Binding of simian virus 40 a protein to DNA with deletions at the origin of replication.

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Vol.49, No. 1 JOURNALOFVIROLOGY,Jan.1984, p.9-13

0022-538X/84/010009-05$02.00/0

Binding of Simian Virus 40 A

Protein

to

DNA

with Deletions

at

the

Origin

of

Replication

BETSY A. LEWTON, ANGELO L. DELUCIA, AND PETER TEGTMEYER* DepartmentofMicrobiology, State University ofNew York, Stony Brook, New York 11794

Received 18May 1983/Accepted 23September 1983

Previousstudies with wild-type simian virus 40 DNA have shown that thesequence5'-GAGGC-3'directs

the binding of A protein (T antigen). The functional origin of replication contains four recognition pentanucleotides each of which is separated by a single base pair and arranged as two pairs of direct repetitions that areinverted relative to each other. Analysis of A protein bindingto a series of nonviable

mutanftsprogressively deleting these contactsites leadstothefollowing conclusions: (i) stablebinding of subunits of Aproteintothreeoriginpentanucleotides isnot sufficient for the initiation of DNAreplication,

(ii) the stability of DNA binding dependsoninteractions between boundprotein subunits, and (iii)asingle pentanucleotide is sufficient tobind and orienta subunit ofAprotein.

The origin ofreplication in simian virus 40(SV40) DNA has beenmapped to a 60- to65-base-pair(bp) regioncentered at the unique BglI restriction site (1, 3). Binding ofthe A

protein (T antigen) to the origin regulates the initiation of each round ofDNAreplication (10, 12, 14, 16, 18). Accord-ing to Hay and DePamphilis, RNA

oligonucleotides

at

variouspositionson theearly strand within theorigin prime

thesynthesis ofthe firstforwardarmofDNAreplication (7). Studieswithwild-type SV40DNA show thataconsensus

family of recognition sequences directs the binding of A

protein (2, 17). Different arrangements ofthe

pentanucleo-tide determine different patterns ofprotein binding in three

adjacent regions at theorigin of replication. Inregion I on the early side of the origin, three contact sites arranged as

direct repetitions lead to high-affinity binding but are not essentialfor DNAreplication(2, 3, 10,17).RegionIII on the late side of the origin contains six recognition sequences

arrangedasdirectrepetitions. Bindingtothese sites in vitro

is stable onlyat low saltconcentrations (2, 15).

Region II corresponds well to the functional origin of replication defined by the mapping of viable deletion mu-tants(3). Itcontains fouridealrecognitionpentanucleotides

that are eachseparatedbyabpandarrangedas twopairsof

direct repetitions inverted relative to each other. Base

substitutions between pentanucleotides are associated with wild-type or even increased levels of DNA synthesis. In contrast,

substitutions

within pentanucleotides reduce, but do not eliminate, replication (11). Deletions ofa part of a

pentanucleotideappear to

eliminate

replication altogether (6, 10).

Inthe presentstudyweanalyze thebinding ofAprotein to

nonviable deletion mutants of region II togain insight into theminimalbinding requirementsfor DNA replication. Our

findingsalsoconfirmthe major features of a model for DNA

binding established with wild-typeDNA (15). MATERIALS ANDMETHODS

Proteinpurification. The SV40 A protein was purified from

productively infected CV-1 cells as previously described

(13).

DNA.Wild-typeas wellas mutant DNAs 6-1(dl6) and 6-17

(d19),

cloned inplasmid pMK16, were a gift from Y.

Gluz-man(5). The deletion mutant 295

(d118),

cloned in pBR322,

*Correspondingauthor.

was given to us by R. Martin.

The

plasmid was grown in

Escherichia coli HB101, and the DNA was prepared as

describedby Kahnetal. (8). Forsirnplicitywerefertothese mutants as dl6,

d19,

and

d118,

thenumbers of which indicate

thenumber ofbp deleted from theoriginofreplication.

DMSfootprinting. SV40 DNA wasdigestedwith NcoI and 3' end labeled with

[32P]deoxyribosylthymine

triphosphate byuseof avianmyeloblastosisvirus reversetranscriptaseas previously described(18). Thetranscriptasewasinactivated byheatingat

70°C

for 10min, and the labeledDNA wasthen

cutwith

Hinfl.

The

origin-containing

fragment, radiolabeled

on the 3' late end ofthe early strand, was purified by gel electrophoresis and was ethanolprecipitated. AfterSV40A proteinwas bound to the DNA for 1 h on ice, the guanine residues were methylated by the addition of 1 [L of 10 M dimethyl sulfate

(DMS)

for 10 min. The DMS reaction was

stopped,and theDNAwasrapidly processed bythe Maxam andGilbertDNA sequencingmethodspecificforcleavage at

guanine residues (9).Theresulting fragmentswere runon an

8.3Murea,8%polyacrylamide gel electrophoresissystem at aconstanttemperature of

50°C.

Footprinting

of the late strand was accomplished by

radiolabelingthe3' early endof the late strandofthe

origin-containing restriction fragment Hinfl-BstNI as previously

described(2).

DNase footprinting. The BstNI-Hinfl

origin-containing

fragment ofeach DNA was radiolabeled on the 3' end of the

earlyor the late strandbypreviously describedmethods (2). SV40 A protein was bound to the

fragment,

and then 0.0005 units of DNase was added. The reaction was stopped, and the samplewas processed by the method ofDeLucia et al.

(2).

Protected

DNA was analyzed by polyacrylamide gel

electrophoresisas describedabove. RESULTS

DMS footprinting. Nucleotides in close proximity to bound protein were

identified

with the DMS protection assay. For convenience we will refer to these as contact sites. In theseexperiments,anorigin-containingSV40DNA fragment was radiolabeled on the 3' end of either the early or

thelatestrand. Theguanine residues were then treated with DMS in the presence or absence of A protein at two ionic strengths to determine the bindinglocations and affinities of the mutant DNAs. Because these experiments were

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Dtein 0 SVASVA

rl i11"..

.005.005 .125

O SVA SVA

I

a..

0*0

G G _

A*: .

G 4[I

G

,I*

G G

G a

G mo _ _

G _w -_ _

.-W

G

G 'd_ Amm W

G ._m ...W 4,

....I

... la,

C

FIG. 1. DMS footprints of theSV40Aproteinboundtoboth strands of the origin regions of wild-type (WT) andmutantDNAs. Restriction

fragmentswereexposedtoDMS in thepresence orabsence ofAprotein in0.005 MNaCIor0.125 MNaCl and processedasdescribed inthe

text.Allguanine (G)residues of region11areplacedonthesamelinewhere guaninesoccuratadjacent positions inthe sequence,andsmall

brackets numbered in an increasing orderidentify recognition pentanucleotides. A circled G representsguanines protected at low ionic

strength, andadottedGrepresentshypersensitiveguanines. (A)NcoI-Hinflorigin fragmentwaslabeledonthe 3' late end of the early strand.

(B) TheBstNI-Hinfl origin fragmentwaslabeledonthe3' early endofthe latestrand.A6,A9,andA18ared16,d19, andd118,respectively.

10

.005.005 .125

0 SVA SVA

.005.005.125

o SVA SVA

V,'A$4." NW-*-*~

*PA

"WV

Pro

A.

II

B.

III

tiG G

1[S

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;....

4

'.'

.,:,::

GG

_

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la,

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10 VW_

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PROTEIN A BINDING TO SV40 ORIGIN DELETIONS 11

signed to compare the strength of binding with various

origins, A protein concentrations were adjusted to protect 50 to 70% of wild-type origins from DMS. Excess concentra-tions of protein would have decreased the sensitivity of the comparative assay.

Protein contact sites on the early and late strands are shown in Fig. 1A and B, respectively. The large brackets to theleft ofthefigure outline DNase protection

regiQns

I, II, and III to be described below. All of the mutant DNAs maintain the wild-type guanine sequenceand DMS

prQtec-tion pattern in regions I andIII. The results are described in detail and correlated with the DNase protection analyses

below. However, it is evident that deletionsof pentanucleo-tides inregion II have little effect on binding to the remaining

recognition sites at 0.005 M NaCl and have variable effects on binding at 0.125 M NaCl, depending on the extent and

location of the deletion.

)Nase footprinting. The limits ofSV40A protein bound to DNAweredefined by the use of DNase footprinting. In this assay each molecule of DNA is cut once producing a ladder

offragments displayed by gel electrophoresis. When the DNAis firstboundwithprotein and thencut, gaps appear in the ladder representing the region of DNA protected by T

A.

DNA

Protein

WT A6 A9 A18

0 SVA 0 SVA 0 SVA 0 SVA

antigen. Double-stranded restriction fragments containing

theoriginofreplicationwereradiolabeledatthe3' endof the early or late strands (Fig. 2A

and

B). The figure shows

DNase protection patterns obtained under low salt condi-tions. A protein protects wild-type DNA in the regions

indicatedbythebracketstotheleft of thegels(Fig. 2). Each regionis flankedbyDNasehypersensitive sites and each has

acharacteristic stabilityatdifferentsaltconcentrations(15).

Nevertheless, the boundary between regions I and II is somewhat arbitrary because A protein bound to region I sometimes protects DNA in theearly endofregion IIfrom

DNase(15). As shown inFig. 2, deletions inregion II have little if any effectonbinding ofAproteintoregions IandIII. Protein protects all of the undeleted region II of mutantsd16

andd19 but fails to protecttheearly endof regionIIofd118.

The hypersensitive sites flanking each end of region II are

gradually lost with increasing deletion size. Thus, these sites mustbe determined, in part, by binding to region II.

The DNaseprotection appears to be more complete than DMS protection even though the assays were carried out underthe same conditions. This difference is typical and can be explained in several ways. First, protection from the larger DNase molecule is indeed more efficient because of

B. DNA

Protein

WT *6 *9 A18

0 SVA 0 SVA 0 SVA 0 SVA

I *

I:

_

D

__

Se-e

a-

_OeO

.-0Sh

rr*

a

a"

III

11

r

ma

_0 as

mm _ 1 . S.:"

o _ _ Y:

4* _S

_s

0 *,

do

_0d

* * 4'

[image:3.612.71.528.328.692.2]

_ - ...

FIG. 2. DNase footprints ofthe SV40 A protein bound to both strands of the origin regions in wild-type (WT) and mutant DNAs.

Restrictionfragmentswerecutwith DNase in thepresence orabsence of A protein in0.005 M NaCl and processedasdescribed in thetext.

(A)TheBstNI-Hinfl origin fragmentwaslabeledonthe 3' late end of the early strand.(B) Thesamefragmentwaslabeledonthe 3' early end

of thelate strand. *6, *9, and *18ared16, d19, and d118, respectively. II

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12 LEWTON, DELUCIA, AND TEGTMEYER

the size of the probe. Second, protection by subunits ofA protein bound to adjacent pentanucleotides provides do-mains of overlapping DNase protection.

DISCUSSION

To understand the process ofinitiation of DNA replica-tion, wechosetofocus onthebindingofAproteintoSV40 DNAdeletedattheBglI restriction site in theorigin. Allthe

mutants are nonviable in permissive host cells. We used

DMS footprinting to define close contacts between origin DNAand A protein and DNasefootprintingtodeterminethe

broader limits of the DNA-protein interaction. The results from both studies aresummarized andinterpretedinFig. 3.

In the case of wild-type DNA, A protein protects all circledguanines (Fig. 3)withinthe fourpentanucleotides of region II and two of three guanines between the

pentanu-cleotides. Weak protectionofanearby guanine isindicated byparentheses andhypersensitive guaninesaredotted(Fig.

3). Protein alsoprotectsthe entire region from DNase. The

arrowbelow each pentanucleotide shows ourinterpretation

of the DMS and DNase protectionresults(15). Inourmodel,

each pentanucleotide directsthebinding ofamonomerofA protein inanorientation thatprotects20bpofDNAatthe 5'

endof the 5'-GAGGC-3' sequence and 10bp at the 3' end

(15).

Mutant dl6 deletes site 2 completely without alteringthe

single-bp spacing

between theremaining sites. Protein

bind-ing

protects all the remaining pentanucleotides from DMS and induces

hypersensitivity

oftheoutlyingguanines of the

mutantDNA in thesamepattern asin thecaseof

wild-type

DNA.

Protein

also protects the entire undeleted portion of

region

IIfrom DNase. Both DMS(Fig. 1) and DNaseassays

(data

not

shown)

indicate that binding isonly slightly

sensi-tive to0.125 MNaCl. These resultsare consistent withthe

predictions

of our

binding

model (15). The loss of site 2

would decrease the number of protein subunits bound to

region

IIbutwouldnotaffect other interactionswith

pentan-ucleotides.

Mutantd19 deletes allofsite2butonly apartof site 3 so

that the

remaining

pentanucleotides are separated by four rather than one

bp.

At low salt concentrations, protein protects sites 1 and 4 from DMS and the entire span of

undeleted

region

II from DNase. It is interesting that this

deletion also creates a new 5'-GAGGC-3' sequence that

overlaps

site1 andis separated fromsite 4 byasingle bpas

shown

by

thebracketsinFig.3.The DMS protectionpattern

II

-- -- -- -- -- --- -- -- -- -- -- ----

--30 20 10 0 10 20 ₃₀

WT I 2 3 4

C T A C T T C T G G A A T A G C T C A@A ( CC A0@0tGI C C T C6@C C T C|T G A T A A A T A A A A A A A A T T A G T GA T G A A G A C C T T A T C O A(G)T|C T C C C T C C CC C A ACO T A T T T A T T T T T T T T A A T C A

I

r

AOk5D1) 1 4 3 4

C T A C T T C T G G A A T A G ,C T C A A(G(GC G|QC C T C C C T C|T G C A T A A A T A A A A A A A A T T A G T

GA TG A A GA C CT TAT CO A(G)T|CT C CC CCCAC CTCAfAA CO TAT T TAT T T T T T T AT C A

A9

*9(6-1)

(6-17)

.

1

1 4

...4

AI

C C T Cj©X C C T C:T G C A T A A A T A A A A A A A A T T A G T GAT GA AGA C CTTAT C GATG T cL ATAAGJC:C AACO TAT T TAT T T T T T T AT C A

I a -...-.-...

*18

(295)*

~~~~~...

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

Ala

(295S)

4 .

FIG. 3. Methylationand DNaseprotectionresults. TheregionIIsequences ofwild-type(WT)andmutantDNAsareshownand numbered

by themethod ofFierset al.(4). Theupper and lower strandsarethe earlyandlate strands,

respectively;

thecurvedarrowaboveeach

sequence specifies the location of the deletion. Boxesidentifyrecognitionpentanucleotides numbered inan

early

tolatedirection. Dotted boxes indicatesites thatareincompletely protectedat0.125 M NaCl. Circledguanines(G) indicateDMS

protection,

and theparentheses signifyweakprotection. Largeand small dotsaboveguaninesindicate differentdegreesof

hypersensitivity.

The

elongated

arrowsbelowthe

recognitionsequencesrepresentourinterpretationoftheDNaseprotectionresults. Eacharrowcorrespondstothe DNase

protection

domain ofamonomerofproteinboundto asinglepentanucleotide (15).Thebracketed pentanucleotidesiteinmutantd19representsa

newly

formed

contactsitethatisapparently unused. A6, A9,and A18aredl6,d19,andd118, respectively.

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PROTEIN A BINDING TO SV40 ORIGIN DELETIONS 13

indicates that A protein prefers bindingto site 1 ratherthan

tothe new site. Furthermore, bindingtosite1 is more stable

than binding to site 4 at 0.125 M NaCl. These results are consistent with ourpreviousfindingthatbindingtoregion II occurs in a sequential process in an early to latedirection

(15). We conclude that thehigheraffinity of bindingtosite 1 does not depend onadjacentrecognition pentanucleotidesin

region II.

Mutantd118deletes all of sites 1, 2,and 3 so that site 4 is theonly siteremaining in region II; it occupiesthe original position of site 1 relative to region I but in the opposite orientation. A protein protects this single recognition

se-quence atlow, but not at high, ionic strength. Thus, high-affinitybinding to site 1 maydepend notonly onsequences orprotein-proteininteractionsontheearlyside ofregion II, butalso onthe orientation ofthepentanucleotide. Bindingto

site 4ind118provides the first direct evidence that confirms

ourmodel for the mechanism ofAproteinbinding.Asingle

recognition sequence directs binding of a unit protein and

results in asymmetricDNaseprotection.Theprotectionofa guanineadjacenttosite4raisesaquestionastowhether the

recognition sequence isapentanucleotide or a hexanucleo-tide. We prefer the formeralternative because aguanine in the sixth position is not consistently present and more importantly isnot alwaysused. Forexample,theguanine at thecenteroftheregion II palindrome isnotprotected

by

A protein(Fig. 1Aand 3).

The deletions in region II change notonly thenumberof

contact sites but also the distance and rotational angles between remaining sites. Yet, the A proteincan

accommo-date these changes and engage all available sites under low salt conditions. These results can be easily explained ifthe

primary binding form is a monomer. Binding ofpreformed dimersor tetramers toaltered DNAwouldrequire consider-ableflexibility of proteinsubunits. Studiesusingquantitative

transmission electron microscopy (I. A. Mastrangelo, unpub-lished data) demonstrate that A protein monomers are frequent binding forms in region II, although dimer and tetramerforms are also readily seen at higher protein

con-centrations. Even though monomers may be the primary binding form, protein-proteincontactswouldaccountforthe

higher affinity binding evident when recognition sequences

correctly position monomersrelative to oneanother. The 27-bp palindrome in the origin of replication could

have a number of additional functions as aconsequence of

protein binding. For example, binding to the palindrome

could induce a cruciform structure, initiate melting in the

adjacent AT-rich sequences,oralterconformationin

anoth-ermanner.Interestingly, oneofthemajorinitiation sitesfor RNA priming ofDNA replication appears to occur within thethird pentanucleotide in theprotein-binding palindrome (7). Theproduction ofaprimercouldrequire either de novo

synthesis or possibly processing of preexisting early

tran-scripts

by an RNase H-like activity at this site. Thus,

deletions in this region could have multiple effects on the

initiation of replication. Mutant

d16

deletes site 2 but maintains a single-bp space between the remaining sites so that the spacing and rotation of bound protein subunits would not be altered. This positioning effect is likely to

accountfor the stablebindingobserved. Because this mutant isnotviable, we conclude that binding of four monomers of

Aprotein in specific positions and orientations is a

require-ment for successful DNA replication. Additional possible functions of the palindrome in DNA replication, if any,

remain tobe demonstrated.

ACKNOWLEDGMENTS

This work was supported by PublicHealth Service Grants CA-18808 and CA-28146fromtheNationalCancer Institute.

WethankY.Gluzman and R. Martin for theirdeletionmutants. LITERATURECITED

1. Danna,K.J.,andD. Nathans. 1972.Bidirectional replicationof simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:3097-3100.

2. DeLucia,A.L.,B.A.Lewton,R.Tjian,and P.Tegtmeyer.1983. Topography of simian virus 40 A protein-DNA complexes: arrangementofpentanucleotideinteraction sites at theoriginof replication.J. Virol. 46:143-150.

3. DiMaio, D., and D. Nathans. 1980. Cold-sensitive regulatory mutantsofsimian virus 40. J. Mol. Biol. 140:129-142. 4. Fiers, W., R.Contreras, G. Haegeman, R.Rogiers,A. Van de

Voorde,H.VanHeuversewyn, J.VanHerreweghe,G.Volckaert, andM. Ysebaert.1978. Complete nucleotidesequenceof SV40 DNA. Nature(London) 273:113-120.

5. Gluzman, Y.,R.J. Frisque, and J. F. Sambrook. 1980. Origin-defective mutants ofSV40. ColdSpringHarborSymp. Quant. Biol.44:293-299.

6. Gluzman,Y., J. F. Sambrook, and R. J. Frisque. 1980. Expres-sion of earlygenesof origin-defectivemutantsof Simian Virus 40. Proc. Natl. Acad. Sci. U.S.A.77:3898-3902.

7. Hay, R. T., and M. L. DePamphilis. 1982. Initiation ofSV40 DNAreplication in vivo: location and structure of5' endsof DNAsynthesized in theori region. Cell28:767-779.

8. Kahn, M., R. Kolter, C. Thomas, D. Figurski, R. Meyer, E. Remaut, and D. R. Helinski. Plasmid cloning vehiclesderived from plasmids ColEl, F, R6K and RK2. Methods Enzymol. 68:268-280.

9. Maxam, A. M., and W. Gilbert. 1980. Sequencingend-labeled DNA withbase-specific chemicalcleavages.Methods Enzymol. 65:499-560.

10. Myers, R. M., and R.Tjian. 1980.Constructionandanalysisof simian virus 40origins defective in tumorantigen binding and DNAreplication. Proc. Natl. Acad.Sci. U.S.A. 77:6491-6495. 11. Shortle,D., and D.Nathans. 1979. Regulatorymutantsof simian virus 40: constructed mutants with base substitutions at the origin ofreplication. J. Mol. Biol. 131:801-817.

12. Tegtmeyer, P. 1972. Simian virus 40 deoxyribonucleic acid synthesis: the viralreplicon.J. Virol. 10:591-598.

13. Tegtmeyer, P., and B. Andersen. 1981. Partial purification of SV40A protein anda relatedcellularprotein from permissive cells. Virology 115:67-74.

14. Tegtmeyer,P., B.Andersen,S. B.Shaw,and V.G. Wilson.1981. Alternative interactions of the SV40 A protein with DNA. Virology 115:75-87.

15. Tegtmeyer, P.,B. A.Lewton, A. L. DeLucia, V. G. Wilson, and K. Ryder.1983.Topography of simian virus40 Aprotein-DNA complexes: arrangement of protein bound to the origin of replication. J. Virol. 46:151-161.

16. Tenen,D.G.,L. L.Haines,and D. M.Livingston. 1982.Binding ofananalog of the simian virus40 Tantigen towild-type and

mutantviralreplication origins.J. Mol. Biol. 157:473-492. 17. Tjian,R.1978.Protein-DNAinteractionsatthe origin of simian

virus 40 DNA replication. Cold Spring Harbor Symp. Quant. Biol. 43:655-662.

18. Wilson, V. G., M. J. Tevethia, B. A. Lewton, and P. Tegtmeyer. 1982. DNAbinding properties of simian virus40 temperature-sensitive Aproteins.J. Virol.44:458-466.

VOL.49,1984