Structural changes in simian virus 40 chromatin as probed by restriction endonucleases.

JOURNAL OF VIROLOGY, Sept.1979,p.718-732 Vol.31, No. 3 0022-538X/79/09-0718/15$02.00/0

Structural

Changes

in

Simian Virus

40

Chromatin

as

Probed

by Restriction Endonucleases

GEORGE L. LIGGINS,t MICHELE ENGLISH,4 ANDDAVTD A. GOLDSTEIN§*

Receivedforpublication13June1979

The structure of simian virus 40 (SV40) chromatin wasprobed by treatment withsingle-andmultiple-sitebacterial restriction endonucleases. Approximately the same fraction of the chromatin DNAwascleavedbyeach of threedifferent single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard tospecific nucleotide sequences within the population of chromatin molecules. However, theextentofdigestionwasfoundtobestrongly influenced by saltconcentration. At 100 mMNaCl-5mM

MgCl2,

only

about 20% of the simian virus 40 (SV40) DNA I in chromatinwasconvertedtolinearSV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01

M),

an additional20 to30% of the DNA wascleaved.Theseresults suggest thatat100 mMNaClonly theDNA betweennucleosomeswasaccessibletotherestriction enzymes,whereas atthelower saltconcentrations, DNA within thenucleosome regions becameavailable for cleavage. Surprisingly, whenSV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was

digested to limit digest fragments, whereas only a small fraction (9 to 15%) receivedtwo or more cuts. Instead, theprincipaldigestfragmentwasfull-length linear SV40DNA III. The failureto generatelimit digestfragmentswas not a consequence ofreduced enzymeactivity in thereaction mixtures orofhistone exchange. When the position of the principal cleavage site was mapped after HpaIdigestion,it wasfound that this sitewas notunique.

Nevertheless,

allsites were not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest thatthe initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We proposethatthismaybeaccomplished byeither"winding" ofthe internucleosomal DNA into the body of the nucleosome, or as suggested by others,bysuccessiveright-hand rotation of nucleosomes.

Polyomaandsimian virus 40(SV40) DNA can beisolatedfrominfected cellsandpurified virus in association withcellularhistones (13, 16, 17, 19, 26, 30, 32-34). In the electron microscope, these nucleoprotein complexes are seen as cir-cularmolecules havingnucleosomes character-istic of cellular chromatin (11, 21, 58). These molecules have been designated as SV40 mini-chromosomes or, morerecently, SV40 chroma-tin.

To understand the arrangement of nucleo-somes in relation to the DNA base sequences, SV40chromatin has been used as a substrate for

tPresent address:Hyland Division, Travenol Laboratories, Inc.,Round Lake, IL 60073.

tPresent address: Department of Biology, University of California,SanDiego,CA 92093.

§Presentaddress:DepartmentofMicrobiology, The Med-icalCollegeofPennsylvania,Philadelphia, PA 19129.

bacterial restriction endonucleases. The as-sumption inherent in this approach has been that DNA cleavage sites which are covered or are strongly associated with histones will be resistant to the endonucleases. Several groups have studied SV40 chromatin, but the conclu-sionsarrived atby the various workers have not always been in agreement. Polisky and Mc-Carthy(42) foundthatonly20% of the DNA in SV40 chromatin was protected from nuclease digestion and that the protected regions were randomly distributed. However, the SV40 nu-cleoproteincomplexwhichtheystudied was pre-pared by alkaline degradation of virions, and thistreatment hasbeen shown to cause partial removal ofnucleosomes anddegradationof his-tones (3, 7, 34, 56). In contrast, Cremisi et al. (11),who studiedthe more"native"intracellular

718

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form of SV40chromatin, found that 73 to 85% of the DNAwas resistant to digestion by EcoRI. Theysuggested that the cleavage was occurring only in the internucleosomal DNA regions. Nevertheless, Persico-Dilauro et al. (41) found that 50% of DNA within intracellular SV40 chro-matin was cleaved by both staphylococcal nu-cleaseand EcoRI nuclease. From their analysis ofthe HindlIl digest patterns, they proposed that thenucleosomes are distributed randomly in the population of chromatin, but are not randomly arranged within the SV40chromatin molecule. Ponder and Crawford (44) on the other hand, proposed that the arrangement of nucleosomes is not completely random within the wholepopulation,but that eachnucleosome canoccupy oneofalimited numberofpositions. In this paper, we describe the results of our experiments using intracellular SV40chromatin purified by hydroxyapatite chromatography(34) as asubstrate for bacterial restrictionenzymes. TheSV40chromatin purified by this procedure hasa ratio ofproteinto DNA greaterthan one (22,34) and hasaboutthesamecomplementof histones asis foundinwholevirionsand in

Hi-depleted cellular chromatin. In our study, we found that thesusceptibility of SV40 chromatin DNA torestriction endonucleaseswasstrongly affected by the exact experimental

conditions,

particularly

thesaltconcentration, andthis

find-ing may provide a partial explanation for the discrepancies in some of the previous studies. Anadditional

significant

findingwasthat relax-ation of the

topological

constraintsinSV40 chro-matinDNA Ialteredthat structureof theSV40 chromatin such that the chromatin DNA be-camehighly resistanttothe action ofthe restric-tionenzymes.

MATERIALS AND METHODS

Cells and viruses. Plaque-purified SV40 virus

(strainRH911)waspropagatedinthe TC7subline of

CV-1aspreviouslydescribed(22, 34).

PurificationofSV40 chromatin. Theprocedure

usedtopurifySV40 chromatin from Tritonlysatesof

infected cellswasessentially thatdescribed in detail

inapreviouspublication(34), with thefollowing

mod-ifications.SV40 chromatinwaseluted from

hydroxy-apatite columns with 0.28 M phosphate buffer, pH

6.8-0.1% Triton X-100afterwashingextensivelywith

0.22 Mphosphate buffer, pH 6.8-0.1%Triton X-100.

Columns and buffers weremaintained at 8 to 100C.

The SV40 chromatinwhich elutedfrom thecolumns

wasconcentrated andthensedimentedat36,000 rpm

in a5 to20%(wt/wt) sucrosegradientcontaining0.1

MNaCl-0.025%Triton X-100-0.001 MEDTA-0.01M

Tris-hydrochloride(pH7.9) for2.5hat40C.Onlythe

leading two-thirdsportion oftheSV40chromatinpeak

was pooled. These fractions were concentrated by

vacuum dialysis and then dialyzed into the appropriate reaction buffer.

The average buoyant density in CsCl gradients of

theSV40chromatin purified by the above procedure

and fixed with glutaraldehyde was 1.44 g/cm3. The

histone complement of thisform of SV40 chromatin is

similar to that found in virions in that it contains

aboutequimolar amounts of H2A, H2B, H3, and H4

histones but is devoid ofHihistone (34). Nonhistone

proteinscould not be detected in the SV40 chromatin

evenafter the proteins were labeled in vitro with125I

to a high specific activity (G. L. Liggins and D. A.

Goldstein,unpublisheddata).

Radioactivelabelingof SV40 chromatin DNA.

Forthe routine preparation of SV40 chromatin DNA,

the DNAwaslabeled at low specific activity to assist

intheidentification and quantitation of the chromatin

duringpurification. ['4C]thymidine, (0.05 to 0.1 ,iCi/

ml; New England Nuclear Corp.) was added at 24 h

after infection and not removed until thecells were

lysed at72to90h afterinfection.

Forlabeling of SV40 chromatin DNA to high

spe-cificactivity with32P,[32P]orthophosphate(25-50,Cil

ml; New England Nuclear)wasadded in

phosphate-free growth medium (Eagle minimum essential

me-dium containing 2.5%dialyzed horseserum) at24h,

and theradioactivitywasremovedjust before Triton

lysisofcellsat 72hafter infection. Naked32P-labeled

SV40 DNA was prepared from purified SV40

chro-matin by removal ofprotein. For this purpose,32p

labeledSV40 chromatinwasincubatedat40°C in 0.1%

sodium dodecylsulfate (SDS) with50ygof pronase

perml for1h.Thereafter, theDNA wassedimented

for2hat45,000 rpm ina 5 to20%sucrosegradientat

roomtemperatureinanSW50.1 rotor(22).39P-labeled

SV40 DNApreparedin this way hadaspecificactivity

of 1x 105to2x 105cpm/jLgof DNA.

Bacterial restriction endonucleases. Restriction

endonucleases R-EcoRI, R-HpaI, R.HpaII, R.

BamHI and R*HindIII were purchased from

Be-thesda Research Laboratories, Bethesda, Md. R.

HindII + III was obtained from Mary Gutai and

Michael Chen. The locations of thecleavagesites for

the variousenzymes usedinthisstudyare depicted

ontheSV40 DNA mapinFig.1.Enzymeswerestored

at-20°C.Unless otherwisespecifiedinthetextorin the legendsto the figures, SV40DNA or chromatin

wasincubated with the enzymesat37°Cinthe

"stan-dard" reactionmixturecontaining10mM

Tris-hydro-chloride (pH7.5), 0.025% TritonX-100,100mMNaCl,

5mMMgCl2and2mM

/3-mercaptoethanol.

All buffers

were autoclaved before use, and all reaction were

carriedoutinsterile vials and tubes. Reactionswere

terminatedbySDS(finalconcentration, 0.1%)-EDTA

(0.05M) and heatedat56°Cfor5min.

Polyacrylamide and agarose gel

electropho-resis.For theseparationofthe DNAfragments

gen-eratedbydigestionofSV40 chromatinwith restriction

enzymes,theproteinwasfirstremovedbyheatingthe

chromatinat56°Cfor10min in0.35%SDS,and the

DNAwaselectrophoresedonverticalagaroseor

poly-acrylamideslabgels.Sampleswereadjustedtoa

con-centrationof 5%glyceroland0.02%bromophenolblue

in avolume of50

1IL

andloadedontoslots of slabgels.

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J. VIROL.

720 LIGGINS, ENGLISH, AND GOLDSTEIN

EcoRI I

Hl

LM

C*iD E KF

Hind M BED C A B

Hin d +-A

HindM

HpaI

A D C E FA

A C B A

Hpao

BamHI -

--MapUnits 0 .2 .3 4 5 .6 .7 .8 .9 1.0

FIG. 1. Restriction enzymecleavage mapof SV40 DNA. The cleavage mappositions assigned to the various enzymes are shown relative to the single cleavage sitefor EcoRI. There are4 cleavage sites forHpaI (12, 50, 66), 6sitesfor HindIII (66), 13 sites

forHindII+III(12, 66), 1siteforHpaII (50, 66), and

1 sitefor BamHI (R. Roberts,personal communica-tion).

Theagarosegelswere 1.4%agarosein 40 mMTris, 5 mM sodiumacetate,and 1mMEDTA(pH 7.8). The

polyacrylamidegelelectrophoresisbufferwas40mM

Tris-20 mM sodium acetate-2 mM sodium EDTA (pH 7.8). Thedimensions ofthegels, the time of the runs, and other conditions varied for each enzyme

reaction. These detailsaregiven in thetextorinthe

legendstothefigures. In allexperiments, bufferswere

circulated between thereservoirs during theruns.

Forradioautographic analysis of 32P-labeled DNA

digest fragments, slab gels were removed from the

glass plates at theconclusion of the electrophoresis, thewetgelswerewrapped tightly in Saran Wrapto preventdehydration, and thegelswereplaced in

con-tactwithmedical X-ray film. Filmsweredeveloped8 hto 5daysafterexposure. Forquantitative analysis, theexposed filmswerescanned witharecording

den-sitometer. For analysis of DNA digests not labeled with 32p, gelswereplaced inan aqueous solutionof

ethidium bromide (2 ytg/ml) at 4°C for at least 4 h. Thewetgelswereilluminated with UV light,and the fluorescent DNA bands were photographed with a

Polaroidcamera.

Forremoval of DNA fragments fromagarose gels,

gel slicesweredissolvedin2volumes of5Msodium perchlorate at 65°C, and the dissolved agarose was

passed through small columns of hydroxyapatite at

65°C. Columnswerewashed with 10volumes of0.01 M sodium phosphate buffer (pH 6.9),and the DNA

waseluted with0.4 M sodiumphosphate buffer (pH

6.9). DNA wasdialyzed against 0.1 M NaCl-0.01 M Tris (pH 7.9)-0.001 M EDTA, and the DNA was

concentrated byethanol precipitation.

RESULTS

Theoretical considerations. The generally

accepted notion that histones do not bind to DNAwithbasesequencespecificityhasa

theo-retical basis in thefact that the histones as an

evolutionary class of proteins are very highly

conserved. Nonetheless, this does not rule out

thepossibilitythat in vivo there is

"phasing"

of nucleosomeswith respectto a

specific

DNA base sequence as, for

example,

the sequence at the origin of

replication.

If nucleosomes doindeed associatewith the DNAat a

region

suchasthe origin qf

replication

(20, 49), each

subsequent

nucleosomecouldbe "in

phase"

with the

corre-sponding nucleosome in all other SV40 chro-matinmolecules. The

assumptions

inthis argu-ment are that (i) nucleosomes do not move

laterally

along the DNA backbone in vivo and

(ii)thefirstnucleosome addedtothe DNAafter initiation ofreplication establishes the natural spacing of nucleosomes inherent in chromatin. In a casewhere thenucleosomeswould be in phase, digestion by a restriction endonuclease withasingle cleavagesiteshould resultin either cleavage ofallthe molecules (e.g., site not pro-tected byanucleosome) or

complete

resistance (site

protected

byanucleosome). If the nucleo-somesare notinphasewithinsucha

population

but randomly arranged with respecttoa given base sequence, thenan intermediate degree of digestion would be

predicted.

In this case, the resistant fraction would reflect the degree to

which the histones protect the DNA. Neverthe-less, partial digestion might result from other causessuchasreassortmentduring isolation or

digestionorboth,orduringother

perturbations

which would allow access tosites in some, but

not all molecules. Of course, the above predic-tionsare

justified provided

nounforeseen inter-actions take place between the endonuclease andthe histones during treatmentof the SV40 chromatin. However, if the same fraction of chromatin molecules was resistant to cleavage after digestion by other single-site restriction enzymes,thenastrongargumentcouldbe made foranonspecific arrangementof histones. Fur-thermore, chromatin with such a distribution of histonesshouldyield equimolaramountsof each limit digest DNA fragment after cleavage by multiple-site restriction enzymes. It should be pointed out that others (11, 41-44) have pre-sentedsimilararguments inprevious studies on thearrangement of histonesinSV40 and

poly-omachromatin.

Digestion of naked SV40 DNA and SV40 chromatin with single-site restriction en-donucleases.SV40 chromatin and naked SV40 DNAwereincubatedseparately with

EcoRI

and treated with SDS to stop the reaction and to

remove the protein from the DNA, and the

reaction products wereanalyzed by electropho-resis on agarose slab gels (Fig. 2). Bands are observed at threepositions; the top, middle, and lower bandscorrespond to nicked-circular SV40 DNA (DNA II), linear DNA (DNA III), and

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supercoiled DNA (DNA I), respectively. Treatmentof naked control supercoiledDNA

wascharacterized by the conversion of DNA I

tolinear DNA III molecules bywayofa

short-lived,nicked-circularDNAIIintermediate (Fig.

2a).The conversionsequencehasbeennotedby

others(37, 47). Theintermediate doesnotresult from endonuclease contamination, since

pro-longed incubation with EcoRI didnotresult in furtherdegradation of thelinearform and alkali

denaturation of the linear molecule failed to

reveal significant amounts of single strands

shorterthanunit-length SV40 DNA.

Incontrast tonakedSV40 DNAI,whichwas

entirelyconvertedtoDNA III, SV40 DNA I in chromatin was partially resistant to digestion

evenafterprolonged incubation with theenzyme

DNA 11

DNA III

DNA I

DNA 11

DNA III

DNA I

Controls1 3 6 9 12 15 30 45 60

MINUTES AT 37`C

FIG. 2. Kinetics of digestion ofSV40 DNA and SV40 chromatin by EcoRIat 100 mMNaCl. SV40 DNA (5.5pg) andSV40chromatin (8.3pg) were in-cubatedseparatelyinavolumeof120,ul ofthe "stan-dard" reaction buffer (see text) to which 2.5 Uof EcoRIwasadded. At the times indicated, 10,Il of reaction mixture wasremoved and diluted

immedi-atelyinto30,l ofasolutioncontaining10mM

Tris-hydrochloride (pH 7.5),50 mMEDTA,and 0.1% SDS

tostopthereaction,and thesampleswereanalyzed

byagarosegelelectrophoresis. Thedigestion

prod-ucts werelocatedby stainingthegelwith ethidium bromideatafinalconcentrationof2pg/ml. (a)SV40

DNA; (b) SV40 chromatin. The controls represent

portions ofSV40DNA andSV40chromatinremoved

fromthe reactionmixturebeforeadditionofthe en-zymeandincubatedat0°C (slot 1)orat37°C(slot 2)

for60min.

(Fig. 2b). A large fraction remained as DNA II andwas notconverted to DNA III. To demon-stratethat the partialdigestionpatternobserved with DNA from chromatin was not due to lim-itingenzyme orinteraction of the histones with enzyme, additional reactions were carried out in the presence of a large excess of EcoRI. Even underthesereaction conditions, only 23% of the DNA in chromatin was converted to DNA III (Table 1),whereas control naked SV40DNA I wasentirely converted to DNA III within 1min (data not shown). In addition, the amount of DNA IIinitiallypresent inSV40chromatin did notdecrease,but

actually

increased slightly (Ta-ble 1).

In view of the finding that treatment with EcoRI resultedin only partial cleavage ofthe SV40 chromatin, the tentative conclusion would be thatnucleosomesare not inphase within the population. However,theexperimentsdescribed abovegive little informationas to the distribu-tion or coverage by nucleosomes within any givenSV40 chromatinmolecule, since as pointed out above, partial cleavage could be a conse-quenceofanumber of factors.

To further investigate this distribution, SV40 chromatin was digested with twoother restric-tionenzymes, BamHIandHpaII, each of which cleaves SV40chromatin at adifferentsingle site. These sitesarelocatedatfractional-lengthmap positions0.14(61) and0.735(50) for BamHI and HpaJJ,

respectively,

relativetothesingle EcoRI site, whichisarbitrarily assignedthe zero posi-tion(12).

Separate kinetic experiments were done on thesamepreparation of

32P-labeled

SV40

chro-TABLE 1. EcoRItreatmentofSV40 chromatin in

100mMNaCla

Distributionof DNA(%)b Tine(min)

DNA I DNA II DNA III

0 91 9 0

1 80 16 4

3 71 14 15

6 68 13 19

15 66 13 21

60 62 15 23

aFive units of theEcoRIwerereacted with 380ng

of3P-labeled SV40chromatin. The reactionwasina

volume of60

Id

ofthestandard buffer (seetext).At

the timesindicated, 10

pl

wasremoved fromthe

re-action mixture, and the samples were analyzed by

agarose gel electrophoresis. Under these conditions,

naked SV40 DNA I usedas a control was

quantita-tively convertedtoSV40DNA III within 1 min.

b Valueswerecalculatedfromdensitometertracings

ofexposed X-rayfilmsplacedincontactwithagarose

slabgels.

w

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722 LIGGINS, ENGLISH, AND GOLDSTEIN

matin by using each of the three enzymes.Before digestion, theSV40chromatinpreparation

con-tained90% DNA I and 10% DNA II. The results ofonesuchexperimentaresummarized in Table 2.Only 19 to 22% of the DNA inSV40chromatin was converted to DNA III by each of the

en-zymesunder the conditions of this reaction (100 mM NaCl, 5 mM MgCl2, 0.025% Triton X-100, 10mMTris, pH 7.5,at37°C).The salientfeature of thisexperiment is that all three enzymes gave approximately the same degree of double-stranded cleavage, even though the respective sensitive sites are located at considerable dis-tances apart on the genome. Therefore, these results indicate that there isnopreferential dis-tribution ofnucleosomes within any oneregion of the genome. Instead, it appears that the nu-cleosomes are arranged along the DNA inde-pendentof nucleotidesequence.

Thevalue of19to22%for thefraction suscep-tible to double-stranded cleavage agrees well with the findings of Cremisi et al. (11), who digested SV40 chromatin with EcoRI and ex-amined the distribution of linear and circular molecules in the electron microscope. These workers interpreted thecleavage as representing digestion in the naked DNA "bridges" or "spacers"extending between nucleosomes, since

asimilar sensitive region is found upon digestion ofcellular chromatin (1, 10, 39, 52, 55, 57).

Effect of salt concentration on endonu-clease cleavage of SV40 chromatin. In an

[image:5.507.263.453.202.463.2]

electron microscopic study, Griffith (21) found that the SV40 chromatin went from a highly condensed circular structure with a contour length of about 210 nm to an extendedbeaded form with a contour length of about 514 nm when the salt concentration was lowered from

TABLE 2. Comparison of EcoRI, HpaII,and BamHI treatmentofSV40chromatinin100mM

NaCla

Restriction endo- Distribution of DNA(%) nuclease DNA I DNA II DNA III

EcoRI 65 16 19

HpaII 70 14 16

BamHI 66 12 22

a:32P-labeledSV40 chromatin was incubated in

sep-aratereactionmixtures with either 4 U of EcoRI, 2 U ofHpaII, or2U of BamHI in the standard reaction buffer. As acontrol,32P-labelednakedSV40 DNA was incubated at the same ratio of DNA to enzyme. Under these conditions, the control DNA was converted

quantitativelytoDNA IIIwithin 3 min.

bValueswerecalculated from densitometer tracings

of the 20-min reaction products, at which time the reactions were 90%complete.

J. VIROL.

0.15 to 0.015 M NaCl. On the basis of this

observation, it might be predicted that such a

configurational change would cause the SV40 chromatintobemoresusceptibleto endonucle-asecleavage.

Figure 3 shows the kinetics of cleavage of

SV40 chromatin upon digestion with EcoRI

(Fig. 3a) orBamHI (Fig. 3b) in the presence of 50mMNaCl-5 mM MgCl2. Naked SV40 DNA

I is

completely

converted to DNA III in 1 min

(as indicated by the arrow in Fig. 3), whereas the reaction of enzyme with chromatin is not

100-

I0

'

_(a)

75-enX

w U

-J 50

-0

0

Oj

25-5

z

0

a IOO-

(b)

0

7575

z w

25

-0

1 2 3 5 10 15 20

[image:5.507.59.250.487.560.2]

MINUTES AT 37°C

FIG. 3. Kinetics of SV40 chromatin digestion by

EcoRI and BamHIat50mMNaCl.32P-labeledSV40

DNA and32P-labeled SV40 chromatin, each

contain-ing 55,000 cpm (1.52 x 105 cpm/lg of DNA), were

mixedseparately with either: (a)8UofEcoRIor(b)

4 Uof BamHI in reaction mixtures containing 50

mMNaCl,10mMTris-hydrochloride (pH 7.5),5mM

MgCl2, 0.025% TritonX-100, and2mM

/3-mercapto-ethanol. Reaction mixtures were incubated at 37°C and,atthetimesindicated, sampleswerewithdrawn and thereactionswereterminated.SV40 naked DNA I(arrow) wascompletelyconverted to DNA III within 1minunder these conditions.Sampleswereanalyzed

byelectrophoresisin 1.4% agarosegels, and the

diges-tionproductswere revealedbyexposure ofthe slab

gels toX-ray films. The percentages of the various

forms of SV40DNA were calculatedfromthe areas

under thepeaksobtainedfromdensitometric tracings

ofthefilms. Curvesaredrawn in thisfigureonly for

DNAfromSV40 chromatin. Symbols:U, DNA I; A, DNAII;0,DNAIII.

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complete untilsometime between5and 10min. It is clear thatthere is asignificant increase in the susceptibility of SV40chromatinupon low-ering the saltconcentration. Whereas 19 to 22% of themoleculeswereconvertedtoDNA III at 100 mM NaCl (Table 2), a decrease in concen-tration to 50 mM increasedyieldsof DNA III to 47 to 50% (Fig. 3). There was no significant increase in yields of DNA III when the NaCl concentration was lowered even further to 10 mM (datanotpresented).

Examination of the kinetic data in Fig. 3 re-veals that digestion is atwo-phase process. Dur-ing the firstminute, 25% of the chromatin DNA is digested, whereas the remaining 25% is cleaved during thesubsequent 10-min period.The first fraction,presumablycorresponding to the inter-nucleosomal fraction,appears tobe quite acces-sibletothe enzyme,sincecontrol nakedDNA is

also

completely digested

during the first minute.

This isconsistent with the electronmicroscopic data ofGriffith (21),which showed naked inter-nucleosomal DNA regions exposedatthelower salt concentrations. The additional25% which is cleaveduponprolonged incubationsuggests that regions presumablynear, orperhaps within the nucleosome, are lessaccessible for cleavage by the enzyme. Importantly, the total susceptible fraction of50% corresponds wellwith the esti-mate of coverage as determined by digestion with nonrestricted endonucleases (8, 9, 32, 41). Resistance ofSV40 chromatin containing DNA II to cleavage by single-site endonu-cleases. In most ofthe experiments described above,uponpurification from cellextracts, chro-matin preparations contained about90% DNA I andabout 10% DNAII. Since the fraction of DNA IImoleculesremainedapproximately con-stant orincreased uponendonucleasetreatment (Fig. 2band 3;Tables1and2),it appearedthat the chromatincontainingDNA II wasresistant todouble-strandedcuts atall of thesalt concen-trationstested.

Todetermine whetherthisresistancewas per-haps a property of DNA II chromatin derived fromanin vivo mechanism (14)or,instead,was a general characteristic ofDNA II chromatin generated byrandomsingle-strandcutsofDNA Ichromatin, thefollowing experimentwasdone. SV40 chromatin labeled with 32Pwasstored for several weeks at

40C,

duringwhich time random single-strand nicks were introduced by 32P de-cay. This chromatin and nakedSV40DNA pre-pared from the chromatin were used as sub-strates in separate reactions with BamHI and EcoRI. Figure4showsresults of anexperiment inwhich about 25% of the chromatinemployed

as asubstratefor theenzymeBamHIcontained

Naked DNA Minichromosomes

DNA E - _ me_ SW

DNA

M

impqnO

- _

am

DNAI _i

_1U

_ _m

0 1 3 5 15 0 1 3 5 15

MINUTES AT370C

FIG. 4. Resistance of SV40 chromatin DNA II to

BamHI.32P-labeled DNA and chromatinwerestored

at4°C for4weeks,during which time about25%of

theDNAwas found to be in the linearDNA III form,

presumably converted to this form by decay of the32p.

SV40 chromatin and DNAwerereactedwith BamHI

under conditions similar to those described in the

legendtoFig. 3, except that theNaCl concentration

was 10mM insteadof50mM.

DNA II. In contrast to naked DNA, in which DNA I and DNA II areboth rapidly converted toDNAIII, the fraction initiallypresent as DNA II remained essentially unchanged. This SV40 chromatin preparationrepresents onlyoneofa number of different SV40 chromatin prepara-tions inwhichthe amountofSV40 DNA IIdid notdecreasesignificantlyupon incubation with thesingle-site restriction enzymes.

Digestion of SV40 DNA and SV40 chro-matin with multiple-site restriction endo-nucleases. The apparent resistance of the nicked-circular form of SV40 chromatinto cleav-age suggested that, upon relaxation of the closed-circular DNA, a conformational change mayhaveoccurred,causing theDNA tobecome resistant to digestion by restriction endonucle-ases.Thus itwould followthat,

regardless

of the number ofpotentialrestriction enzymecleavage sites, theremaining sitesmight be rendered in-accessible afterreceiving thefirstnickor

chop.

To testthis

hypothesis,

32P-labeled SV40 chro-matin and 32P-labeled naked SV40 DNA were reacted separately with the

multiple-site

en-zymesHindll+IIIforvarious timeperiods,and the resultant

fragments

were

separated

on 4% acrylamide slab

gels.

The location of

cleavage

sites in SV40 DNA for

HindII

+ III and the otherenzymesused in this

study

areshown in Fig. 1. The digest patterns areshownin

Fig.

5.

SV40 DNAyielded 11

fragments

within 15min after exposure to the enzymes. Not shown are

twovery smallfragments,30and20base

pairs,

respectively, which run off the

gels

under

con-ditionsrequiredforresolution of the other

frag-ments (66). The naked SV40 DNAused inthis

experiment

wasobtained

by

theremoval of

pro-tein from intracellular SV40

chromatin,

but it

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724 LIGGINS, ENGLISH, AND GOLDSTEIN

~*

--f,/ I :1.."

,

I-

t

l.. .,4'.._.11 11-1-I

.z '.7!..

FIG. 5. HindII+IIIdigestion ofSV40 naked DNA andSV40chromatin.Naked32P-labeled SV40 DNA (1.3 jig)and32P-labeledSV40chromatin (2.5jug)were

incubatedseparatelyat37°C with HindII+III (5 U)

in a volumeof 250 ,ul ofabuffercontaining50mM

NaCl,6mMTris, 6 mMMgCl2, 0.6 mM

,8-mercapto-ethanol, 0.6%glycerol, and 0.01% TritonX-100 (pH 7.5). At the times indicated, 50-jilsamples were

re-movedfrom each tube, the reactionswereterminated,

and the samples were electrophoresed on 4% poly-acrylamide slab gels (40 by0.3cm)for21.5h at100 V,53mA.To locatethe DNA bands, gelswereplaced

incontactwith X-ray filmsfor 72 h.

gave the same digestion pattern as DNA from

purified virus.

In contrast tothe 11 fragments of the naked

SV40 DNA digest pattern, the majority ofthe

DNA in chromatin was resistant to multiple cleavagebyHindII+ III(Fig.5). Even4h after incubation,mostof theSV40DNA in chromatin

had received only one cut and migrated to a

position characteristic of linear full-lengthDNA.

Furthermore, asignificant fractionwasnot

sus-ceptible to cleavage and, instead, failed to

mi-gratein the

gel.

Only

about 10% of the molecules received more than one chop, as indicated

by

the mixture of incomplete digest

products,

whereaslessthan 1%correspondedtoanyof the 11 limit digest

products-in

this case,

only

to

fragment A.Ifallthecleavagesiteswereequally susceptible,onthe average, theSV40 chromatin molecules shouldhavereceivedsixtosevencuts

under these reaction conditions. The fact that most ofthe cleavage sites in SV40 chromatin were protected from

digestion

after the initial cut provided additional evidence that a

rear-rangement or a

configurational change

orboth tookplace suchthat

potentially susceptible

sites became covered

by

histones. To what extent

these

configurational changes

could be influ-enced by the reaction conditions was next

ex-amined.

It is known that rearrangement andexchange of histonescan occuratcertainsalt anddivalent ionconcentrations (8). Thus,oneofthefactors studiedwastheeffect of

magnesium

concentra-tion onthe

susceptibility

ofSV40 chromatin to

HindII + III. Inthe

experiment just

discussed (Fig. 5), the concentration ofmagnesium chlo-ridewas6mM.Shown inFig.6 arethedigestion products of SV40 chromatin DNA at 0, 1, 20, and100mMmagnesiumconcentrations. At very low concentrations ofmagnesium (less than 1

mM), control naked SV40 DNA was only par-tially convertedtolimitdigest products,sothese reactions with SV40 DNA and chromatin are not shown (also, thereactions had to be incu-bated forlong periodsat37°C,conditions which could cause degradation and denaturation of chromatin).

Atall of themagnesiumconcentrationstested, SV40DNA inchromatinwaseither resistantor was converted mostly to linear DNA III, indi-cating that the majority of the molecules had

one cut or less. An interesting feature of this

experimentwasthatSV40 chromatinDNA was digestedto a greater extent at the lower mag-nesium concentration (1mM), whereas the op-positewas truefor the control nakedSV40 DNA (e.g.,Fig. 6, slots3and4 compared with slots 7 and 8). These reactionswerecarriedout at37°C for4.5 h, so it isdifficult to conclude that this effectwassolely the result ofa magnesium-de-pendent configurational change, since it may

have been caused by partial denaturation or

degradation ofproteins. However, it is the op-posite effect that would be predicted for ex-changeorremoval of histones, since these

phe-nomenaappeartoincreasewith increasing

con-centration of divalent cation (8). It should be noted thatat100mMmagnesium concentration, both theSV40DNAand chromatinwere resist-ant, probably due to the aggregation of DNA,

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[image:7.507.70.244.74.428.2]

1

l 2 3 4 5 6 .7 6 9

19

RI3...

_0

to

g4

1_

_I

_ _ _

C D

E

F

G-H

K-FIG. 6. Effect of magnesium concentration onthe

digestionof SV40 DNA and chromatin byHindII+

III. Naked 32P-labeled SV40 DNA or 32P-labeled SV40chromatinwereincubatedat37°C for4.5 hwith

HindII+ III(2 U) inavolumeof72,l ofa buffer

containing80 mMNaCI,8mMTris,

mM/3-mercap-toethanol, 0.02% TritonX-100(pH 7.5), andvarious

concentrationsofMgC42.Reactionswereterninated, and 50-pi samples were electrophoresed on a 4%

polyacrylamideslabgel for19.5hat120V, 48 mA.

Each slot represents a different reaction mixture. Slots1through6,SV40 DNA;slots7through9, SV40

chromatin; slot 10, SV40DNAplus EcoRIcontrol.

Slot 1, 0 MMgCl2, no enzymes; slot2, 0MMgC12, enzymes;slots 3 and7,1mMMgCl2, slots4and8, 20

mMMgCl2;slots 5and9, 100mMMgCI2;slot 10,5

mMMgC12.

chromatin,enzyme,or acombination of these.

Further attempts were made to detect the

removalorexchangeofhistones underthe

stan-dardenzymereaction conditions by sedimenta-tion analysis. 3H-labeled SV40 chromatin was mixed with32P-labeled SV40 naked DNA at a weight ratio of chromatin to DNA of 16 to 1, incubatedfor 1 h in 50 mMNaCl-6mM

MgCl2-1mM

,6-mercaptoethanol-0.01

MTris (pH

7.5)-0.01% Triton X-100, andsedimentedin asucrose gradientcontaining the same buffer. Except for asmallshoulder on the leading edge of the DNA peak, therewasno evidence of a change inthe sedimentationpatternof theDNA or chromatin (Fig.7).Thus, the conclusion from the two afore-mentioned experiments is that protein exchange is not adeterminingfactor inthe acquisition of resistance of SV40 chromatin upon the initial cleavage of SV40chromatin.

Reactivity of restriction enzymes with SV40 chromatin. Another possibleexplanation for the inability to generate limit digest frag-ments by multiple-site restriction enzymes is thatthechromatin or some other component in the reaction mixturemay cause adrastic reduc-tion or change in the enzyme activity so that only sufficient activity remains to generate a

^-- 4

0

x3 "2

0.

=u

4^

0

3,X 2 0

a.

cm

I M

4^-..

0

3 X CL

0~

,.4

0 10 20 30 40

Fraction Number

FIG. 7. Sedimentation ofmixturesof SV40 chro-matin withSV40DNAathighratiosofchromatinto DNA.A2-pgamountofSV40 [3HJchromatin(20,000

cpm/pg)wasmixed with 0.125pgof32P-labeledSV40 DNA (122,000 cpm/pg) in a volume of50 ,ul and incubated as described in the text. Samples were

layered onto 5 to 20% sucrose gradients and sedi-mentedfor2 h at36,000rpm in a SpincoSW50.1 rotorat4°C. Radioactivity representstrichloroacetic acid-insolubleproducts. (a)DNAalone;(b)DNAplus chromatin.Sedimentationwasfrom righttoleft. A

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726 LIGGINS, ENGLISH, AND GOLDSTEIN

single cleavage inmost of the molecules. Con-sequently, SV40 chromatin and naked SV40 DNA were incubated togetherin various

com-binations withHindIII to determinetheextent

of enzyme activityduring the courseof the

re-action. Such an experiment is shown in Fig. 8 which shows the reaction of 3H-labeled SV40 chromatin and SV40 [32P]DNA with HindIII endonuclease. HindIII cleaves SV40 DNA into six fragments (12, 66). Incubation of HindIII with SV40 chromatin for 2 h (slot 2) did not

generatelimitdigestfragments.Instead,the

ma-jor fraction was cleaved only once toyield full-length linear DNA (position of RI marker), whereas the remainder of the DNA either did not migrate into the gel (this fraction was a

mixture of closed-circular and nicked-circular

DNA)or wasconverted intopartial digests (8%).

The addition of fresh enzyme after 2 h and incubation foran additional 2hdid not signifi-cantlychange thedigestpattern (slot3,Fig. 8). Todemonstratethat the enzymewasindeed in

excess and still active after 2 h of incubation,

[32P]DNAaddedtothe SV40 chromatin reaction mixture at this time and incubated for2hmore

wascompletelydigested(slots4and 4B). When SV40 DNA and chromatin were mixed before the additionof enzyme, thecompositeresultwas

the sameasthat obtained when DNA and chro-matin were incubated separately (slots 7 and 7B).These experiments show that the resistance of the SV40 chromatin to limit digestion by HindIIIdoesnotresult fromachangeor

reduc-tion in enzymeactivity.

Initial cleavage sites of HpaI in SV40 DNA chromatin. Since the majority of the SV40 chromatin molecules could only be cut

once by the multiple-site enzymes, it was of interest to ascertain whether or not the initial cleavageoccurred at asingle site or at preferred sites on the SV40 genome. The position of the initial cleavage site was determined by restric-tion enzyme mapping. To simplify the analysis, themultiple-site restrictionendonuclease HpaI

was used to generate linear DNA III, since it chops SV40 DNA at only four positions along the genome (12, 50, 66). Two of these sites are situated very close together, being only 0.004 map units apart (66), and for the purposes of mapping, theycanbetreated as a single site.

A time course ofdigestion of control naked SV40 DNA by HpaI is shown in Fig. 9. Limit digestion generated by cleavage at all sites, i.e.,

0.755(actually,0.753and0.757), 0.375, and 0.175

produces three major fragments: (A) 41% frag-ment; (B) 38%fragment; and (C) 20% fragment (thepercentages denote the fractional length of the DNA). Incomplete digestion caused by

Rl-

A-

B-

C-

[image:9.507.266.458.74.300.2]

D--E

-.

FIG. 8. HindIII digestion of mixtures of SV40

chromatin and nakedSV40 DNA. SV40 chromatin

and32P-labeled nakedSV40DNA wereeither incu-batedseparatelyormixedtogetherinapproximately

equalamounts(2 ,ug) anddigested with HindIII(2 U)

in 100jil ofabuffercontaining 85 mMNaCl,8.5mM

Tris, and 15 mM MgCl2 (pH 7.5). Reactions were

terminatedafter4h. Samples were electrophoresed

on a4%polyacrylamideslabgel (0.15by10cm)at60

Vand70mAfor9h. Slot1, chromatin,noenzyme;

slot 2, chromatin with HindIII; slot 3, chromatin,

HindIII (2 U), 2 h, additional HindIII (2 U) added

and incubated for 2 more h; slot 4, chromatin,

HindIII2h, DNA added and incubatedfor2

addi-tional h; slot 5, DNA, HindIII; slot 6, DNA, no

enzyme; slot 7, chromatin, DNA, no enzyme, 2 h,

HindIII added and incubated for2 additional h.

Slots 1 through 7 show ethidium bromide-stained

gels. Slots 4b through 7brepresentautoradiograms

of slots4 to 7.

cleavage at onlytwo sites yields the following "partial" digest fragments: an 80% fragment (0.175 and 0.375); a 62% fragment (0.755 and 0.375);and a58%fragment (0.755and0.175).In

Fig. 9, the 58% and 62%fragmentsare notclearly resolved.

Figure 10 shows a time course of SV40 chro-matindigestionby HpaI. Unlike the SV40 DNA, which is cleaved to the limit digest fragments within1 h, digestion of SV40 chromatin results inless than 3% of any limit digestfragment, even after prolonged incubation in a vast excess of enzyme for6h.Instead, the major digest product

was SV40 DNA III. Thisis consistent with the previous data obtained after digestion with HindIII and HindIl + III.

J. VIROL.

.91LAINk

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STRUCTURAL CHANGES IN 727

SV40 DNA SV40 Chromatin

DNA 11 am

_M

-DNA III

80% fragment. DNA 58% fragment

A_

B-,.U_4_._ _

DNA 11

El_

DNA III

S*m_ *. ,,

.~~~ ~ ~ ~~~~z

0 p n

:..*.#.

S.

DNA I

A

B

W

C-s

0.25 3 5 15 20 30 60

Minutes

FIG. 9. Kinetics of SV40 DNA digestion by HpaI.

32P-labeled SV40 DNA (2 pg) was incubated with

HpaI(2U) inavolumeof 50 ,il ofabuffer containing

50mMNaCl, 6mMKCI, 15 mM Tris, 5 mMMgCl2,

1 mM,8-mercaptoethanol, and 1%glycerol (pH 7.5)

andincubatedat25°C. At the times indicated, 5,ul

was removed and the reactions were terminated.

Sampleswereelectrophoresedat100V,48 mA on a

1.4% agaroseslab gel (0.3 by30cm)at48 mA, 100V

for7h. DNAfragmentsweredetectedby

autoradiog-raphy. FragmentsA, B, and C represent the limit

digestfragments forHpaIdigestion ofSV40DNA,

whereas 80% and 58% representpartialdigest

frag-mentsproducedby cuttingthe genomeatonly2sites.

There is also a 62%fragmentwhich isnotresolved

fromthe58%fragmentonthisgel.

About 12% of the chromatin DNAappeared

as

partial

digest

fragments-a

62%

fragment

and

a58%

fragment.

These partial

digest fragments

migrateveryclose-totheposition ofDNA Iand are notresolved in these gels. However,no sig-nificant amount ofDNA could be detected at the position of the 80%partial

digest fragment.

Since

cleavage

at 0.755 is required to

produce

the62and 58%

fragments,

itappeared that0.755 was more

probable

as afirst(or

second)

cleavage

site.

Todeterminethesite(s) ofthe firstcleavage, linear DNA III was eluted from the gelsafter digestionofSV40 chromatin by HpaI, andthe DNA IIIwasredigestedwithrestrictionenzymes whichcleaveSV40DNAat

only

onesite. Since thecleavage positionfor eachsingle-siteenzyme isknown,thepositionof the first sitecleavedby HpaI can be calculated from the size of the

C

Control 1 5 15 30 120 360

Minutes at 370C

FIG. 10. HpaI digestion of SV40 chromatin.

32p-labeledSV40 chromatin

(3jg)

wasincubated at 37°C

withHpaI(1.5U) in 175IlIofabuffer containing75

mMNaCl,3mMKCI,0.7mM/3-mercaptoethanol,3

mMMgCl12, 13mM Tris(pH 7.5), 0.02% Triton X-100,

and 0.02mM EDTA. At various times between 1 and

120min, 25-,dsampleswereremoved andthe

reac-tionswereterminated.After120min, 1.0 Uof HpaI

enzymewasadded,andthe remainderofthe reaction

mixture wasincubatedfor anadditional4h. Asa

control,naked32P-labeled SV40DNAwasincubated

under similar reactionconditionsfor120min.

Sam-pleswereappliedto a/.4% agarose slabgel (22by0.3

cm)at45mA,100Vfor6h. DNAdigestion products

were detected byexposure ofthe slabgels toX-ray

films.

resulting DNA fragments. As a

control,

naked

SV40DNAwas

partially digested

with

HpaI

to

thestagewhere theDNAhad

only

a

single

chop. HpaI-derived DNA III from naked SV40 DNAandSV40 chromatinwere redigested with BamHI,EcoRI, andHpaII (Fig. 11). Inspection of therightpanelofFig.11shows thatthe initial cleavage of the SV40 chromatin

by HpaI

was

not at aunique site, forhad thisbeenthe case, redigestion with BamHI should have

yielded

onlytwodistinctfragments.

Instead,

redigestion

yieldedsixfragments,five shown in

Fig.

11(right panel,slotc)andonevery small

fragment

which can only be resolved on a

higher-percentage

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728 LIGGINS, ENGLISH, AND GOLDSTEIN acrylamide gel (notshown in Fig. 11). Indeed, comparison of the naked SV40 DNA III and SV40 chromatin DNA IIIdigestionpatterns

by

BamHI shows that thesepatterns arequite sim-ilar(Fig. 11,middle andright

panels,

slotc).

Quantitative

analysis

of the BamHI

rediges-tionof theHpaI-derivedDNA IIIrevealed that theinitial cut inthe naked DNA as wellas in thechromatin ismore

likely

to occuratthe0.755 position thanatthe othertwosusceptible sites (Table 3). For theDNA, initialcutsoccurabout 1.5timesmorefrequentlyatthissite thanatthe othersites, whereasinthechromatin, initialcuts aremadeabout2.5timesmorefrequently than atthe other sites (Table 3). The 0.755position containstwoveryclosely spacedHpaI cleavage sites,sothat it is reasonable that the initialcuts should be more

frequent

in this region. For instance, ifit wereassumed thatcleavageoccurs withequalprobabilityateach ofthe four HpaI sites, there should have been twice as many DNA fragments generated by cleavage at the 0.755sites relativetoeachofthe othercleavage sites.However(Table 3), thefrequencyof cleav-age at the 0.755 site is significantly higher for cleavage of SV40 chromatin and significantly

:'4a. Naked DNA

4 ?7C.ce o"i

.A I

J. VIROL.

less frequentfornaked SV40 DNA. These data indicate that theprobability ofcleavage is dif-ferent for each site and is different in naked DNAcompared with that in chromatin. Unfor-tunately, these experiments were done with a singlepreparation of SV40chromatin, and fur-ther work would be needed to determine the significance of these differences.

DISCUSSION

In thisstudy, thesusceptibility of SV40 chro-matintorestriction endonucleaseswasfoundto bedependentonthe ionic environment andon the topological configuration of the chromatin DNA. The SV40 chromatin employed in these experiments was devoid of Hi histones, so the observed

dependence

was

presumably

due to conformational changes between the nucleoso-mal histones and theSV40 DNA.

Recent biochemical dataindicate that there are about 200DNA base pairs associated with eachSV40orpolyoma nucleosome andastretch of about50DNA basepairsconnecting adjacent nucleosomes (4, 6, 32,43). Consistent with this model isourfinding that only about 20% of the SV40 chromatin DNA was attacked by the

re-HpaIderived 'a de Naked DNAmA' : NA d e

I)NA

DNA11t DNA vit"sA

-_.

-VW

*ri. i.

i * tox

.)f!

FIG. 11. Cleavage ofHpaI-derivedDNAIII bysingle-site enzymes.32P-labeledSV40DNA III was removed

from preparativeagaroseslab gels after limit digestion ofSV40chromatin or after partialdigestion of naked

SV40 DNA. The DNAIII wasthendigestedbyeither EcoRI, HpaII, or BamHI in a buffer containing 70mM

NaCl,4mMKCI, 14mM Tris (pH7.5),3.5mM

MgCl,,

and 0. 7 mM

II-mercaptoethanol.

DNA fragments were

separatedon1.4% agarose slabgels.Left panel,SV40DNA digest products as markers: (a) untreatedDNA;

(b, c,andd) partial digestionbyHpaI; (e)HpaII digestion;

(t)

HpaI+ HpaII. Middle panel,HpaI-derived

DNAIIIfrom partial digestionof naked DNA: (a) untreated DNA; (b) EcoRIdigestion; (c)BamHIdigestion;

(d)HpaI digestion. Rightpanel,HpaI-derivedDNA IIIfromlimit digestion of chromatin: (a) HpaII digestion

(verylowactivity); (b) HpaIandHpaII(HpaII, very lowactivity); (c) BamHI digestion.

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TABLE 3. BamHIdigestion of HpaI-derivedSV40 DNA IIIa

Digestion

Fragment MapPOSitiOn Predicted DNA Chromatin

size' a oiinPeitd

Wt(%) No. of mole- Wt( NO.OfmOle Wt( No. of

mole-cules(% cules(%)ls %

96.5 0.175 24.4 14.3 24.1 14.5 22.2 12.9

76.5 19.3 14.3 26.2 19.9 16.9 12.4

23.5 0.375 5.9 14.3 6.2 15.3 5.2 12.4

62.5 31.5 28.6 27.6 25.6 34.7 31.0

37.5 0.755 18.9 28.6 15.9 24.6 21.0 31.3

aDatawerederived fromdensitometric tracings of gel patterns. Values represent averages for duplicate runs.

bFragment size represents percentage of the length of SV40 DNA. For each initial cut made by HpaI,

redigestion by BamHI will generatetwofragments. Thus, the 76.5 23.5 pair is generated by BamHI digestion

of DNA III terminatedatthe0.375mapposition, etc. A 3.5% fragment runs off this gel and is not included in

thisanalysis; thus, the96.5fragment is lacking its complement.

"Predictionswerebasedonthe assumption that each of the fourHpaI sites is cleaved with equal probability.

Since0.755represents adoublesite, cleavage at the site should be at twice the frequency of cleavage at the

othertwosites.

striction enzymes at 100 mM NaCl, suggesting that nucleosome-associated DNA was entirely resistant tothe enzymes atthis salt concentra-tion. However, ourkineticexperiments indicate that the susceptible DNA, presumably in the "spacer" regions between nucleosomes, didnot exist inastate

analogous

tofreeSV40 DNA.For example, inthe short timerequired to convert control naked SV40 DNA to its limit digest products,only about 4% ofthechromatinDNA wascleaved (Table1).Complete digestionof the susceptibleDNArequiredincubationtimes6 to 15timeslonger.

Atlower salt concentrations, however,about 50% of the SV40 chromatin DNA wasdigested by the restriction enzymes. This value is in agreementwith values foundin studies in which chromatin was digested with nonrestricted en-donucleases (8, 9, 32, 41). Of this

susceptible

fraction, about half of the DNA was cleaved rapidly, i.e., duringatimeperiodcomparableto

that

required

for limit

digestion

of

equivalent

amounts of control naked SV40 DNA

(Fig.

3). From these experiments, we propose that the internucleosomal DNA has very little associa-tion with the histonesatlow saltconcentrations. In contrast, an incubation period at least 10 timesgreater wasrequiredtodigestthe remain-ingsusceptiblechromatin DNA.Thisfractionof thechromatin DNA may representsiteswithin nucleosomes whichareonly exposedatthelower saltconcentrations but nevertheless remain

par-tially

associated with histones. The fraction

to-tally resistanttodigestionatthe lowersalt

con-centration probably contains sites within the

140-base-pair nucleosome core (1, 2, 10, 52, 53, 55, 57).

SV40 chromatin containing DNA in a topol-ogically relaxed configuration, i.e., with DNA having either a single- or a double-stranded break, was highly resistant tofurther cleavage by restriction enzymes. The resistance ofSV40 chromatincontaining nicked DNAwasrevealed in experiments with single-site enzymes, in which itwasfoundthat DNA IIinitiallypresent in chromatinfailedto be converted to DNA III (Fig. 3 and 4). Atthehigher salt concentration (100 mMNaCl) andatlowenzyme-to-chromatin ratios, the quantityof DNA IIactually increased during the course of the reactions (Fig. 2 and Table 1). In these experiments, the SV40 chro-matin used as asubstrate for the enzymes con-sisted

initially

of mixtures of chromatin with DNA I and II present, so that the possibility thatDNA II was anintermediatebeing formed and then converted to DNA III could not be ruledout.Nonetheless, kinetic

experiments

such

as those shown in

Fig.

3 and 4 in which the fraction of DNA II remained essentially un-changedthroughoutprolongedincubationwhile DNA Iwasquantitatively convertedtoDNAIII

are notconsistent with suchamechanism.

Theconclusion thatasingledouble-stranded break can render SV40 chromatin resistant to

furtherdigestion by multiple-siteenzymes was

based onexperiments such astheoneshown in Fig. 5 in whichmostof the DNAwasconverted toDNA III and only 1% of the DNA could be detected atpositionscorrespondingtolimit di-gestfragments.Thesmallamountoflimit

digest

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730 LIGGINS, ENGLISH, AND GOLDSTEIN

products was not due to insufficient enzyme activity, sincenakedSV40DNA added to reac-tionmixtures before orduring thereactions was rapidly digested tolimit digestfragments (Fig. 8). In contrast to theseresults, Persico-DiLauro etal. (41)found that20 to25% ofthe chromatin DNA wasdigestedtolimitdigestfragments after exposure to HindIII. The discrepancy may be related todifferences in the purification of the chromatin and differencesinthe specificactivity andsource of enzymes.

Upon observing that most of the DNA in SV40 chromatin receivedonlyasinglecleavage after digestion with multiple-site enzymes, we attempted to determine whether this initial cleavage occurred at some preferred site(s) on the chromatin. Consequently, the initial cleav-age sites were mapped after digestion withHpaI. Subsequent experiments revealed that,innaked SV40DNAaswellas inchromatin, all potential siteswerecleaved,but notallsites were attacked withequalprobability (Table 3). For instance, it was shown that certain sites, specificallythose sites whichmap at position 0.755 on the SV40 genome, were cut at a much higher frequency thanthe other two sites.However,thefrequency wasnothighenough to justify a conclusion that these sites were devoid of histones. Had this been thecase,the ratio ofcutsat these sites to those atthe othersites should have been even greater, no DNA I ought to have remained un-digested, and limit digest fragments A and B should have beengenerated athigherlevels and in equal amounts (see Fig. 10

an4i

Table 3). Nonetheless,the results of ourexperimentwith HpaI demonstrate that thedistribution of his-tones in SV40 chromatin is not random. This conclusion is inagreementwith the data found for the

distribuition

ofhistones in polyoma chro-matin (44). It also raises the possibility that moreextensiveanalysisofchromatinby restric-tion enzymes might reveal certain sites which precludethebinding ofhistones.

One type ofconformational change likely to render

internucleosomal

DNAresistant to diges-tion after relaxation of the SV40 DNA I in chromatin is one whichcauses the exposedDNA to associate with nucleosomes. Before relaxa-tion,SV40DNA I inchromatin is constrained in ahigh-energystate (64, 65), but upon relaxation the DNA canbecome free to rotate. In a model suggestedbyWorcel and Benyajati(63), succes-sive right-hand rotations of nucleosomes can cause

internucleosomal

DNA to coil bringing adjacent nucleosomes together. In this model, the spacer DNA forms a coil in the groove created by the contiguous nucleosomes. Alter-natively, it is conceivable that grooves exist or

are formed upon rearrangement of the core structure. The extent ofcoiling andspacing of nucleosomes would of course depend on other factors,such as saltand divalent ion concentra-tion. An association between spacer DNA and nucleosomes similar to that proposed in this modelcanbe concluded fromrecent endonucle-ase studies which revealthat certain repeating elements found in nucleosomes extend intothe spacerregion (31, 44). Indeed, othershave sug-gestedthatnative chromatinordinarily existsin acompact structurewith nucleosomes almost in contact(15, 21,59).

The two proposed models ofchromatin, i.e., thecompactform and thebeaded-string config-urationappeartorepresent aconversionof one form to the other,dependingonthe conditions of observations. WithregardtoSV40chromatin, Griffith (21) observed thecompact form under physiological salt conditions and the beaded form atlow salt conditions. In

subsequent

ex-periments, Christiansen and Griffith(6) showed -thatmagnesium ions can condense SV40 chro-matinevenatlow salt concentrations intoaform which ressembles compact chromatin. This would suggest that, under the conditions em-ployed in our experiments (i.e., 1 to 10 mM

MgCl2),

theSV40 chromatin became condensed

when the DNA was relaxed, whereas, had the magnesium concentrations been reduced fur-ther, the internucleosomal DNAmayhave still remainedpartially extended. Inagreementwith this prediction, we noted above (see

Results)

that SV40 chromatinwas digestedto agreater extent atthe lower concentrations ofmagnesium (Fig. 6).

Additional data relate to the mechanism of action of the restrictionenzymes.Ruben and co-workers(47) and Modrich and Zabel (36) found that the reaction of EcoRI with DNA takes place in two distinct steps; a single-stranded break is made

rapidly

inone

strand,

followed by aslower reaction whichgeneratesabreakinthe

complementary

strand. When EcoRI was

re-acted withSV40 DNAat alow concentration of enzymerelativetoDNA,anicked-circular DNA IIintermediatewasgeneratedbeforeconversion to DNAIII (Fig. 2a). Incontrast, whenEcoRI was reacted with SV40 chromatin under the

same conditions, a significant amountof DNA II was generated, but itfailed to converted to DNA III. In agreement with this observation, Persico-DiLauroetal.(41)observed a 15 to 25% increase in DNAII

during

digestionwith EcoRI. In ourwork, however, this increase inDNA II

didnotoccur athighconcentrationsofenzymes

(Fig. 3).If we are correct inassumingthat a nick inchromatin DNA can cause structuralchanges

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whichsequestercleavage sites,apossible

expla-nation for the failureto convertDNA IItoDNA

IIIatlowenzyme concentrations would be that

these changes occur before the enzyme can

cleave the complementary strand. We intendto

further investigate this possibility in more

de-tailed studies.

ACKNOWLEDGMENTS

We thank Michael Chenand Mary Gutai for theirgenerous

gifts of the Hindll+IIIenzymes,Abraham Worcel for his usefuldiscussions and hisinterest in this work, and Samuel Litwinfor his help with the analysisof the data.

This workwassupported by Public HealthServicegrant

no.CA11151from the National CancerInstitute. G.L.L. held Public Health Service fellowshipno. CA 05442, and D.A.G.

wasthe recipient of Public HealthService Research Career Development award no. CA 32425,both from the National Cancer Institute.

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