dna replication and chromatin structure of simian virus 40

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1984. p. 1499-1507 Vol. 4. No. 80270-7306/84/081499-09$02.00/0Copyright © 1984. American Society for Microbiology

DNA Replication and Chromatin Structure of Simian Virus 40Insertion Mutants

JEFFREY W. INNIS AND WALTER A. SCOTT*Department of Biochemistrx, University of Miami School of Medicine, Miami, Florida 33101

Received 23 February 1984/Accepted 21 May 1984

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replicationefficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin ofreplication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfectioninto COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure wasretained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the oni site.New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves withinthe ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficientfunction of the viral origin of replication is correlated with its proximity to an altered chromatin structure.

Replication of simian virus 40 (SV40) DNA in monkeycells is dependent on a short segment of the viral genome(the ori site) (2, 8, 18, 20, 39, 40). Bergsma et al. (2) haveidentified additional sequences which are necessary for themaximal replication rate of recombinant SV40/pMK2004plasmids in COS-1 cells (a line of SV40-transformed monkeycells which provides all necessary trans-acting functions forreplication of plasmids containing an SV40 ori site [15]).Nucleotide sequences responsible for replication enhance-ment were mapped to a region of the SV40 genome near oriwhich contains the tandemly repeated 21-base-pair (bp)sequences. This region has also been implicated in both earlyand late gene promoter function (1, 4, 9, 11, 12, 19, 26, 30,31).The ori site lies at one end of a segment of the viral

genome which is preferentially sensitive to endonucleasecleavage in SV40 chromatin (6, 34-36, 41, 42, 44). Thisnuclease-sensitive chromatin structure is determined bymultiple genetic elements (13, 14, 25, 47), and its positioncorrelates with a nucleosome-free gap in a subpopulation ofchromatin molecules as visualized by electron microscopy(24, 35). By using duplicated mutants of SV40, it was shownthat deletion of sequences within ori had little effect on theoverall nuclease sensitivity of this region. Since deletionsand point mutations within ori abolish replication (8, 38, 39),it was concluded that replication from that origin was notnecessary for the establishment of nuclease sensitivity.We are interested in defining the relationship between

SV40 chromatin structure and the ori site replication func-tion. We constructed a series of insertion mutations in whichthe distance between ori and the 21-bp repeats is increased,and we evaluated the effects of these mutations on DNAreplication and on the nuclease-sensitive chromatin struc-ture.

MATERIALS AND METHODSCells and viruses. COS-1 cells (15) were grown in minimum

essential medium (MEM, Earle salts; MA Bioproducts)supplemented with 10% fetal calf serum (FCS) (KC Biologi-cals or Flow Laboratories) at 37°C in an atmosphere of 5%CO2. BSC-1 (African green monkey kidney) cells were

* Corresponding author.

grown in the same medium. Stocks of wild-type SV40 (strain776) and in(Or)-1411 (a double-origin mutant of SV40) (37)were grown in BSC-1 cells. Virus infections were performedat 37°C at multiplicities of 0.5 to 3.Enzymes. BamHI (Bethesda Research Laboratories) and

all other restriction enzymes (New England Biolabs) wereused under conditions suggested by the suppliers. T4 DNAligase and Escherichia coli DNA polymerase I were pur-chased from New England Biolabs. The Klenow fragment ofDNA polymerase I was purchased from New EnglandNuclear Corp. RNase A (type R) and DNase I (type D) werepurchased from Worthington Diagnostics, and stock solu-tions were made as previously described (23).

Construction of pJ1l and plasmid insertion mutants. Plas-mid pJIl was constructed as described by Innis and Scott(23) and purified by centrifugation to equilibrium in cesiumchloride-ethidium bromide gradients. Insertion mutationswere constructed by digesting pJIl with NcoI, incubating theDNA with the Klenow fragment of DNA polymerase I, andreligating without added SV40 DNA (to form the 4-bpinsertion) or in the presence of an AluI digest of wild-typeSV40 DNA (purified by centrifugation to equilibrium in acesium chloride-ethidium bromide gradient, followed bycentrifugation in a neutral sucrose gradient [7]). Plasmidsfrom tetracycline-resistant clones were screened for insertson the basis of size (determined by agarose gel electrophore-sis). Accurate sizing of the inserts was performed by restric-tion enzyme digestions and polyacrylamide gel electrophore-sis. Appropriate fragments of plasmid DNA were sequencedby the chemical degradation procedure of Maxam and Gil-bert (29).

Construction of viral insertion mutants. Plasmid insertionmutant DNA (3 ,ug) was mixed with 3 ,ug of a plasmid (pJI2)containing the large PstI fragment of in(Or)-1411 inserted inthe PstI site of pBR322. The mixtures were digested withPstI. PstI was removed by addition of sodium dodecylsulfate and EDTA, followed by phenol and chloroform-isoamyl alcohol (24:1) extraction. The DNA was precipitatedwith ethanol. resuspended, and incubated overnight at 16°Cwith T4 DNA ligase. The mixtures were chloroform steril-ized, and one-half of the DNA was used to transfect 60-cm2dishes of BSC-1 cells (80 to 90% confluent). After transfec-tion for 1 h at 37°C in the presence of 200 ,ug of DEAE-

1499

1500 INNIS AND SCOTT

dextran per ml (molecular weight. 500.000: Sigma ChemicalCompany), the fluid was removed, the cells were washedonce with 5 ml of MEM containing 10% FCS, and 10 ml ofthe same medium was added. The following day, the mediumwas replaced with medium containing 0.9% agar. Ten dayslater virus plaques were visualized by staining with neutralred. Plaques were isolated, and virus stocks were grown inBSC-1 cells. Viral DNA prepared from 32P-labeled infectedcells by the method cf Hirt (21) was digested with restrictionenzymes and fractionated by electrophoresis in 6 or 8%polyacrylamide gels to determine the structure of the viralDNA.

Transfection with plasmid DNA. Plasmid DNA was intro-duced into COS-1 cells by using DEAE-dextran as describedby Innis and Scott (23). Briefly, cells were washed withMEM (containing no serum) and incubated with plasmidDNA (0.005 to 0.3 [Lg/ml) and DEAE-dextran (200 ,ug/ml) forthe times indicated in the figure legends. Cells were washedwith MEM containing 5 c FCS and then incubated either inmedium containing 100 FLM chloroquine and 5% FCS (28) forthe times indicated in the legends or in fresh medium with5% FCS. Chloroquine-treated cells were subsequently incu-bated in fresh medium with 5% FCS.

Isolation of nuclei, DNase I digestion, and recovery ofplasmid or viral DNA. Nuclei were isolated from plasmidDNA-transfected COS-1 cells or virus-infected BSC-1 cellsand digested with DNase I as described by Wu et al. (48)with slight modifications (23). Form III (full-length linear)plasmid or viral DNA was recovered as previously described(23) and digested with restriction enzymes to localize DNaseI cleavage sites.Endogenous endonuclease and Bgll digestion of viral chro-

matin from infected cells. Chromatin from virus-infectedBSC-1 cells was extracted 43 h after infection by the TritonX-100-EDTA extraction procedure of Green et al. (16) asmodified by Scott and Wigmore (36). The endogenous endo-nuclease in this nuclear extract was activated by the additionof MnCl to 2 mM and incubation at 37°C for 10 min. FormIII 32P-labeled viral DNA was purified and digested with arestriction enzyme to localize endogenous endonucleasecleavage sites.

In some experiments, the Triton X-100-EDTA nuclearextract was layered onto a 5 to 20% linear sucrose gradient inTENT buffer (10 mM Tris-hydrochloride [pH 7.4]. 0.5 mMEDTA, 50 mM NaCl, 0.17% Triton X-100) and centrifuged at35,000 rpm for 2.5 h (4°C) in an SW41 rotor. Gradientfractions containing -P-labeled viral chromatin were pooledand dialyzed against TENT buffer with several bufferchanges. This preparation was incubated with BglI underconditions suggested for the enzyme by the supplier, andform III viral DNA was purified and redigested as described.

Blotting, hybridization, and autoradiography. The DNAwas transferred to nitrocellulose and hybridized in thepresence of 50% formamide for 2 to 3 days at 37°C (43) withthe 32P-labeled probe(s) (prepared by nick translation [33])indicated in the figure legends. The blots were washed threetimes for 5 min each in 2x SSC (1x SSC is 150mM NaCl and15 mM sodium citrate [pH 7.0])-0.1% sodium dodecylsulfate at room temperature and twice for 15 min each in0.1x SSC-0.1% sc'dium dodecyl sulfate at 37°C. Afterdrying, each blot was placed directly on Kodak XRP-1 X-rayfilm with a Du Pont Quanta III intensifier and exposed at-70°C for 1 to 3 days or without an intensifier for up to 6days. Bands on unintensified autoradiograms were scannedwith a Zeineh scanning densitometer.

RESULTSConstruction of plasmids containing insertion mutations.

Insertion mutations were introduced into the SV40 se-quences in plasmid pJII (Fig. 1). This plasmid (23) containsthe small PstI fragment of SV40 variant inz(Or)-1411 harbor-ing an inserted SV40 origin region and adjacent sequences(37) at the 3' ends of the early and late transcriptional units.The non-SV40 portion of pJIl consists of a derivative ofpBR322 constructed to remove sequences inhibitory toreplication in COS-1 cells (27. 32). Inserted DNA segm ts

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FIG. 1. Physical maps of plasmid insertion mutants and in(Or)-1411 derivatives. (A) pJ1l and plasmid insertion mutants. PlasmidpJI1 contains the small Pstl DNA fragment from in(Or)-1411 (thickline) in the single Pstl site of the pBP.322 derivative. pBR-d/1329-2517 (thin line) (23). Segments of DNA were inserted into pJIll at theunique Ncol site as described in the text. Plasmid insertion mutantsare designated pJIl-inX where X is the size of the insert in bp. pOrand vOr refer to the pBR322 and SV40 origins of replication.respectively. The stippled box labeled A indicates a fragment ofpBR322 DNA used to prepare hybridization probe A. (B) in(Or)-1411 and insertion mutant derivatives. in(Or)-1411 is a nondefectivedouble-origin mutant of SV40 constructed by T. Shenk (37). l and Ilare nuclease-sensitive regions seen in in(Or)-1411 chromatin afterextraction from mutant-infected cells (14). Early and late refer totranscr ption units of the virus. Unlabeled wedge-shaped extensionsfrom tht; circle indicate deleted sequences in i:(Or)-1411 by compar-ison with wild-type SV40. The stippled boxes represent DNAsegments used to prepare hybridization probes B and C. in(Or)-1411derivatives containing insertion mutations in region ll are designat-ed inX, where X is the size of the insert in bp.

MOL. CELL. BlOt.

REPLICATION AND CHROMATIN STRUCTURE OF SV40 MUTANTS 1501

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FIG. 2. Nucleotide sequences of inserted DNA segments. Nucleotide sequences for the insertion mutations shown in Fig. 1 weredetermined by using fragments of plasmid DNA spanning the inserted segment. The insertion in pJI1-in56 and in(Or)-1411 derivative in56corresponds to SV40 sequences from nucleotide 1581 to 1634 (SV numbering system for SV40 [3]), and the insertion in pJI1-in132 correspondsto nucleotides 2523 to 2651 from SV40. The inserted DNA segments in pJI1-in42 and pJI1-in9O [as well as in(Or)-1411 derivatives in42 andin90] do not correspond to SV40 DNA, and their origin is unknown. All insertions were introduced between nucleotides 37 and 38 (numberingis from the center of the 27-bp palindrome in the SV40 sequences of pJIl with positive values in the late direction [23]). The DNA sequences ofinserts in pJI1-in260 and pJI1-in390 were not determined. The sizes of these insertions were estimated with an accuracy of ca. + 10 bp from re-striction enzyme fragments, and it was determined by restriction enzyme analysis that neither insert contains the SV40 origin.

were placed between the 21-bp repeated sequences and theSV40 ori site in pJIl1. The nucleotide sequences of theseinserted segments are shown in Fig. 2.

Replication of recombinant plasmids in COS-1 cells. Wecompared levels of supercoiled (form I) pJIl and plasmidinsertion mutant DNA at various times after transfectioninto COS-1 cells (Fig. 3). Most of the input DNA did notpersist as form I for longer than 24 h and was presumablydegraded to relaxed (form II) and linear (form III) molecules(27, 32); however, in cells transfected with pJIl or theshorter insertion mutants, form I DNA reappeared andincreased in amount between 24 and 48 h after transfection.To determine whether replication was responsible for thisaccumulation, samples of DNA from cells transfected bypJIl and by pJI1-in132 (48-h time points from the experimentshown in Fig. 3A) were incubated with Bcll. The form IDNA was completely digested in both of these samples,whereas input DNA was resistant to cleavage by this en-zyme due to adenine methylation in dam' E. coli (data notshown).We used the levels of form I DNA at 48 h as a basis for

comparing replication efficiency between plasmids. Thegreatest reduction in replication efficiency was seen with thelargest insertion mutants. Cells transfected with pJI1-in260or pJI1-in390 accumulated form I DNA to a level less than1/10 of that in cells transfected with pJIl (Fig. 3B). PlasmidspJI1-in42, -in56, -in90, and -inl32 showed moderate reduc-tion in replication efficiency. The effects of these insertionmutations were somewhat more pronounced when only 10ng of plasmid DNA was used to transfect each dish (Fig. 3B),in agreement with the results of Lusky and Botchan (27).There was no difference between the replication efficienciesof pJIl and pJI1-in4.

Superinfection of plasmid-transfected cells with wild-typeSV40 did not correct the replication defects in the plasmids(Fig. 3C), indicating that these mutations act in cis.

In summary, replication from the SV40 origin is depressedin plasmids containing inserted DNA between nucleotides 37and 38, and the degree of depression is dependent on the sizeof the insertion but independent of the sequence inserted.The most likely explanation for this phenomenon is thatsequence element(s) on the late side of the site of insertionis required for maximum replication efficiency and that thecontribution of this element(s) is reduced when it is movedaway from the ori site.

Construction of viral insertion mutants. Since a moresensitive analysis of chromatin structure is possible whenDNA molecules are introduced into cells by virus infectionrather than by transfection, we used the plasmids described

above to reconstruct nondefective viral genomes. Plasmidscontaining each insertion mutation were mixed with plasmidpJI2 [which contains the remainder of the in(Or)-1411genome]. The mixture was digested with PstI, incubatedwith DNA ligase, and transfected into BSC-1 cells. Virusplaques were isolated and stocks were prepared. The ge-nomes present in these stocks were the same as in(Or)-1411except that they contained inserted DNA between nucleo-tides 37 and 38 in region II (Fig. 1B).

Cleavage of mutant chromatin by endogenous endonucleaseor DNase I. We compared the frequency of cleavage in thetwo nuclease-sensitive regions of chromatin from in(Or)-1411 and the insertion mutants (Fig. 4). Cleavage of chroma-tin by endogenous endonuclease in region I or II, followedby isolation of full-length linear DNA and redigestion withEcoRI, yielded four clusters of fragments. (Only the largertwo clusters are shown in Fig. 4. The band extending from3.4 to 3.7 kilobases [kb] corresponds to cleavage withinregion I, whereas region II gave fragments 4.0 to 4.3 kb insize.) At the level of resolution in this experiment, insertionof up to 90 bp ofDNA had no significant effect on the ratio ofspecific cleavage in region I to that in region II.When the location of cleavage sites was examined at

higher resolution (Fig. 5), a systematic effect of the insertionmutations became apparent. The characteristic pattern ofcleavage seen in region II of in(Or)-1411 was retained in theinsertion mutants, but the sites were displaced in the latedirection. Endogenous endonuclease cleaves primarily atfive positions in region II of in(Or)-1411, generating frag-ments of ca. 0.94, 0.98, 1.03, 1.12, and 1.2 kb. This charac-teristic pattern was retained in the insertion mutants; howev-er, except for the band at 0.94 kb, the fragments were largerby approximately the size of the inserted segment. From thiswe conclude that cleavage occurred in the same sequences inthe insertion mutants as in in(Or)-1411 on the late side of thesite of insertion; however, on the early side, cleavage oc-curred in new sequences. This is illustrated in Fig. 5 by thevertical bars to the right of lanes containing digests from thelarger insertion mutants. These bars show where the insertedDNA would lie if the DNA sequence of this region werealigned with the cleavage pattern. The band at 0.94 kbcorresponds to cleavage within the same sequences near theBglI site in all of the mutants.The patterns of cleavage by DNase I and endogenous

endonuclease were different in detail, although each enzymeselectively cleaved within the nuclease-sensitive region (adirect comparison of the cleavage patterns is shown in Fig.6B). The effect of the insertion mutations on the pattern ofDNase I cleavage was similar to that seen with endogenous

VOL. 4, 1984


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FIG. 3. Replication of pJIl and plasmid insertionpJl1 -in4 -in42 -in56 -1in9o mutants. (A) Subconfluent dishes (20 cm2) of COS-1

cells were transfected for 7 h with 100 ng of super-c b c b c b c b c b coiled plasmidDNA in a volume of 2 ml. At the timesr-l I I m m1 m--- rI ii__ i

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 indicated (hours after the start of transfection) the cellswere lysed. and the cellular DNA was precipitated bythe method of Hirt (21). DNA in the supernatantsolution was fractionated by electrophoresis through a1.2% agarose gel, blotted to nitrocellulose paper, andhybridized with probe A (Fig. 1). Lanes: a, pJIl; b.pJI1-in4; c, pJIl-in42; d, pJ11-in56; e, pJI1-in90: f.pJI1-in132. I, II, and III (in panels A, B, and C) refer tothe supercoiled, relaxed circular, and linear forms ofplasmid DNA, respectively. (B) Subconfluent dishes(20 cm2) of COS-1 cells were transfected for 7 h with10 ng of plasmid DNA in a volume of 2 ml. At 24 (lanes1), 48 (lanes 2), and 69 (lanes 3) h after the start oftransfection, the cells were lysed, and plasmid DNA inthe supernatant solution was fractionated by electro-phoresis through a 1.2% agarose gel, blotted, andhybridized with probe A. (C) Subconfluent dishes (20cm2) of COS-1 cells (lanes c) and confluent dishes ofBSC-1 cells (lanes b) were transfected with 50 ng of

w**-* +plasmid DNA for 2 h and then superinfected with wild-type SV40. At 24 (lanes 1) and 48 (lanes 2) h after thestart of transfectiqn, plasmid DNA was extracted bythe method of Hirt (21), fractionated in a 1.2% agarosegel, blotted, and hybridized with probe A.

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FIG. 4. Comparison of the frequency of cleavage by endogenousendonuclease in region I versus region 1I in viral insertion mutantchromatin. A nuclear extract was prepared from 32P-labeled. mu-tant-infected BSC-1 cells. Endogenous endonuclease was activatedby addition of MnCI. to 2 mM. Form III viral DNA was isolated bypreparative agarose gel electrophoresis and digested with EcoRI.and the resulting fragments were fractionated by electrophoresis in1.2% agarose. Autoradiography was performed on the dried gel. Iand I1 indicate digest products resulting from initial cleavage byendogenous endonuclease in region I or 11 of the viral genome (Fig.1). The two largest bands for each mutant were scanned with adensitometer, and the areas which correspond to specific cleavageover regions I and II were determined by integrating the densitome-ter tracing after first graphically subtracting a background of randomcleavage. For each lane the ratio between the number of moleculesspecifically cleaved in region I to those specifically cleaved in regionII (F,/F,,) was calculated and is given at the bottom of the figure.This calculation is a modification of the quantitative proceduredescribed by Wigmore et al. (47) in which the DNA is assumed to beuniformly labeled with 32P and the relative number of molecules isdetermined from the total radioactivity and the molecular weight.M, Size standards from in(Or)-1411 DNA.

endonuclease. DNase I digestion of nuclei from infectedcells gave rise to two major bands after redigestion withEcoRI [ca. 0.96 and 1.1 kb in in(Or)-1411; Fig. 6A]. Thesebands were larger in the insertion mutants by approximatelythe size of the inserted segment. The lower band in each caseresults from cleavage within new sequences (illustrated bythe vertical bars which show the position of the insertedsequences within the cleavage pattern).To determine the pattern of DNase I cleavage within

chromatin formed by insertion mutants too large to bepackaged into virions, plasmids containing these insertionmutations were introduced into COS-1 cells by transfection.Full-length linear plasmid DNA, recovered after DNase Idigestion of nuclei from the transfected cells, was redigestedwith EcoRV, and the sizes of the resulting DNA fragmentswere determined by electrophoresis (Fig. 6C). These resultsconfirm the trend seen with virus infections analyzed witheither DNase I or endogenous endonuclease. The two majorbands seen after EcoRV digestion (ca. 1.7 and 1.8 kb forpJII) were increased in size in the insertion mutants byapproximately the size of the inserted segments. From theseresults we infer that the determinants for DNase I cleavagemust lie on the late side of nucleotide 37 (the site ofinsertion). Results with endogenous endonuclease andDNase I are summarized in Fig. 7.

In summary, DNase I and endogenous endonucleasecleaved SV40 chromatin at different positions within thenuclease-sensitive region; however, inserted DNA segmentsbetween nucleotides 37 and 38 displaced the pattern ofcleavage for each enzyme away from the ori site in thedirection of late transcription. Sequences on the late side ofnucleotide 37 provide genetic determinants for the adjacentchromatin structure which lies on the early side of thatposition.

Cleavage of mutant chromatin by BglI. The BgII restrictionsite lies near the origin, and BglI cuts once in each of the twoorigin regions in the mutants. Sucrose gradient-purifiedchromatin was digested briefly with BglI, and moleculeswhich had been cut only once (form III DNA) were isolated.The relative frequency of cleavage at the two BglI sites wasdetermined by redigestion with EcoRV (Fig. 8). Moleculescleaved in region I gave a band at 4.5 to 4.6 kb, whereasthose cleaved in region II gave a band at 3.2 to 3.6 kb.Accessibility to BglI in region II was reduced in thosemutants containing 56- and 90-bp insertions. This result isconsistent with our observations that the altered chromatinstructure is displaced away from the BglI restriction site inthese mutants.

DISCUSSIONWe have presented evidence showing that plasmids con-

taining large insertion mutations between the 21-bp repeatedsequences and the ori site have reduced ability to replicate inCOS-1 cells. The magnitude of this reduction is dependenton the size of the insert but is independent of its nucleotidesequence. Maximum replication efficiency of plasmid DNAwas not restored when plasmid-transfected cells were super-infected with wild-type SV40.Bergsma et al. (2) have reported that the 21-bp repeated

sequences provide an auxiliary replication function whichenhances replication efficiency in a dose-dependent manner.Those authors also reported that deletion of sequences onthe late side of the 21-bp repeats had no effect on replication.Mapping of a replication function to the 21-bp repeat regionwould explain the recent observation (19) that mutants

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FIG. 5. High-resolution mapping of endogenous endonucleasecleavage sites in mutant chromatin. Form III viral DNA. generatedby endogenous endonuclease cleavage of chromatin, was digestedwith EcoRI as described in the legend to Fig. 4, fractionated througha 2% agarose gel, blotted, and hybridized with probe B (Fig. 1). Onlythe lower part of the gel containing region 11 is shown. M. DNAmarkers: EcoRI to Bcll. EcoRI to BglI. and EcoRI to BawnHIfragments from in(Or)-1411. Dark vertical bars placed to the right ofsome lanes indicate the positions of the inserted DNA segments inthe cleavage patterns.

VOL. 4, 1984


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FIG. 6. DNase I cleavage of viral and plasmid chromatin. (A)Nuclei from mutant-infected BSC-1 cells were isolated 42 h afterinfection and digested with DNase I at 25 U/ml (37°C for 3 min).Form III viral DNA of each mutant was isolated by preparativeagarose gel electrophoresis, redigested with EcoRI, fractionatedthrough a 2% agarose gel, blotted, and hybridized with a mixture ofprobes B and C (Fig. 1). M refers to the DNA markers described inthe legend to Fig. 5. I and II refer to nuclease-sensitive regions in theviral chromatin (Fig. 1). Dark vertical bars indicate the positions ofthe inserted DNA segments in the cleavage patterns. (B) Compari-son of the patterns of cleavage in region II of in(Or)-1411 chromatinby DNase I and endogenous endonuclease. Portions of form IIIin(Or)-1411 DNA isolated from DNase I (D) or endogenous endonu-clease (EE)-digested chromatin (prepared 42 h after infection) wereredigested with EcoRI, fractionated in a 2% agarose gel, blotted, andhybridized with probe B as in Fig. 5. M refers to the DNA markersdescribed in the legend to Fig. 5. (C) COS-1 cells were transfectedwith plasmid DNA for 3 h in the presence of 200 ,ug of DEAE-dextran per ml, washed with MEM, and further incubated withmedium containing 5% FCS and 100 ,uM chloroquine. This mediumwas removed after 2 h of incubation at 37°C and replaced with MEMcontaining 5% FCS. Nuclei were isolated 42 h after the start oftransfection and digested with DNase 1 (25 U/ml) at 37°C for 3 min.Form III plasmid DNA was isolated by preparative agarose gelelectrophoresis and fractionated by electrophoresis in 1.6% agarosein the absence of further treatment (U) or after digestion withEcoRV (RV). The DNA was blotted to nitrocellulose and hybridizedwith probe A (Fig. 1). M refers to DNA markers prepared from pJIlDNA. Vertical bars indicate the positions of the inserted DNAsegments in the cleavage patterns.

lacking this region have a growth deficiency that cannot becomplemented by temperature-sensitive helper viruses.

Several possible mechanisms for replication enhancementcan be suggested. (i) Proteins bound in the 21-bp repeatregion may interact directly with proteins bound at the orisite. This possibility is not easy to reconcile with ourdemonstration that replication is enhanced to an intermedi-ate extent even when these two regions are separated by upto 90 bp of inserted sequences. (ii) The 21-bp repeat regionmay provide a favored binding site for the replicationmachinery. The ability of such an entry site to enhancereplication might decrease as the site is moved away fromori. (iii) Successful initiation of transcription may have astimulatory effect on replication from the ori site. It has beenshown in several laboratories (1, 9, 11, 12, 19, 26, 31) thatsequences in the 21-bp repeat region play a major role indetermining the amount of early transcription. Moreau et al.(30) demonstrated that insertion ofDNA sequences betweenthe 21-bp repeats and the start of early transcription (be-tween nucleotides 34 and 35 in the numbering system used inthis paper) resulted in reduced T-antigen expression. (iv)

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Some of the initiation sites for DNA synthesis may notfunction when the 21-bp repeated region is separated fromthe minimal ori site. Hay and DePamphilis (20) have shownthat, in wild-type SV40, sites for initiation of DNA synthesislie within the 21-bp repeated sequences as well as within ori.Based on our investigation of the nuclease-sensitive chro-

matin structure of these mutants, we favor a fifth possibili-ty-that the 21-bp repeat region contains determinantswhich create an altered chromatin structure over ori and thatdisplacement of this chromatin structure reduces access forT-antigen and other replication proteins to their bindingsites. We have shown that insertion mutations between the21-bp repeated sequences and the ori site displace thenuclease-sensitive chromatin structure away from the origin.Accessibility of the relocated nuclease-sensitive region toendogenous endonuclease was not decreased in these mu-tants; however, accessibility of the ori site to DNase I,endogenous endonuclease, or BglI was reduced. We con-clude that determinants for the nuclease-sensitive region lieon the late side of nucleotide 37 and that the nuclease-sensitive chromatin structure which occurs on the early side

REPLICATION AND CHROMATIN STRUCTURE OF SV40 MUTANTS

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FIG. 7. Summary of DNase I and endogenous endonuclease cleavage sites in parental and insertion mutant chromatin. Regions ofcleavage by DNase I (hatched boxes) or by endogenous endonuclease (stippled boxes) are shown for pJIl. region 11 of in(Or)-1411 and ih4 (A).pJI1-in42 and in42 (B). in56 (C). in9O (D). pJI1-in132 (E). and pJ11-in260 (F). The edges of the regions of cleavage are shown with a precision ofca. +8 bp. Vertical heights of the boxes shown for a given enzyme represent relative frequencies of cleavage. Thick. dark lines designateinserted DNA segments. The darkened triangles indicate joints of the inserted origin region (37). The 21-bp repeated sequences and the 72-bpsegment which is repeated in wild-type SV40 but not in either segment of in(Or)-1411. pJll. or the insertion mutants are shown by numbersand open boxes. A/T refers to the Goldberg-Hogness sequence. ori refers to the minimal replication origin of SV40.

".\ef (0 0

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kb

4.5-4.6- _I*_ _

3.2-3.3 - doom

FMF=

1.4 1.4 1.7 2.0 2.0

FIG. 8. Frequencies of BgIl cleavage in region I versus regionin chromatin of in(Or)-1411 and viral insertion mutants. Viralchromatin was extracted from 32P-labeled infected cells, isolated bysucrose density gradient centrifugation, and incubated with Bgllunder conditions which gave partial digestion. Form III viral DNAwas purified by preparative agarose gel electrophoresis. AfterEcoRV digestion, the resulting DNA fragments were fractionated byelectrophoresis through a 1.4% agarose gel. I and lI refer to digestproducts resulting from EcoRV cleavage of DNA from chromatinwhich was cleaved by BgIl in regions I and II, respectively. EcoRVcleavage yields four fragments of different sizes; only the two largestfragments are shown for each mutant. As described in the legend toFig. 4, the ratio between the number of molecules cleaved at each ofthe two sites was calculated by using the total radioactivity in eachof the two bands (determined from a densitometer scan of a lighterexposure of the autoradiogram) and adjusting for the difference inmolecular weight. The results are expressed as the ratio between thetwo cleavage frequencies, F,/F,, (47).

of that position is induced by those determinants. In thewild-type SV40 genome, a nuclease-sensitive structure oc-curs over ori; in the larger insertion mutants. this feature isinduced over the inserted segments (irrespective of nucleo-tide sequence), and the oni site lies outside of the alteredchromatin structure.

Previous studies have located determinants for the nucle-ase-sensitive chromatin structure to at least two regions (13,14, 25, 47). One component lies in or near the 72-bp repeatedsequences (13, 25), and a second maps in the 21-bp repeats(25; R. D. Gerard et al., manuscript in preparation). Deletionof sequences in either of these regions reduces the frequencyof cleavage by BglI (Gerard et al., in preparation) and byinference reduces access to the on-i site.By using electron microscopy, a nucleosome-free region

has been observed near the origin of replication in SV40chromatin isolated from infected cells (24, 35). Reconstitu-tion experiments suggest that primary DNA sequence playsa dominant role in determining the location of nucleosomes(22, 45). From our data we predict that the determinants forthe nucleosome-free region lie on the late side of nucleotide37.

Functioning promoters usually occur within nuclease-sensitive sites in chromatin (5, 10, 46). The 72- and 21-bprepeated sequences have well-established, but as yet unde-fined, roles in viral promoter function (1, 4, 9, 11, 12, 17, 19,26, 30, 31). One function provided by these sequences maybe to create an altered chromatin structure around theGoldberg-Hogness sequence and transcription start sites.Using insertion mutations which separate genetic ele-

ments, we have established a correlation between the abilityof a DNA molecule to replicate efficiently and the proximityof the replication origin to an altered chromatin structure.Eventual elucidation of the mechanisms of replication andtranscription in SV40 will depend on an understanding of therole of chromatin structure in these processes.

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VOL. 4. 1984 15()5

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ACKNOWLEDGMENTSWe thank C. Walter for valuable technical assistance. Y. Gluzman

and N. Muzyczka for COS-1 cells. and T. Shenk for mutant in(Or)-1411.

This research was supported by Public Health Service grant Al-12852 from the National Institute of Allergy and Infectious Diseases.W.A.S. is the recipient of Research Career Development awardAM-00549 from the National Institute of Arthritis, Diabetes, Diges-tive and Kidney Diseases.

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MOL. CELL. BIOL.


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VOL. 4, 1984

dna replication and chromatin structure of simian virus 40

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