418 to 528 inSV40Tand

that this element


makecontact withthea-phosphate of ATP. In


with other


this model also


thatamino acid Glu-510 would



with the ribosemoiety and that Glu-473 with Asp-474 would bepartofthe

magnesium phosphate (MgPO4) binding pocket



ATPmolecule(MgATP; see






wemademutations inthe T gene

in anattemptto makediscrete defects in the




Inthisreport, wedescribe theeffect ofmutagenizingthe

acidic residues in the predicted MgPO4 binding pocket ofT to alanines. Presumably, this change would maintain the

structure of the MgATP


site but eliminate the


charges that hold the


in position. Consistent with


of otherTgenesmutated in the ATP-binding


wefound thatthismutantgenome,designated SV2905,



defective for viral DNAreplication butwas

able to transform mouse cell lines. Investigation of the




showed that the ATPase and helicase activitieswere-10%thatofwild-typeT.Mostinterestingly,

ATPmodulationof both the ATPaseactivity andthe




themutation. TheATPase


of the mutant T was not stimulated by incubation with


as was thecase for wild-type T. Assay of the


activity showed that the mutant protein boundto SV40DNA with the same affinity as wild-type T.


modulation of bindingto the minimalSV40origin DNA (site II) by ATP was absent in the mutant T binding

reaction. These results indicated that a defect in the

inter-action ofTwith ATPdirectlyaffected the active state of the T



Cells and viruses. The African green monkey kidney cell


CV-1P and COS-1 (29) were cultured in Dulbecco's

modified Eagle's medium (DMEM) and 10% newborn calf serum at 37°C in 5% CO2. The human 293 cell line was cultured in DMEM and 10% fetal calf serum. Spodoptera frugiperda cells (Sf9) and Autographa californica nuclear

polyhedrosis virus (AcNPV)were obtained from Max

Sum-mers (Texas A&M University, College Station). The Sf9 cellswere grown in either monolayeror spinnercultures at

27°C either in Grace's insect medium (GIBCO) supple-mented with 0.33% yeastolate, 0.33% lactalbumin

hydroly-sate(Difco), and 10% heat-inactivated fetal calfserum(Flow Laboratories)orin Excell-400(JRHScientific),aserum-free defined medium. Hybridoma mouse cell lines producing T-specific monoclonalantibodies PAb419 and PAb423 were

obtained from E. Harlow, the cell line producing PAb2O4

was fromD. Lane, and the cell lineproducing PAblOl (31)

wasfromtheAmericanTypeCultureCollection(Rockville, Md.). These culturesweregrowninDMEMwith5% horse

serum and 5% fetal calf serum, and the antibodies were

purifiedasdescribedpreviously (33).

Plasmids.Theplasmid pSDL13-1.13 used for mutagenesis

was a gift fromE. Fanning. It contains thecomplete SV40

genome inserted into the BamHI site of pSDL13 (57).

Plasmid pSDL13 contains the ColEl and M13 origins of




asdescribedby Levinsonet

al. (38). TheSV40




from C. Cole. dlA1209has adeletion from nucleotides 4862 to5191resulting intheloss of



as acontrol intransfectionassaysfor viralDNA


(78). dlA2462 has adeletionresultinginthe loss of Leu-509 in T(17). Theplasmid pSVori (77)wasused as thetemplate for DNA


assays. It contains a 203-bp

HindIII-SphI restriction


from the SV40origin (nucleotides

5172 to 132) cloned into the


pML-2 (40).


pONwt, and


were used in gel bandshift assays.

pONwt and p1097 were


from E.



consists of a synthetic 19-bp sequence from binding site I

cloned into the BamHI site of pAT153 (54), and p1097

consists of the whole SV40 genome, containing a 31-bp deletion of site I, cloned into pAT153 (24, 34).

Oligonucleotide mutagenesis.Asatemplate, weused puri-fiedsingle-stranded pSDL13-1.13. A19-meroligonucleotide

was synthesized,



con-tained three changes from the wild-type sequence of SV40 nucleotides3407 to3389,asunderlined.Reagentsand

meth-ods used have been reported previously by Schneiderand

Fanning (57). Double-stranded clonedDNA was sequenced



the presence of the mutations and the correct

surroundingsequences(Sequenase; United States Biochem-icals).

Transfection of SV40DNAinto monkey cells.Transfections

were carried outaccordingto the method of


et al.

(70),except thattheincubation solution contained4 x 106to 6 x 106 cells per ml and 500 ,ug ofDEAE-dextran per ml. Aftermixing, the solutionwasrocked gently at 37°Cfor40

min, washed, and plated at 1.5 x


to 2 x


cells per 60-mmtissue culture dish(usually five dishes).To assayfor

theefficiency of transfection, onedish ofthe cells wasfixed

with ethanol-acetone (1:1) at 48 h posttransfection and

immunostained forthe presence of T with PAb419 (7). Detection of viral DNAreplication in vivo. The assay for the

production of viralDNA by the mutant SV40 genome was carried out asdescribed previously(52, 70).

Expression and stability of the mutant T. After the

trans-fection of2 x 106 CV-1Pcells with 10




the wild-type or mutant SV40 genome) and incubation at 37°C for 48 h, the cells were washed and J. VIROL.

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incubated in methionine-free medium for 1 h.

[35S]methio-nine (250


was used to label the monolayers for 30min at


One set of duplicate plates was harvested in 0.5 ml of lysis buffer (9), and the remaining plates were washed and refed with fresh medium without


and incu-bated for 2 and 6 h before harvest. Samples of each extract were immunoprecipitated with 2


each of PAb419 and PAb423 and 15


of protein A-Sepharose (Sigma). The immunoreactive-radioactive peptides were isolated and vi-sualized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography.

Production of recombinant baculovirus. TheSV2905 muta-tion in the SV40 early gene was present in the


restriction enzyme DNA fragment. This fragment was cloned into the baculovirus transfer plasmid pAc373T2 con-taining the wild-type T cDNA sequences (48). The exchange of plasmid sequences was verified by dideoxy-DNA se-quence analysis of the double-stranded plasmid pAc373-SV2905T. The recombinant baculovirus


was produced and isolated as previously described (48). The presence of the mutation in the recombinant viral genome was confirmed by the purification of recombinant viral DNA and temperature-dependent hybridization with the original oligonucleotide used for the mutagenesis (75). The size and immunological integrity of the protein produced by the recombinant virus was compared with those of wild-type T by immunoprecipitation with amino- and carboxy-terminal specific monoclonal antibodies.

Production and purification of T protein. S. frugiperda

(Sf9) cells in a spinner culture were infected with either vAc373T2 orvAc373-SV2905T at a multiplicity of infection of 10. Cells were harvested at .40 h postinfection by


in a solution containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 9.0, 0.5% Nonidet P-40, 0.2 M sodium acetate, 1 mM dithiothreitol (DTT), 10% glycerol with fresh additions of 50


of phenylmethylsulfo-nyl fluoride (PMSF) per ml, and 10


of leupeptin per ml. Purification of T was performed by a modification of the method of Simanis and Lane (60), using either PAb419 or


conjugated to protein A-Sepharose. T was eluted from the column with 20 mM triethylamine (pH 11.3) in5%

glycerol and neutralized with one-tenth volume of 0.5 M HEPES (pH


M potassium acetate-10 mM DTT-5% glycerol-50 jxg of PMSF per ml. Protein concentrations were determined by spectrophotometry at A280, with 1 optical density unit equaling 1 mg of T monomer per ml or 12.3,uM

T monomer. The initial fractions eluted from antibody col-umns had the highest specific activity forSV40 DNA repli-cation in vitro, and only those fractions were used in subsequent assays.

DNA replication in vitro. Assays forSV40 DNA replica-tion in vitro were performed as described previously (48). Cell extracts were prepared from 293 cells according to the method of Yamaguchi and DePamphilis (77).

ATPase. ATPase assays were performed, as described previously (11), in a


reaction mixture containing 50

mM Tris-HCl (pH 7.8), 100 mM


1 mM DTT, 2% glycerol, [-y-32P]ATP (specific activity,


Ci/mmol), and total ATP concentrations as indicated.Reactionswere

incu-bated at


and analyzed after 15, 30, 45, 60, and 90min. Test samples (1


were applied to polyethyleneimine

cel-lulose plates (Brinkmann) for thin-layer chromatography in

0.75 M NaH2PO4. Phosphate release (in picomoles) was

measured by analysis of the developed plates onaBetascope 603 (Betagen Corp).

Activation of the ATPase activity by preincubation with


adenosine diphosphate(AMPPCP) wasassayed asdescribedpreviously (6).Tprotein (5



pH 7 and incubated with and without 2 mM AMPPCPand5 mM MgCl2for 30minat


Freenucleotide wasremoved bygelfiltration throughBiogel P10(Bio-Rad) equilibrated in ATPase buffer. ATPase activity wastested at 5 and 10 puM

ATP asdescribed above.

Helicase.Helicaseassays wereperformed according to the method of Stahl et al. (64) by using single-stranded, circular DNA annealed to a complementary 19-bp oligonucleotide. Hybrid DNA was radioactively labeled with Klenow en-zyme. This DNA template (-8 ng) and purified T were combined with 2 mM ATP in buffer (20 mM Tris-HCI [pH

7.5], 10 mM MgCl2, 0.5 mM DTT, 100 ,ug of bovine serum

albumin per ml) and incubated for 1 h at 37°C. Release of

radioactive oligonucleotide from DNA was analyzed by

polyacrylamide gel electrophoresis andquantitated by excis-ing the bands from the dried gel for scintillation counting.

Gel bandshift. DNAfragments, described in the legend to

Fig. 5, were radioactively end labeled with T4 DNA poly-merase and gel purified with NuSieve agarose (FMC Bio-products) and Geneclean (Bio 101). Purified T was incubated with 2 ng of radioactive DNA fragment in 10,ul of asolution containing 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES, pH 7.0), 1 mM DTT, 0.1 mM EDTA, 5% glycerol, 0.1


of bovine serum albumin per ml, 150 mM

NaCl, andpoly(dI)-poly(dC) (25 ,ug/ml) (Pharmacia) for 1 h



The reaction mixtures were resolved immediately in 1.5% agarose gels by electrophoresisfor 1 hat100V at room temperature in 15 mM sodium phosphatebuffer, pH7.0. The apparent Kd, or


for the protein-DNA interaction was determined by titration of theinput proteinaccordingto the method of Carey (13). Percent shifted DNA was quanti-tated by excising the free and the slower-migrating DNA bands from the dried gel and by scintillationcounting. When specified, 4 mM ATP (neutralized topH 7) was added to the buffers and incorporated into the gel.


Mutagenesis and analyses of themutated SV40 genome. In

our three-dimensional model for the ATP-binding site in SV40 T, we have identified a series of residues that are

predicted to come in contact with the nucleotide (11). The

negatively charged amino acids Glu-473 and Asp-474 would

be near the MgPO4 group ofMgATP (Fig. 1). The position was corroborated by the binding of fluorosulfonylbenzoyl 5'-adenosine (FSBA), a covalent affinity analog for ATP

known to cross-link totheMgPO4pocketin theATP-binding site (6). We used


mutagenesis to

change the Glu-Asp pair of amino acids to Ala-Ala and

designated the genomeSV2905 according toassigned SV40 mutant terminology (Carcinogenesis Branch, National Can-cer Institute). From the predicted secondary structure

sur-rounding the site, the exchange was thought to be

conserv-ative and least disruptive to the folding ofthe


in a

region known to be highly sensitive to mutation (23,


Several discrete mutations resulting in replication-defective but transformation-competent T have been


to the ATP-binding region (CllA, dIA2462, etc.) (17,



The aim of our studieswas toconstructmutantTgenes


stable, replication-defective proteins that



un-ravel the complex biochemical behavior of SV40 T. DNA replication and virion production by SV2905. We compared thereplication of viral DNA and virionproduction

by the mutantSV2905 and by the wild-type genome.

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r'473 D474

SY2905TA i


FIG. 1. A model of theproposed tertiarystructureof the

ATP-bindingsite inSV40T. Thisschematicdiagramhas been introduced

previously by Bradley et al. (11). Residues 418 to 528 have been

predicted to form a p-sheet with at least three parallel central strands (shownas arrowspointingtotheir Ctermini). Thep-sheet has been depicted as a flat structure, and the strands have been labeled alphabetically according to their primary sequence order fromtheN tothe Cterminus oftheprotein (Ato E).Anenlarged MgATPmolecule has beenplacedin the site, with itsa-phosphate

withintheglycine loopwhich extends from the centralp-strand A

(G-426toG-431).From otherdata,thearrangementofthep-strands has been determined to be BCADE (6, 11). Note that the


approaches the negatively charged residues glutamic acid (E-473) and aspartic acid (D-474) at the C terminus of p-strand C. The mutations in SV2905 produce codons foralanine replacement of both acidic residues.

cation of viral DNA was assayed by the transfection of CV-1Pmonkey cells with 1 to 100 ngof DNA(Fig. 2A and Table 1). To assure that the monolayers were equivalently

transfected,theprocedure was carriedoutby using cells in suspension, and the transfected cells were aliquoted into tissue culture dishes as described(seeMaterials and

Meth-ods). Themonolayerswereincubatedat 37°C for 96 h,after which Hirtextracts were made and analyzed by restriction endonuclease digestion and Southern blotting (52). The

assaywassensitiveenoughtodetect thereplication of 0.1ng





d e

f g



a b c d e f


FIG. 2. Analysesofviral DNAreplication invivo by wild-type

andmutantSV40genomesinCV-1Pand COS-1cells. Both panels A and BshowSouthernblotsprobedwith35S-labeledSV40DNA. (A) Analysis ofviral DNA replication in CV-1P cells harvested 96 h posttransfection with wild-type and SV2905 DNA. Lanes a to f contained restriction enzyme-digested Hirtextractsfrom transfec-tionwith 100ng(a), 10ng(b),or1ng(c)ofSV40DNAorwith 100 ng(d), 10ng(e),or1ng(f) ofSV2905DNAper60-mmdish.Lanes

gand hcontained100ngofmethylatedSV40plasmid DNA digested withBamHIalone(g)orplusDpnI(h), both in thepresenceofHirt extractsfromCV-1P cells. (B) Example of analyses of viralDNA

replication in COS-1 cells harvested 24 and 48 h posttransfection withnoDNA(lanesaandb),SV40wild-typeDNA(lanescand d),


SV2905DNA(laneseandf),orSVdIA1209DNA(lanesgand h).

TABLE 1. Summary of the analyses ofSV2905in vivo Result for: Assay

SV4Or SV2905b SV2462C

Viral DNAreplication + -

-Virion production at:

320C + -

-370C + -

-39.50C + -

-trans-Dominance - + +

Transformation + + +

Protein stability + + +

SV40 originDNAbinding + + +

PAb204-sensitive ATPase + +

aSV40 referstothe SV40wild-type genome. Each of the genomesanalyzed

wastransfected into animal cells, and theexpression ofTin those cellswas confirmed byimmunostaining.

bSV2905 is themutantgenomeof SV40containingmutations in theearly genewhich result inchanges of Glu-473 and Asp-473toalanines.

cSV2462 is SVdIA2462, the mutant genome of SV40 constructed and

analyzedby Cole et al. (17). It containsadeletion in theearlygeneresulting in the loss of Leu-509.

ofinput wild-typeDNA.Under theseconditions,noprogeny DNA was detected in cells transfected with SV2905.

Virionproductionwasmonitored after the transfection of CV-1P cells with wild-type or SV2905 DNA. Transfected cells were incubated at 32, 37, and 39.5°C and scored for virus production ifa monolayer showed cytopathic effects

(CPE). Wild-type DNA produced 80% CPE at9, 5, and 5

daysat 32, 37, and 39.50C, respectively. Monolayers trans-fected with anequal amountof SV2905 DNA were held for over 6weeks without CPEbeingdetected. Theexpressionof Tencodedby the wild-type andmutantgenomes was equiv-alent,asjudged bythe numberof nuclei stained per

micro-gram of DNA transfected in an immunoassay. Repeated transfection with SV2905 DNA and incubation at 37°C produced no CPE, nor did a reinoculation of fresh CV-1P

cells with supernatant fluids harvested from SV2905

DNA-transfectedcells thathad beenfrozenand thawed.

Dominant-defective replication by the SV2905 gene product.

Several replication-defective mutants of SV40 T, including

onesmapped to theATP-binding region, have been shown

to be dominant defective or trans-dominant over the wild-type protein in replication assays (28, 78). We assayed whether SV2905-encoded T expression (2905T) interfered

with wild-type Treplication activity by transfecting COS-1

cells with SV40 mutant and wild-type genomes. The wild-type T is constitutively produced by COS-1 cells and

repli-cates SV40origin DNA transfected intothose cells.

Trans-fection of a wild-type genome containing an origin of replication and an additional T gene produces high levels

of replicated viral DNA. As a control for the basal level of replication by the COS-1 T, we used an SV40-contain-ing plasmid that did not express the early genes but was the same sizeasthewild-typegenome, SVdlA1209 (78). As a control for the trans-dominant defect, we used a plas-mid containing a replication-defective SV40 genome,

SVdlA2462, having amutation inthe ATP-binding site (17). In our studies, a comparison was made by transfecting

COS-1 cells with pSV2905 and the control plasmids

pSVdlA1209, pSVdlA2462, andpSVL13.13. Hirt extracts of thetransfectedcellsweremade at 24, 48, and 72 h

posttrans-fection, and the extracts were assayed in the same manner asviralDNA replicationwasanalyzed. The largest amount

ofprogeny DNA was produced by the wild-type genome. J. VIROL.

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[image:4.612.316.557.90.213.2] [image:4.612.104.258.446.597.2]

Transfection of SVdlA12O9 into COS-1 cells produced a significant amount of replicated SV40 DNA, as expected (Fig. 2B shows a comparison at 24 and 48 h). However, the two mutant genomes SV2905 and SVd1A2462 (data not shown) produced fewer progeny than the T negative control

SVdlA12O9. These results indicated that the T protein en-coded by the SV2905genome was trans-dominant.

From these data, we concluded that theSV2905 genome was completely defective for viral DNA replication. This mutant T was able to interfere with thereplication ofSV40

DNA bya wild-type T produced by COS-1 cells, as was the case for other T genes mutated in the ATP-binding site (28, 78).

Analyses of the mutant SV2905 gene product. The T

ex-pressed bySV2905 DNA transfected into CV-1Pcells wasas stable as wild-type T, as assessed by [35S]methionine label-ing of the cells. Thirty-minute labeling times werefollowed by the immediate extraction of the cell monolayer or by refeeding with nonradioactive medium for 2 and 6 hbefore the extraction (data notshown). 2905T,immunoprecipitated from the extracts, was compared with wild-type T and the

stably expressed mutant T gene SVdlA2462 (17). No

signif-icant differences were seen. These results also showed that

metabolically labeled 2905Tand wild-type T migrated

simi-larly in SDS-polyacrylamide gels.

Oncogenic transformation of mouse cell lines by SV2905. SV2905 was competent fortransforming NIH 3T3 and A31

mouse cell lines, as determined by assaying for growth in

soft agarand focus formation (data not shown).Transformed cell lines wereproduced bycotransfectionofplasmidDNAs containing theSV40and theneomycingenes. Cell lines were

cloned by selecting for neomycin expression in G418 (12) andanalyzed for expression of T by the immunostaining of

fixed cellmonolayers. SV2905-transformed mouse cell lines from these studies were used as a source of mutantprotein (2905T) for preliminary biochemical analyses of its

DNA-binding andATPase properties.

Biochemical analyses of the 2905T from transformed-cell extracts. We prepared extracts from SV2905-transformed mouse cells and incubated them with radioactively labeled fragments from SV40 DNA according to the method of

McKay (45). After immunoprecipitating the proteins with

PAb419, we analyzed the DNA fragments bound to the protein by electrophoresis in agarose gels. The mutant

protein 2905T bound the SV40 origin fragment in the pres-ence ofnonspecific, competitor DNA.

We also usedtransformed cell extracts toinvestigate the

ATPase activity of 2905T. We performed these assays in

parallelwithassays ofextracts containing wild-type and the

ATPase-defective T encoded by SVdlA2462 (17). After

immunoprecipitationofT from cellextractsbyusingpurified PAb419 antibody and protein A-Sepharose, we washed the

immune complexes with ATPase buffer and performed the assay as described inMaterialsandMethods. To ensure that the ATPaseactivity detected was Tspecific, weassayed for inhibition by PAb2O4, a monoclonal antibody that inhibits

the ATPase activity of SV40 T (14). Both 2905T and wild-type T immunoprecipitates contained PAb204-sensitive ATPase activity, while 2462T was essentially negative, as

reported previously (17).

Tosummarize(Table 1), although the SV2905 genome was

defective for viral DNA replication, the gene product was able to transform mouse cells, as was the case for other

mutants mapped to the ATP-binding region (17, 41, 42).

SV2905 encoded a stable protein when transfected into

CV-1P cells, and the gene product produced in transformed

mouse cells was able to bind specifically to SV40 DNA

containing the origin of replication. Surprisingly, the protein retained ATPase activity even though the mutation was in theATP-binding region. A more complete biochemical com-parison with wild-type T required analyses of the purified protein.

Expression of 2905T in the baculoviral system. We didnot

expect to find ATPase activity associated with the mutant 2905T; therefore, we prepared to make purified protein for

direct biochemical comparison with wild-type T. Since we

needed large amounts of protein for kinetic studies and the

SV2905T gene was completely defective for viral DNA

replication, we chose the recombinant baculovirus as an

expression system. The recombinant baculovirus produced T that was biochemically equivalent to that produced by

SV40-infected monkey cells and produced it in quantities 10-fold higher per infected cell (48).

Weconstructed the 2905T baculovirus transfer plasmid by exchanging aPflMI-to-PstI restriction DNA fragment

con-taining the mutant sequences from SV2905 with the same

fragment in the baculovirusplasmidcontainingthewild-type T cDNA pAC373T2 (48). Construction and purification of the recombinant baculovirus was as described in Materials and Methods(7,68). T wasextractedfromSf9cellsinfected

with recombinant virus at 10 PFU per cell and harvested

when the rate of Tproductionhad just reached themaximum at -41 h postinfection. Both 2905T and wild-type T were produced at equivalent amounts (1 to 10 ,ug of T per 106 cells). Variations in protein productionfrom the polyhedrin promoter were due to themetabolic state of theinfectedhost

cells. Since T can exist in a variety of oligomeric and posttranslationallymodified forms, severaldifferentextracts

and two different types of monoclonal antibody columns

were used for the purification of the protein by immunoaf-finity chromatography (60). The results presented in this report were repeated several times with a number of dif-ferent preparations of T. Therefore, it is likely that we have compared T molecules representing the wild-type and mu-tant proteins rather than two subpopulations ofT.

Assay forSV40DNAreplicationin vitro by purified 2905T.

Although we already knew that the SV2905 genome was

defective for viral DNA replication in vivo, we tested the purified 2905T in an SV40 DNA replication assay in vitro

(48). Our assay was sensitive enough to detect SV40 DNA

replication by less than 200 ngofT (Fig. 3A). As expected,

we did not detect any origin-specific DNA replication

di-rected by 2905T (Fig. 3A) evenwhen16,ugofmutant


was added to the assay (data not shown).Interestingly,when 2905T was titrated into an assay mixture containing

wild-type T, a decrease in DNA replication was seen


with the results of an assay withwild-type Talone(Fig. 3B).

In this and other assays, the addition ofa concentration of

2905T -1.5-fold that of wild-type T reduced replication

-10-fold. This is likely to be the same dominant-defective

effect seen in vivo when SV2905 genome was transfected

intoCOS1cells constitutively




(28, 66,


Analysis ofthe ATPase activity ofpurified 2905T. In order to investigate further the ATPase


associated with

2905T in transformed-mouse-cell extracts, we


and assayed purifiedwild-type and2905T




The purified mutant protein was able to


ATP. How-ever, the maximum level of

hydrolysis (Vmax)

for 2905Twas

approximately 10-fold less than that of the

wild-type protein

(Table 2). On the other




for ATP in the hydrolysis reaction


was the same for both


on November 10, 2019 by guest





jiL T Al 0 0 003 05 1 24 0 / 3 4 7.0 10I

30min expostore



Aq3 T-1 Af3 WT 2 0 O 2 O 2d O 210 9W)(} O C 0 0 7 3'. 7O

FIG. 3. (A) Assayof 2905T forDNAreplicationactivityin vitro. Samples contained0 to2.4,Igofwild-typeT(left panels)or0.7to

10.0 jg of 2905T (right panels). The top and bottom panels are autoradiographs from thesameexperiment exposedtofilm withan

intensifyingscreen at-70°C for differenttimes,asindicated.(B)To testfor interference by the defective 2905T,increasing concentra-tions of the mutant protein were added to a replication-positive reaction containing 2 ,ug of wild-type T. The arrow marks the position of DpnI-resistant, formIIIpSVori in thegel.

Thesekineticparameters ofATPhydrolysis by



and 2905T were calculated from double-reciprocal plots of increasing substrate concentration (from 0.5 ,uMto 1.0 mM

ATP)versuspicomoles of phosphate released by


Further analysis showed that the turnover rate


and catalytic efficiency


oftheenzymatic reactionwere

both10-to15-fold lower forthe mutant thanforwild-typeT. Because the mutation in 2905T had lowered the ATPase

activity oftheprotein but notthe affinity of theenzymefor

thesubstrate,itwaspossiblethat the 2905Tproteinwasnot

efficient atbecoming ATPase active. From previous work,

weknew that ATPase was associated with dimeric T (9). In

addition, wefoundthatpreincubation withMgATPaffected

the ATPaseactivity ofT, probably byinducingtheformation of dimers (6). It was possible that 2905T, mutated in the ATP-binding site, wouldbe defective inthe ATPactivation

of enzymeactivity.

Assay for activation of the 2905T ATPase activity. We

TABLE 2. Comparison of wild-type and mutant T ATPaseactivities





S) Activation'

Wild-type T 20 0.30 600 2.1

2905T 21 0.02 38 1.1

a Km and


were calculated from the slope of the double-reciprocal plot of the ATP concentration versus the amount of phosphate released, where x = l/Kmand y= 1/Vmax.

bKcatlKmis thecatalytic efficiency.Kcatis the turnover number and is equal to Vmaxdivided by the enzyme concentration.

cActivation was measured by comparing T ATPase activity after prein-cubation with and withoutMg2+andAMPPCP, as described in Materials and Methods.

jIg T

FIG. 4. Helicaseactivityforwild-typeTand 2905T. The helicase

assay was described in Materials and Methods. The percent of

radioactive oligonucleotide dissociated from the single-stranded DNA plasmidwas determined foreach protein in parallel

experi-ments. The 100% value was equivalent to the oligonucleotide releasedbyheat denaturation. Amounts of 4to6,ugofwild-typeT

were able to dissociate 100% of the oligonucleotide (data not shown).

investigated whetherwe wereabletostimulate the ATPase activity of wild-type T by preincubation with MgATP (6). The effect was not dependent upon hydrolysis, since the nonhydrolyzable ATP analog AMPPCPalso stimulated the ATPase. We preincubated wild-type and mutant T with 5 mM


and2 mMAMPPCPat30°C. Aftergelfiltration to removethe freenucleotide,weassayed theATPaseactivity

of bothproteins. Whilewild-type Tactivitywas stimulated twofoldaspreviously reported,theATPaseactivity of 2905T

wasnotstimulated significantly by the preincubation (Table 2). As expected, themutantprotein appearedtobe defective inits interaction with the


and nucleotide.

Assay forDNAhelicase. Since the DNA helicaseactivity of Trequires ATP hydrolysis (64),wealsomeasured theability

of the mutant proteinto act as a helicase. The assay used detected the release ofaradioactively labeled

oligonucleo-tide which had been annealed to single-stranded, circular DNA. Wild-typeTshowed significant helicase activity ata

concentration of0.1 ,ugofproteinperassay, with 0.8,ug of Treleasing 50% of the oligonucleotide during a 1-h incuba-tion(Fig. 4). Inaparallelassay,themutantprotein had only 10% of the helicase activity of wild-type T. 2905T (10 ,ug) dissociateda maximum of 20% of theoligonucleotide.

Although 2905Twasdefective, the protein retained -10%

of theenzymeactivity, whichwassimilartothe defect in the ATPase. Wefound that themutantprotein did associate with the helicase substrate in immunoprecipitation assays (data notshown). Therefore, the inefficiency of the 2905T helicase reactionmayalso beexplained byadefect in the interaction

oftheprotein with ATP.

Analyses of the DNA-binding properties of purified 2905T by gelbandshift. Since the data from DNA immunoprecipi-tation assays already indicated that 2905T was able to recognize the origin region specifically, we compared the

binding affinities ofthe mutant andwild-type proteins. We

0 co




0 la




cmo O0


on November 10, 2019 by guest


[image:6.612.318.552.77.282.2] [image:6.612.]

SV40 origin


I sp


site I site Il














-FIG. 5. Map ofDNAfragments used in gel bandshiftassays. The position ofthe Tbinding sites in the SV40origin of replication is

indicated byarrowsrepresentingpentanucleotiderepeats.IR,the invertedrepeat;AT,theAT-rich region. A 231-bp EcoRI-to-SphI restriction fragment from pSVori contained thecomplete SV40 origin (orn). A352-bp SphI-to-NheI restrictionfragment from pONwt contained the synthetic 19-bp sequence coding for two pentanucleotides considered to be equivalent to the complete SV40 site I (54). A 169-bp HindIII-to-SphI fragment from the plasmidp1097containedadeletion ofbinding site I (nucleotides 5178to5208) butacompleteSV40binding siteIIwith thesurrounding IR andATsequences.

examined the Tbinding to regions of the DNA associated with transcription (site I) and replication activity (site II) separately by using the DNA fragments shown in Fig. 5. These fragments allowed us to analyze the ability of T to

recognize the DNApentanucleotidesequence(GAGGC) and

the DNAstructureof the origin region together (50, 54,56). We set upgel bandshift analyses at limiting DNA

concen-trations, where




according to

Carey (13). The amount ofprotein necessary to give half-maximal binding of the DNA in a gel bandshift assay has

been showntobeequivalentto theapparent disassociation constant


This is a stringent assay that

underes-timates the affinity constant.

Both wild-type and mutant T showed


in the

range of 400 nM for binding to 2ng of the complete origin

DNA fragment (ori). Experiments were repeated with

sev-eralpreparations of the proteins. Table 3 shows the results of

one parallel experiment for direct comparison. Binding

re-actions performed at 4 and 37°C showed similar


butahigherpercentageof bound fragmentwasachievedat 4°C. Similaramountsofwild-type andmutantT(7to9 pmol) shifted 100% of ori at 4°C; see an analysis ofwild-type T

and 2905T in the top panels ofFig. 6 and 7, respectively. Bandshifting of oriwasdetectedatconcentrationsaslowas

0.05 pmol of T. Sometimesa specific band of shifted DNA was seenatintermediate concentrations which changed into

abroad, undefinedpattern astheproteinconcentrationwas

increased. The pattern of the gel bandshift was similar in

analyses of bothwild-type andmutantT.

Our gel bandshift analyses included investigation of the origin site I DNA. Site I has been associated with

transcrip-TABLE 3. Kd(app)forTbindingtoSV40DNA Kd(app)(nM)a Protein

ori Sitelb SiteII

Wild-typeT without ATP 320 310 690

Wild-typeTwith ATP 420 370 1,410

2905Twithout ATP 470 530 560

2905Twith ATP 360 230 480

aNanomolar concentration ofT-Agthat shifted50o of the DNAfragment

(2 ng). The datawerefitted toa curveby usingacomputerprogramfrom SPSS, Inc., developed accordingtothe method ofMorganetal.(47).

bWithoutATP, only'609oofsite I DNAwasshiftedbyeitherTprotein. TheKj(app)wascalculatedaccordingtothe maximum shift.

tional regulation of theSV40 earlygenes(32,53). Theaffinity

ofT for site I has been shown tobe higher for the whole

origin than for the minimaloriginfragment site11(56). Inour

repeated assays,the


of wild-type Tfor site I DNA

wasthesame asfor ori. 2905T consistently shifted site I with

aloweraffinity than wild-type T [i.e., therewas anincrease



seeexample in Table 3]. However, bothproteins

shiftedonly s60% of the total site I DNAfragment. Exam-ples of the interaction ofwild-type andmutantTwith site I

areshown inpanel I of Fig. 6 and 7. Concentrations of either

protein higher than those shown shifted nonspecific DNA fragments tested in parallel (datanotshown). All of the site Ibandshifted patterns showedone, distinct band.

For site II, containing theminimum origin of replication, wild-type T bound the DNAwithaKs/app) from 500to700 nM (seeone example in Table 3). This affinitywas

consis-tently lower than that for ori and for siteI.Although 100% of

thefragmentwasshifted, thepatternof theshifted DNAwas notdefined(Fig. 6,toppanel II). The site II shiftwasalways a broadsmearthat moved furtherup thegel astheprotein

concentration was increased. In parallel studies, mutant T had a


similar to that ofwild-type T, and the two proteins showed similarpatterns in theirrecognition of the minimal SV40 DNA origin by these analyses (Fig. 7, top panel II).

Determination of the K1(app) for wild-type T binding to DNA in the presence of ATP. Even though there were no

large affinityorpatterndifferences in DNAbinding between wild-type and mutant T, we suspected there may be some

significant differences in the presence of ATP. First, we

needed to establish the effects of ATP on the wild-type

protein in our assay. T and the DNA fragments were

incubated for 1 h at 4°C with neutralized 4 mM ATP. No

Mg2+wasaddedtoreduce thepossibility of ATP hydrolysis. Electrophoresiswasperformed withATPaddedtoboththe geland therunningbuffer(seethebottompanelsinFig. 6). Thesegel bandshift analyses showed that thepresenceof ATP produced no net change or a slight inhibition of

wild-type T binding to ori. However, when the individual DNA-binding siteswere examined wefound that the addi-tion ofATPincreased wild-typeT bindingto site I so that 100%of the DNAwas shifted (Fig. 6, bottompanel I). The K,(app) remained similar because the determination was

basedon100% total DNAshifted, comparedwith 60% inthe

absence of ATP. The pattern of shifted DNA was still a

singleband. Thisimpliedthatnochangewasinduced inthe way T bound to site I. Rather, it appeared that ATP




I 1 1f 0

on November 10, 2019 by guest


[image:7.612.149.480.74.173.2] [image:7.612.64.305.603.683.2]



.... -.. . w

.. = ...






.-..s s w_

_...._.:._ ... _ .N W_.. _ W | |. _ XF.- ! s- iE w

:''.',r:.' ,;

.:' :.


pmoles T 12 9 6 5 35 2 4 1.2 0 18 12 6 1 2 0 18 15 12 9 6 5 3.5 0

FIG. 6. Analyses ofwild-typeTbindingtoSV40originDNAfragments by gelbandshift. Shown in the topsetofpanelsisananalysisof purifiedprotein combined with 2 ng oforiDNA, siteI,orsiteII. After1hat40C,thecomplexeswereseparatedbyelectrophoresisin1.5% agarosegelsrun in 10mM NaPO3, pH7.0. Thegelswere dried andautoradiographed. The last lane in eachpanel shows theresults of incubating DNA in the absence of T. The bottomsetof panels shows the results of the incubation andelectrophoresis ofTandDNA in the presenceof 4 mMATP(seeMaterials andMethods). The number ofpicomoles ofwild-typeTadded is shown below each lane andindicates the amountof protein addedtotheexperiments in the top and bottompanels.

increasedthe amountof thesubfraction ofTable tobindsite I.

In contrast, the addition ofATP resulted in a dramatic

inhibition ofTbindingtositeII(Fig. 6,bottom panel II,and

Fig. 8). At less than saturating concentrations of T, the


was increased twofold in the example shown in

Table 3 and, therefore, the binding was decreased signifi-cantly. Otherinvestigators have shown that theadditionof ATPtogel bandshiftandimmunoprecipitation assays inhib-itedtheDNA-binding activity ofT(19, 71). IfweaddedATP


to our assay during the preincubation step, but not to the

electrophoresis, no inhibition was seen. The inhibition de-pended on the continued presence ofATP, indicating that the effect was reversible. Vogt et al. (71) made a similar observationon the basis ofimmunoprecipitationassays.

Determinationof the


for2905T binding to DNA in the presence of ATP. We tested the comparative binding

affinities of 2905T and wild-type T for DNA in the presence ofATP (Fig. 7, bottom panels). Similar to what was seen with the wild type, theKd(app) of 2905TbindingtooriDNA




[image:8.612.72.545.75.275.2] [image:8.612.68.549.482.680.2]

p15ol4s 2905T 171385

6.85 3.417


... W.

16 12 7.7 -4 1 19 o

1 ... .7.56 2

17 13 8-5 7-6 6 5-1 4.2 O FIG. 7. Analysesof2905T binding to SV40 origin DNA fragments by gel bandshift. These experiments were performed in parallel with thoseinFig.6.The number of picomoles of 2905T added is shown below each lane and is the amount of protein for both the top and bottom panels. Note that the data for determining the Kd(app)were derived from scintillation counting of the gel material cut on the basis of the autoradiographs, notfromdensitometric tracing of these autoradiographs.




on November 10, 2019 by guest










-80 60 40


-0 2










4 6 8 10 12 14 16

pmoles WT T

4 6 10 12 14 16


pmoles 2905T

FIG. 8. Comparison ofSV40 site II binding activities of

wild-typeTand 2905T. Thepercentgel bandshiftof the DNA fragment bywild-type T (top panel) and 2905T (bottom panel) is shown in

grey. They were similar in repeated assays performed in parallel.

Conditionswerethesame asinFig.6and 7. The black hatchedarea

inthetoppanel showsthereduced bindingofwild-typeTtositeII

DNA inthepresenceofATP. For 2905T, the black hatched areais

similarifnotindicatingaslightly higherpercentageof bindingtosite


wasnot changed significantly by the addition ofATP(Table 3), although the pattern of the shifted DNA was altered.

Also, 2905Twasstimulatedtoshift 100% of siteI DNAwhen 4mMATPwasadded. These results showed that themutant

protein could respond to ATP if it was present during the entirereaction, including the electrophoresis.

Therewasnosignificant inhibition of 2905T bindingtosite

II as compared with the dramatic decrease in binding seen

withwild-type T(Table 3 and Fig. 8). Some changes in the smearpatternwere seen(Fig. 7), but the addition of ATP did notchangethe


for 2905Tbindingto siteII DNA.



change in theMgATP binding site ofTinorderto study the roleof ATPbinding andhydrolysis in viral DNA synthesis. Mutating the MgPO4 binding site in T produced a protein,

2905T, that was incapable ofparticipating in DNA

replica-tion both in vivo and in vitro. Not only was 2905T itself

inactive, but it inhibited viral DNAreplicationby wild-type protein (a dominant-defective phenotype). The SV2905 ge-nomeoncogenically transformed rodent cells, and the 2905T

proteinproducedby those cellswasactiveas anATPaseand

as anorigin-specific DNA-binding protein. Afterplacing the

mutant Tgene into a recombinant baculovirus for

overex-pression, the protein was purified and retested for its

bio-chemical activities. Purified 2905T was only 10% as active as wild-type T for both ATPase and helicase. This biochemical defect was consistent with the mutation in the ATP-binding siteof 2905T, but it was not necessarily sufficient to explain the complete absence of detectable DNA replication activ-ity. We didfind that the interaction of 2905T with ATP, other than forhydrolysis,was defective compared with the

inter-actionof the wild-type protein with ATP. Incubation of the mutant protein with MgATP did not stimulate the ATPase (6). In addition, although 2905T bound specifically to the replication origin DNA (siteII) as efficiently as wild-type T, 2905T did not change its affinity in the presence of ATP. Together, these data indicated that this mutation in the

ATP-binding site affected more than one active state of the

protein. The MgPO4 binding region in the ATP-binding site in T has the potential to be involved in initiating the events

ofviral DNA replication that precede helicase activity (5, 50), as well as inducing the formation of the helicase or ATPaseactivity (6).

Although the mutation in the MgATP binding site pro-duced a protein that hydrolyzed ATP at a lower efficiency than did wild-type T, the affinity for the substrate in the

hydrolysis reaction (Kin) was unchanged. One explanation

forthisresult isthatfewer2905Tmolecules becameactive as ATPases. Inthis regard, we have shown previously that the ATPaseactivity of T was associated with a dimeric

subfrac-tion ofthetotalprotein(9). The ATPase activity of T can be stimulated by binding


andnonhydrolyzable nucleotide (6),presumably byinducing the formation of ATPase-active dimers and higheroligomers. 2905T was not activated under

conditions of preincubation and gel filtration that were

sufficient to stimulate the wild-type T. This suggested that theinteractionbetween the mutant protein and MgATP did notpromote the formation of ATPase-active dimers. Since thehelicase activity requires ATP hydrolysis (64), it was not

surprising that the mutant protein was defective as a heli-case. Likethe ATPase,ithas beensuggestedthat oligomer-ization of T is required for helicase activity (30). The

ATPase-active molecule would form after T bound to the

helicase substrate. Thus, the mutation in 2905T may be

responsible for altering MgATP-modulated protein-protein interactions important for efficient formation of the

enzymat-ically activeprotein.

Without using antibodies, we compared the affinities of mutant andwild-typeTbinding to SV40 DNA sites I and II

in gel bandshift assays. Both proteins bound the DNA

fragments with similar affinities. These measurements were madeaccordingtothemethodofCarey (13) by using a fixed

concentration ofDNA and titrating the protein. This

elec-trophoretic method does have the potential to disturb the

equilibrium constant, and for that reason the results are referred toas a


Thatis, it is a stringent assay that underestimates the actual affinity. Note that our affinity





higherthanthose derived from

nitrocellulose binding assays orfrom other means bywhich

theprotein concentration is heldconstant. Althoughwe did

not detect significant differences in DNA binding between themutant andwild-type proteinsin repeatedexperiments,

wedid notesignificantdifferencesinTinteractionswith site Iversussite II DNAfragments.The patternsofgel bandshift

for the twoDNAfragments were distinctlydifferent. Site I DNA formed a specific complex, while site II


producedasmearthatincreasedwithprotein concentration.

Bothwild-type andmutantTboundonly -60%of the site I DNAfragment,comparedwith100% of siteII.Theseresults

wereconsistentwith theformationofT-Tcomplexesonthe

on November 10, 2019 by guest




DNA, and they indicated that there were different subfrac-tions ofT that bound toeach of thetwo DNAfragments.

We report here that the addition ofATP to gel bandshift

assaysstimulated the binding ofT to site IDNAfragments. It has been proposed that SV40 site I is recognized by preformed dimers ofT (21). If this is so, the constitutive

presenceofATP inour assays induced theformation of the site I binding form of T, which is presumablyadimer. This proposal is consistent withour previous finding that only a

7S or dimeric form of the replication-defective mutant T

from SV80 cells bound to SV40 DNA (8). Inother studies,

we have found that ATPinduced the oligomerization ofT

and the loss of the replication active form (71a) and that binding of ATP stimulated the ATPase


of T (6). ATP-induced


binding maybe the

prereq-uisite forTbindingtositeI DNAandfor itsATPaseactivity, althoughwedonotknow whether the active dimersfor these

twoarethe same.



of wild-type T binding to site II DNA was

significantly increased inthe presenceof ATP. Binding ofT tothecompleteori wasonly slightly affected by ATP, which is consistent with it being a combination of two opposite reactions. These results appeared to contradict the reports

by others (4, 22, 71). However, Dean et al. (19) reported a

decrease in T binding to DNA in a gel bandshift assayand thereafter used glutaraldehyde fixation to freeze the

com-plexesformed after incubating the protein andDNA. Wedid

not cross-link T to the DNA and therefore detected the


of the components by electrophoresis in the presenceofnonspecific


DNA,asshown. We do

not propose that the Tleaves the origin region in vivo, as seen in the gel bandshift assay. Rather, these results are

consistent with the

ATP-modified protein

shifting thenature

of its


reaction from



to the recognition ofa structural deformation in the early


(5)orinvertedrepeat(50).Tcomplex formation in this adjacent region appears to start the opening of the



(3). To complete the opening of the origin, T must become a helicase ata fork, followed by the entryof cellular replication initiation enzymes such as single-stranded


protein, topoisomerase, polymerase, and primase (3, 37, 65).

2905T bound to site II with the same affinity as did

wild-type T, but its ATP-modified binding was defective. That


inourassay2905Tremained tightly boundto siteII DNA in the presence of ATP whereas wild-type T was

dissociated by electrophoresis. If ATP was required for

producing high-affinity, sequence-specific recognition, then 2905Twouldnotbe expectedtobindtothe origin in the gel

bandshift assay aswell as wild-type T. These data support



given above that there is atleast a two-step

interactionbetween T and DNA at theorigin (site II). If the

replication defect of2905T is due to the formation of an

inappropriate and static complex at the origin of DNA

replication, then the defect would be compounded by the

inefficient formation of the helicase- and ATPase-active

formsofthemutantprotein. This would be one explanation

of the trans-dominant defect for replication. 2905T would

block theentry of wild-type T to the origin. We are

exam-ining the interaction of2905T and DNA in greater detail,

since themutantprotein is probablyunable to open (5, 50) or

untwist (20) the originDNA efficiently after it binds to the

GAGGC sequences in preparation for the unwinding and

formation ofDNAforks (3).



of complex biological activities by the


andhydrolysis of nucleotide, such as thatdescribed

above,isa commonmechanism. Inparticular, ATP has been shown to affect the manner inwhich proteins interact with

DNA substrates in replication, recombination, and repair

(35, 43, 44, 49, 58, 69, 72). For replication of E. coli oriC, therearethreetofourATP-dependentsteps in the initiation process (59). The regulatory role of ATP in T-dependent SV40 replication isjust being uncovered. We present here

genetic evidence suggesting that the MgPO4 region in the

ATP-binding site in T is important in that regulation,

influ-encing the interdomaincommunication of theprotein.

Bind-ing of MgATPto Tappearstoleadtothe coordinationof the

DNA-protein with protein-protein interactions, resulting in theformation ofanactive


complexatthe SV40 origin.


Wethank EllenFanning,ChuckCole, and John Bodnar for advice onthemanuscript.

This workwassupported bygrantCA38069from NIH. REFERENCES

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2. Auborn, K., M. Guo, and C. Prives. 1989. Helicase, DNA-binding, andimmunological properties ofreplication-defective simianvirus 40mutantTantigens. J. Virol. 63:912-918. 3. Borowiec, J. A.,F. B.Dean,P. A.Bullock,andJ.Hurwitz.1990.

Bindingandunwinding-howTantigenengages theSV40origin of DNAreplication. Cell 60:181-184.

4. Borowiec, J. A., and J. Hurwitz. 1988. ATP stimulates the binding of simian virus40 (SV40) large tumor antigento the SV40originofreplication. Proc.Natl. Acad. Sci. USA 85:64-68.

5. Borowiec, J. A.,andJ. Hurwitz. 1988. Localized meltingand structuralchangesin the SV40originofreplicationinducedby T-antigen. EMBO J. 7:3149-3158.

6. Bradley, M. K. 1990. Activation of ATPase activity ofSV40 largeTantigen bythe covalentaffinityanalog of ATP, fluoro-sulfonylbenzoyl5'-adenosine. J.Virol. 64:4939-4947.

7. Bradley,M. K.1990.Overexpressionofproteinsineukaryotes. MethodsEnzymol. 182:112-132.

8. Bradley, M. K., J. D. Griffin, and D. M. Livingston. 1981. Phosphotransferase activities associated withlarge Tantigen. ColdSpringHarbor Conf. Cell Proliferation 8:1263-1271. 9. Bradley, M. K., J. D. Griffin, and D. M. Livingston. 1982.

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Livingston. 1984. Specific in vitro adenylation of the simian virus 40 large tumor antigen. Proc. Natl. Acad. Sci. USA 81:6574-6578.

11. Bradley,M.K.,T. F. Smith,R. H.Lathrop, D. M. Livingston, and T. A. Webster. 1987. Consensus topography of the ATP bindingsiteof the simian virus40andpolyomavirus large tumor antigens.Proc. Natl. Acad.Sci. USA84:4026-4030.

11a.Bradley,M.K.,and B. M.Weiner.Unpublished data. 12. Brown, M.,M.McCormack,K. G.Zinn, M. P. Farrell,I.Bikel,

and D. M. Livingston. 1986. Arecombinant murine retrovirus for simian virus 40largeTcDNAtransformsmousefibroblasts toanchorage-independent growth.J. Virol. 60:290-293. 13. Carey, J.1988.Gel retardation at low pH resolves trp

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14. Clark, R.,D. P.Lane,and R. Tjian. 1981. Useof monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J. Biol. Chem.256:11854-11858.

15. Clark, R., K. Peden, J. M. Pipas, D. Nathans,and R. Tjian. J. VIROL.

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Weissbach,andJ. Hurwitz. 1987. Simian virus40(SV40)DNA replication: SV40 large T antigen unwinds DNA containing the SV40originof replication. Proc. Natl. Acad. Sci. USA 84:16-20.

19. Dean, F. D., M. Dodson, H. Echols, and J. Hurwitz. 1987. ATP-dependent formation of a specialized nucleoprotein struc-tureby simian virus 40 (SV40) large tumor antigen at the SV40 replication origin. Proc. Natl. Acad. Sci. USA 84:8981-8985. 20. Dean, F. B., and J. Hurwitz. 1991. Simian virus 40 large T

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21. Deb, S. P., and S. Deb. 1989. Preferential binding of simian virus 40T-antigen dimers to origin region I. J. Virol. 63:2901-2907. 22. Deb, S. P., and P. Tegtmeyer. 1987. ATP enhances the binding

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Table 1). To assure that the monolayers were equivalentlytransfected, the procedure was carried out by using cells insuspension, and the transfected cells were aliquoted into
FIG. 4.assayradioactivewerements.releasedDNA Helicase activity for wild-type T and 2905T
FIG. 5.fragmentHindIII-to-SphIindicatedsyntheticsite Map of DNA fragments used in gel bandshift assays
FIG. 6.theagaroseincubatingpurifiedpresence Analyses of wild-type T binding to SV40 origin DNA fragments by gel bandshift


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