Programmed factor binding to simian virus 40 GC-box replication and transcription control sequences.

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Vol.64, No. 1 JOURNALOFVIROLOGY,Jan. 1990, p.347-353

0022-538X/90/010347-07$02.00/0

Copyright C1990,American Society for Microbiology

Programmed Factor Binding to Simian Virus 40 GC-Box Replication

and Transcription Control Sequences

ROBERTL.BUCHANANt AND JAY D. GRALLA*

Departmentof Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los

Angeles,

405

Hilgard

Avenue,

Los

Angeles,

California

90024

Received6 July 1989/Accepted 22 September 1989

Nuclearfootprinting revealed a temporal programinvolving factor binding to the repetitive GC-box DNA elements present in the simian virus 40 regulatory region. This program specified ordered and directional binding to these tandem regulatory

sequences

in vivoduring the late phase of infection. The program was interrupted by the DNA replication inhibitor aphidicolin or by inactivation of the viral replication factor simian virus 40 T antigen, suggesting a link between viral DNA replication and new factor binding. Measurements of DNAaccumulation in viruses lacking either the distal or proximal halves of the GC-box region suggested that theregion has a dual role in replication control. Overall, the datapoint toimportantrelationships between DNAreplication and factor binding to the GC-box DNA, a multifunctional regulatory region.

Factor binding to mammalian regulatory DNA elements is

of central importance ingenecontrol. The cellular

distribu-tion and activity ofsuchfactors likely contributes to cell-specific patterns ofgeneexpression. Although many

DNA-binding factorshave beenidentified (see reference 26 for an example), the mechanisms that control their distribution and

activity are notunderstood. Many models relevant to such

mechanisms center onprogrammed factorbinding to DNA

and especiallyon theinfluence of DNAreplication on such programs (see, for example,reference 5).

Study of mammalian DNA tumor viruses has revealed well-defined genetic programs that depend directly or

indi-rectly on DNAreplication (45). In simian virus 40 (SV40) and adenovirus,componentsofthereplicationand

transcrip-tion machineryinvolved insuchprograms have beenpurified (14, 26). Most of the protein components are host cell proteins, which suggests that regulation of viral programs has important features incommon with cellularregulation. Among these proteins are cell factors which control SV40 replication and transcription through common DNA ele-ments(10).AcriticalDNA targetof these cellular

proteins

is the repeated motif(GGGCGG)6 orthe GC box which is a

regulatory element adjacent to the viral core origin of replication that influences DNA replication (24, 31, 32), as

wellasearly (1,11)andlatetranscription(4,13).Therole of

thesefactors in uninfected cells is alsolikelytobe involved with gene regulation, since GC boxes are often associated

with regulatory DNA (27, 38).

In situ DNase I footprinting of nuclear SV40 templates isolated from infected CV-1monkey cellsshowed abundant

factorbindingtothe SV40GCboxesat48 h

postinfection,

butneithertheidentity ofthe factorsnortheirfunctionalrole

isknown (6). Inthis report, we

identify

a

genetic

program

involvingthis factor andpresentexperiments

suggesting

that the programmed binding requires templates competent to replicate. Since GC boxesand

repetitive

DNAare

relatively

common in cells, these observations may have

interesting

implicationsfor

programming

interactions with cellular

reg-ulatoryDNA.

*

Corresponding

author.

tPresentaddress:Division ofBiology

156-29,

CaliforniaInstitute ofTechnology, Pasadena, CA 91125.

MATERIALS ANDMETHODS

Viral infections, preparation of nuclei, and DNA purifica-tion. SV40 strain tsA58 and parental strain VA45-54 were

obtained fromPeterTegtmeyer. Ingeneral, CV-1 cellswere

infected with1 to5 PFUofSV40 per cell and maintained in Dulbecco modified Eagle medium plus 2% calf serum plus antibiotics. Enough cells wereinfected to provide approxi-mately0.1 to 0.5 ,ugof SV40DNAperfootprinting reaction. Isotonic nuclei lysed with Nonidet P-40 (Sigma Chemical Co.) wereprepared andnicked with DNase I aspreviously described (6).DNA waspurifiedaspreviously described(6), except that the order ofRNase Aand proteinase K diges-tions was reversed to use endogenous RNA as acarrier in DNAprecipitations.

In some experiments, DNA replication was inhibited

before isolationof infected nuclei. At32 hpostinfection,the

cell culture medium was brought to 5 ,ug of

aphidicolin

(Sigma) per ml andinfections werecontinued forup to 16 h

(29).

Primer extension and gel electrophoresis. The sites of

DNase Inickingweredeterminedby hybridizing

5'-end32P-labeled

oligonucleotide

primers to denatured samples and extending them to breaks with Klenow DNA polymerase essentially as

previously

described (6, 20), except that all

extension reactions weredone at50to

52°C.

Base denatur-ation ofviral DNA

templates

gavealower

background

than heat denaturation for samples isolated before 40 h. Base

denaturationandsubsequent

primer

extension have

already

been

reported

(3).

Quantitationofreplicatedviral DNA. ConfluentCV-1 cells were infected with equal titers ofwild-type SV40 mutant virus strain 776, SV-P7, or PXS7.After 2h, infected

plates

were washed three or four times with warm Dulbecco modified Eagle mediumtoreduce theamountof unattached virus. Viral DNA was

prepared

as described above and

electrophoresed on 1% agarose

gels

after linearization with

EcoRI. Full-length nick-translated

([32P]dATP)

FIII SV40

DNA wasusedto

probe

DNA from 12-to24-h

postinfection

samplesbySouthern

hybridization.

DNAthat

hybridized

to thelabeled SV40

probe

wasvisualized

by

autoradiography

with Cronex film at room temperature. Several

autoradio-gramsatdifferentexposureswerescanned

by densitometry.

Viral DNAsamples

prepared

from

experiments

doneat24 347

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5183 5203 5223 5243/0 20 40 60 80 100 120

I I

EARLYmRNA

4-T AG 1 TAG 2

* 5145 -* 5230

.-

*

L

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TAG3

11 III IV v v I

AATTAGTCAGCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCG GGACTATGGTT

28 31 41 51 61 71 81 91 101

FIG. 1. TheSV40controlregion witholigonucleotide primersused infootprinting experiments.The bidirectional arrowdesignates the linear SV40 DNAsequencewith nucleotide numbers accordingtoTooze(45). The directions ofearlyand latetranscriptionareindicated.

Relevant SV40controlregionsarealsoshown, includingthe centers of threeT-antigen (T AG)-bindingdomains. Three arrowslabeled21

depictthe six GC-boxregulatoryelements.Thewedgelabeled 72 is theorigin-proximal portionof the72-base-pairviral enhancerelement.

Oligonucleotideprimersareshownas arrowswith numbers(5145and5230) indicatingthe 3' ends of theprimers.At thebottom,theSV40 GC-boxregionisexpandedtoshow thenucleotidesequencefromnts28to111. Within thisexpanded region,the GC boxesarenumbered with romannumerals. ORI, Originofreplication.

to65 hpostinfectionwerelinearized andelectrophoresedon

1%agarosegels after dilutionstypically intherangeof 1:10 to 1:1,000.DNAwas visualizedby ethidium bromide stain-ing andphotographed withKodak Tri-X film. Theamountof DNA ineach lanewasdeterminedbydensitometry.Arange ofexposures weretaken toassure linearresponse.

RESULTS

Thelatephase of theSV40lifecycleinvolvesprogrammed production of viral DNA and protein with the goal of assembling infectiousvirus(45). As the latephase proceeds,

more RNA is produced fromthe expanding pool ofDNA,

butthe averagetranscriptional activity ofSV40 DNA tem-plates remainsconstant(18). However, theaverage

replica-tion activity ofSV40 DNAdecreases during this time (24, 33). Thus, instead of the exponential production of DNAthat would accompany uncontrolled replication, the amount of SV40 DNA simply increases linearly. This relationship be-tween replication and transcription apparently ensures a

balanced ratio of protein to DNA. The purpose of these

initialexperiments wastoprobe thein situ interactions with DNA elements nearand within the replication core during

thetimewhentheprogrammeddecrease inaveragetemplate

activity occurs.

The method of probing DNA-protein interactions (6, 20) involves adding DNase I to nuclei isolated from SV40-infected CV-1 cells to lightly nick the endogenous SV40 DNA. The extrachromosomal SV40 DNA is then isolated anddenaturedto produce strands whoseendswere formed

byDNase Inicking insitu.Theattackpatternisrevealed by hybridizing5'-end 32P-labeledDNA primers adjacenttothe region ofinterest and extending them in vitrotorevealstop

sites correspondingtonicks(20).Primer 5145 is designedto probe thecore replication elements, and primer5230 reads throughtheaccessoryGC-box element (Fig. 1). Previously, probing with primer 5230 showed that the GC-box region

was partially protectedfrom DNase Idigestionbyabound factorat40 hpostinfection (6). Thetechniquedetects only the interaction of abundant factors with nonencapsulated DNA;the approximately90% ofSV40 DNAin virionsand previrions is relatively DNase I resistant (see reference 6) and does not contribute significantly to the signal in this assay. Factorbindingtoless thanhalf of the remaining10% of the DNA is difficult to detect with this method(6).

Probingthereplicationcoreregionin vivo.Figure2shows the results of in situ probing of the replication core

se-quences and the adjacent T-antigen-binding site I. Factor binding is assessed by comparing digestion of DNA com-plexedwith protein (nuclei, lanes 2 to 4)with digestion of naked DNA(in vitro, lane1). Therewassignificant

protec-tion ofsequences overT-antigen site I at 32, 40, and 48 h postinfection. However,strongprotectionofsequences

cor-responding to the core elements for replication which are

adjacenttothis sitewasnotobserved(comparelanes 2to4 withlane 1).

These footprinting results are in excellent accord with

long-standingobservations in the literature. At this time in the lytic cycle, the dominant interaction in this region is expected to involve T antigen bound at site I acting as a

repressorofearly transcription (34). Immunological studies indicated thatapproximately 10% of SV40 DNA in vivo is boundby T antigen (42), and this agrees well with strong protection ofapproximately 10% of the templates in these nuclear footprints. The limits of protection seen in these

nuclearexperimentsaresimilarto limits of T-antigen binding to site I in vitro (34, 44). Replicating templates constitute only 1% of SV40 DNA (42), and thus, protection of the replicationcoreorigininthisproportion oftemplates would be well below thelevel ofdetection;noextensiveprotection

wasseen,evenwithlighterexposuresof theautoradiogram. The timecourseofprobingrevealed no changesthat could easily account for the programmed decrease in template

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PROGRAMMED GC-BOX BINDING AND DNA REPLICATION 349

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FIG. 2. Nuclear protection ofthe region near the replication

origin (ORI)was probed onthe late mRNA template strand with

primer 5145 at 32, 40, and 48 h postinfection. The minimum replication origin, which is approximatelycoincident with T-antigen (T Ag)-binding siteII, isindicated, asis siteI. Lane 1 contained

SV40nicked in vitroandprobed with primer 5145as acontrol(C),

while lanes2, 3, and 4 contained samples nicked in nucleiat32, 40,

or48 hpostinfection.

replication activity during late times. The only changeseen

wasthat theinternal A+T-richspacerregion within T-antigen

site Iwasslightlymoreprotectedastheinfection proceeded. Programmed bindingtomultiple GCboxesin vivo.Aquite differentresultwas seen when theaccessoryGC-box DNA elementwasprobed (Fig. 3). When DNA samples from 48 h postinfection wereprobed, strongprotectionwas seen over

the entire GC-box regulatory element. When this pattern (lane 7)wascompared with that of the in vitro control (lane

4), it was found that all bands between approximately nucleotides (nts) 50 and 100 were stronglyprotected from

DNase I digestion in SV40-infected nuclei. The region be-tween nts30 and 50waspartially protected, andonlyweak

[image:3.612.320.563.85.402.2]

protectionwas evidentwithinthe enhancer (seealso refer-ence6). Attheearlier timeof40hpostinfection (lane 6),the patternwassimilar, exceptthattheprotectionwasnotquite

FIG. 3. Nuclear footprintsproducedonthe latemRNAtemplate

orCstrand in theGC-box region.Nuclei from SV40-infected cells were prepared at various times postinfection and nicked with

approximately0.01,ug ofDNase Iper,ulfor2min(lanes 2, 3, and

5to7).NuclearDNAswerepurified, denatured, andusedastemplates

forhybridization witholigonucleotide primer 5230. Lane C depicts controlSV40DNAnickedinvitrowithapproximately 0.0001 p.gof

DNase Iper,ul (lanes1 and4).The numbers above lanes 2, 3, and

5to7indicatethetimes (hours) postinfectionatwhich nucleiwere

probed. SV40 nucleotide numbersareshowntothe left of each gel, along with the locationsof theGC-box 21-base-pairrepeatelements (left panel)andindividual GC boxes (rightpanel).

as strong; this weakening occurredprincipally betweennts 70and 100. Evenearlier,at32hpostinfection (lane 5), this regionbetweennts70and 100wasmuch less wellprotected, andat24hpostinfection, the weak protection of this region

wasdiminishedfurther(lane 2). This loss ofprotectionwas

accompanied bytheappearanceoftwopositions of DNaseI hyperreactivity near nts 75 and 95. As discussed above, strongly protected regions inthisassay aretakentoreflect factorbindingtoabout 10%of the nuclearSV40molecules. This is an order ofmagnitude higher than the fraction of templates actively engagedineitherreplicationor transcrip-tion(15, 42).

Thus, ourviewof thisprogram canbegin at24 h postin-fection,whenreplication activityis stillhigh (24, 33).At that time, only the region surrounding GC boxes 2 and 3 is stronglyprotected. As thetemporalprogram proceeds, GC boxes 4 to 6 are bound progressivelywith the factor. This orderedbindinghas notbeenreportedforanyof thecellular factors known to bind the GC-box region. As reported previously (see Fig.3 in reference6), this factorprotectsthe earlymRNAorGstrand lessstronglyin theGC-boxregion,

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VOL.64, 1990

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350 BUCHANAN AND GRALLA

another property not noted with isolated GC-box DNA transcription factors. This preference for the late mRNAor Cstrand wasmaintainedthroughout the latephase(datanot shown). We conclude that programmed factor binding ex-ists;duringthe time when the pool of templatesbecomesless active in producing DNA, the GC-boxDNAis increasingly boundby anabundant nuclearfactor.

Inhibition of programmed factor binding inhibition of DNA

replication. We beganexperiments toprobe the mechanism underlying this progressive factor binding to the GC-box

DNA region. A key issue involving genetic programs in

SV40 and in cells is whether DNA replication playsadirect role in influencingfactor binding (5, 45, 47, 48). This issue

canbe explored in this system because there is continuous production ofnew templates while the SV40 program

pro-ceeds. Thus, replication can be inhibited early in the pro-gram and it can be observed whether the programmed increase in bindingis also inhibited or whether the patternis otherwise perturbed.

Thepossibility that DNA replication is requiredforfactor bindingwastested initially by inhibiting DNA synthesiswith aphidicolin (29) and monitoring alterations in factorbinding by nuclear footprinting. In this experiment, a series ofplates

was infected with SV40. At 32 h postinfection, half of the

plates were treated with 5 ,g ofaphidicolin per ml. Both

control plates and drug-treatedplates were incubated for an

additional8 or 16 h. At these times (40 and 48 h

postinfec-tion,respectively), nuclei were prepared and the SV40 DNA was footprinted in situ to estimate factor binding. As a

further comparison, control samples from 32 h postinfection were processed in parallel. Thus, the effect of the DNA

synthesis inhibitor aphidicolin on factor binding can be

deducedfrom this collection of data (Fig. 4A).

The results showed that inhibition of DNA replication

with aphidicolin inhibited binding of factors to additional

sites within the GC-box region. When DNA synthesis was

inhibitedat 32 hpostinfection, continuedincubation until 40 or 48 h postinfection did not lead to significant additional

factor binding; that is, the 32-h pattern was nearly frozen

(Fig. 4A, compare lanes 1, 4, and 5). In parallel samples in which DNA synthesis was not inhibited, the expected in-crease in factor binding occurred during this time (lanes 2

and 3). These results also indicate that previously bound

GC-box interactions were stable and that nonreplicating DNA remained bound by factors for at least 16 h in vivo

(compare lanes 1 and 5). Similar results were obtained with

cycloheximide, which is known to inhibit protein synthesis and SV40DNAreplication (data not shown).

Sinceaphidicolin inhibitsbothSV40 (9) and cellular DNA synthesis,theeffect oftemplate replicationonfactor binding was assessed by using a viral mutant in which cellular

processes are not affected. In this experiment, parallel

cultures were infected with either tsA58 virus or

VA45-54,

the wild-type parent of mutant tsA58 (43). tsA58 is a virus with atemperature-sensitive T antigen, and thus, it cannot

replicateat42°C. The twoviruses were grown at 32°C for 40 h to allow replication and partial factor binding to

occur,

both ofwhich occur rather slowly at 32°C. TheVA45-54-and tsA58-infected cultures were then shifted to the nonpermis-sive temperature of41.5°C and incubated for 10 h longer.

Viral DNA replication during the 10 h occurred only in

VA45-54-infected cultures. After this incubation, nuclei were preparedfrom bothculturesandfootprintedtoassess

factor binding (Fig. 4B). This comparison showed that

additional factor binding at 41.5°C occurred when viral

replication

proceeded (VA45-54in lane 1B)and notwhen it

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FIG. 4. Inhibition of GC-box factor binding by inhibition of DNA replication. (A) Nuclei were harvested at 32, 40, and 48 h postin-fection, as indicated above the lanes. A plus or minus indicates whether aphidicolin (5 j±g/ml) was added to infected plates at 32 h postinfection. At the indicated times, nuclei were prepared, nicked withDNaseI,and probed with primer 5230. (B) Cells infected with VA45-54(lane 1) or tsA58 (lane 2) for 40 h at32°C.At 40 h, infected cells were shifted to41.5°C(nonpermissive for tsA58 viral replica-tion) and incubated for an additional 10 h at41.5°C.Nuclei were the prepared, nicked with DNaseI,and probed with primer 5230. The amount of viral DNA present at 40 h corresponded approximately to the amount that appeared in a 37°C wild-type infection at 24 h postinfection.

was blocked(tsA58in lane 2B). That is, the GC boxes were much more protected (lane 1B) when T-antigen-dependent viral replication was allowed to continue. The experiment demonstrated that SV40 T antigen is required for GC-box filling. Taken together with the inhibition of binding by aphidicolin, the data indicate that T-antigen-dependent viral DNAreplication is required for programmed factor binding. Effects of the region containing GC boxes on replication timing and rate. Previous studies have shown that deletions within the GC-box region lead to lowered levels of replicated SV40 DNA (23, 24, 32, 50). Since the data indicate that binding to the GC boxes depends on functional T antigen, it seemed possible that the GC boxes could also influence how J.VIROL.

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FIG. 5. Southernblot analysis of DNAs produced bywild-type SV40 (lanes 1, 4, 7, 10, 13, 16, and 19), SV-P7 (lanes 2, 5, 8, 11, 14, 17, and 20), and PXS7(lanes 3, 6, 9, 12, 15, 18, and 21) from 12 to 24 h postinfection. CV-1 cells were infected, and DNAs were harvested at the indicated times postinfection. Purified DNA was linearized and diluted before electrophoresis on 1% agarose gels. Gels were blotted with nitrocellulose and probed with whole nick-translated SV40 as described in Materials and Methods. Only the major SV40 band is shown.

Tantigenturns on replication. The footprinting procedures arenotsensitiveenough to probe the onset of replication at

earlytimes directly. Instead, we used mutants to investigate

the role of the GC-box region in replication timing.

Trans-fected plasmids are unsuitable for this purpose, since the

time at which they begin replication is altered (8). Thus, mutants built into viable virus must be used. We had

available two such mutantviruses, each ofwhich deletes a

different halfof the GC-box region. Virus SV-P7 lacks GC

boxes 1 to 3 and nine adjacent nucleotides (nt 32 to 73),

approaching but not impinging upon the replication core

(Buchanan and Gralla, unpublished data), and virus PXS7

(16) lacksGC boxes4to 6 (nt 73 to 100). Takentogether with

wild-type SV40, the setof viruses allows assessment of the

function ofthe40-base-pair regionadjacent to the replication coreand the30-base-pair region between this regionand the

enhancer.

In thefirstseries of experiments, the time of the onset of

replicationwasmeasuredinviruses lacking eitherearly-side

(SV-P7) orlate-side(PXS7) GCboxes. DNA was harvested every 2hfrom12to 24 hpostinfection, anditsamount was

estimated by probing Southern blots (Fig. 5). The two mutantviruses begantoreplicate atgrossly different times. SV-P7 DNA was visible at 12 h postinfection (lane 2) and

gradually increased inamount overthe time assayed (lanes 2, 5, 8, etc.). Bycontrast,PXS7DNA(lanes3, 6, 9,etc.)was notvisible until22hpostinfection (lane18), whenit beganto

accumulate. Thus, deletion of thetwohalves of this region hadvery different effectsonthe timing ofDNA accumula-tion. This differential effectonreplication timing is unlikely

to be dueto altered early accumulation ofTantigen, since

deletion of the late-side GC boxes should not be more

deleterioustoT-antigenexpressionthanis deletion of early-side GC boxes.

These sameblotsshow thatwild-typeDNA(lanes1,4, 7, etc.)wasbarelyvisible at 12 hpostinfectionandincreasedin amountby 16 hpostinfection, asexpected (45). During this time interval, the hierarchy in the amounts ofDNA was maintainedasSV-P7>wildtype>PXS7.Weconclude that

thePXS7mutationdrasticallyslows theonsetof

replication,

even

though

thePXS7

early

promoter appears tobe

nearly

asefficient aswild-typeSV40 in vivo(21).Theearlier timeat which SV-P7DNAbegantoaccumulatewas

unexpected

for two reasons. (i) The region deleted is critical for

early

transcription (see above references), and thus, one

might

haveexpectedadelayduetoslower

production

of T

antigen.

(ii)Asdiscussedabove, other GC-box deletionshave ledto lower,nothigher, totallevels ofSV40

DNA,

andthislower ultimate levelofDNAwasalso truefor SV-P7(see

below).

Ourbest

quantitative

estimate is that PXS7 is

delayed

for 6

to8 hand SV-P7accelerated

modestly,

by2or

perhaps

3 h.

10

WtSV40

z 6

SV-P7

0

E

4c

4

2

PXS7

0

10 20 30 40 50 60 70 80

TimePost-infection(hours)

FIG. 6. Accumulation of wild-type (wt)SV40, SV-P7, and PXS7 from 24 to 65 h postinfection. DNAs from infected plates were harvested andquantifiedattheindicated timespostinfection. More generally, the

70-base-pair

region

covered

by

these

twodeletions acts as a

timing

element

influencing

the onset

ofSV40DNA

replication.

The amounts ofDNAmade by these three viruses were also monitored past 24 hpostinfection to observe the

long-term effect on rate ofDNA

production.

All three viruses

accumulatedDNAfrom24 to 65 hpostinfection

(Fig.

6)but atdifferentrates.During thistime, wild-typevirus

produced,

on average, 0.26 p,g ofDNA perh, SV-P7

produced

0.18

,ug/h,

and PXS7

produced

0.05

,ug/h.

Thesedata agree with

those of various studies

showing

lowered DNA

yields

causedby GC-box deletions (16, 24, 32,50)butare

interest-ing

intwo respects.

(i) SV-P7,

which

produced

moreDNA

than did the wild type before 24 h

postinfection

(Fig. 5),

produced less by 65 h

postinfection (Fig. 6).

(ii)

Although

SV-P7 and PXS7, which delete separate

subregions,

had

opposite

effects on the onset of

replication, they

had the

same

qualitative

effecton

steady-state

replication

rates;

i.e.,

both reduced the rate ofSV40 DNA

synthesis. Thus,

the

region contains separatecontrolsfor

replication

timing

and

steady-state

replication

rate

(Fig. 7).

Both halves of the

regionare required for

optimal

DNA

accumulation,

consis-tent with the idea that when

they

are occluded

by

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l I I I I I III IV v vI

H

30

EARL Y DELAYSONSET ACCELERATESONSET

LATE REQUIRED FOR MAXIMUM REPLICATION RATE

FIG. 7. Effect(s) ofSV40GC boxes on viral DNA replication.

SV40 GC boxes are schematically shown as six adjacent boxes.

Belowaredepicted the effects oftwoGC-box subregionsonDNA

replication during the early and late phasesof the SV40 lyticcycle. grammedabundantfactorbindingorwhentheyaredeleted,

replication is inhibited. The two subregions encompassing the six GC boxes affect thetime ofreplication onset differ-ently, implying that before replication onset, an interesting

but unknown genetic program exists which controls the

initiation of DNAreplication. DISCUSSION

These experiments have described a temporal program

involving interactions at the repetitive SV40 GC-box

regu-latory elements. T-antigen-dependent DNA replication is requiredfor assembly ofa complex that protectsthe entire region from DNase digestion in nuclei. This assembly

pro-ceeds by filling the GC boxes in order, from the origin-proximal toward the origin-distal elements. It is not known whether therequirementforT-antigen-dependentreplication implies that this genetic program requires specific

compo-nents of the active SV40 replication complex, which is positioned nearby, or simplynewly replicated DNA.

The GC-box region consists of repeated sequences, and

repetitive elements areknowntobe associatedwith

tempo-ral control by diverse proteins, such as X repressor and T

antigen (34, 37). In X, the bound sequences have a dual

function and serve as activator sites at low factor

concen-trations but mediate repression when occluded at high

pro-teinconcentrations. These datasuggestthattheinteractions with the SV40 GC-box elements are analogous; i.e., the

elements serve as activation sites exceptwhen occludedby factors. TheamountofSV40DNA bound by factorsat late times is much greater than the amount involved in replica-tionortranscription; thus, atthesetimes, the factorappears

not to activate the templates but rather to occlude them. Recall that SV40 GC boxes are not required to activate transcription atlatetimes (24) and that no net activation of transcriptionoccursduringthistime(18).Onthebasisof this

circumstantial evidence, wesuggestthatprogrammedfactor binding at late times progressively occludes the GC-box replication element to program the gradual decrease in

template replication activity.

Inthe GC box and X examples, repeatunits arebound in a specified direction as a program proceeds. Since the

GC-box units are of very similar DNA sequences but are

bound inaspecified order, cooperative interactions maybe

involved. However, the mechanism underlying directional GC-box factor bindingremains unknown, since none of the

factors known to bind this region in vitro were reported as

having this property of directional binding (26). Neverthe-less, the ability of prebound factors to specify the nearby positioning ofnew factors only on templates competent to replicate could have an important influence on

reprogram-ming expression in newly made cells (5, 46, 47).

A second property of the bound complex may also have

interesting implications, since thefactorprotectsoneof the DNA strands more stronglythan theother

(6);

upon strand

separation during

replication,

it is

possible

that this

prefer-ence forone of the strands would be

maintained,

allowing

preferential transmission to one of the two

daughter

DNA

molecules (see also references 41and49).

Several different factorshave beenisolated

(17, 28,

35,

36)

thatbind SV40 GC-boxDNAinvitrosince thediscoveryof SP-1 (11). The abundant GC-box factor bound in

SV40-infected cells is believed to be ofcellular rather than viral

origin (7),

although

a role for late viral proteins cannotbe

excluded. At least two

previously

characterized GC-box-binding factors share the unusual property of strand prefer-ence. (i)Achicken

globin

genefactorbindsG-string

regula-tory sequenceswithastrandpreference similartothat of the SV40 GC-box factor (12). (ii) It has recently been demon-strated that uninfected CV-1 cells contain a factor that preferentiallybinds one strand ofanSV40 GCbox,although

it does not appear to bind double-stranded DNA (17). It is not known whether the monkey cell factordetected in this

studyisanalogoustothesefactors, SP-1,oranyof theother isolated human factors; GC-box-binding proteins have not beenpurified frommonkey cells.Inbothhumanandmonkey cells, GC-box elements are common (27) and are often repeated when present, as in SV40.Thus, cellscontain both components ofthe DNA-factor interaction described here,

and therefore, the interesting properties ofthe SV40 com-plex are likely to be shared by certain cellular genes.

These experiments have confirmed that the SV40GC-box repeat unit influences the SV40 replication rate (see refer-ences in the introduction) and have also shown that subre-gions control the timing of replication onset (Fig. 7). Since mammalian cellularorigins of replication arepoorly charac-terized, it is not known whether some origins are associated with GC boxes, G strings, or other repeat elements. How-ever, it is known that thetiming of cellular DNAreplication

is precisely controlled. The earliest genes to be replicated

aregenerallythe so-calledhousekeeping genes (25). GC-box repeat elements are highly concentrated in this gene type (19, 25). Since some proposals link origins ofreplicationwith transcriptional activation (22), it is possible that these cellu-lar GC boxes influence replication as well asgeneexpression;

recallthat SV40GCboxes have this dual effect (10). The data presented here are most easily interpreted as implying that the onset of viral replication requires removal of a repressor fromearly-region-proximal GC boxes I, II, and III (Fig. 7). This observation could have significant impact on the eval-uation and extension of models for control ofDNA

replica-tion onset ineucaryotic cells (2, 22, 30, 39, 40). ACKNOWLEDGMENTS

This research was supportedby grants from theNational Science Foundationand the American CancerSociety.

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