Hin D restriction mapping of upaired regions in simian virus 40 superhelical DNA I: considerations regarding structure-function relationships.

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Hin D

Restriction Mapping of Unpaired Regions

in

Simian

Virus 40 Superhelical DNA I: Considerations

Regarding

Structure-Function

Relationships

M. CHEN, J. LEBOWITZ,'* AND N. P. SALZMAN

Laboratory ofBiology of Viruses, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, and Department of Microbiology, The Medical Center, University of

Alabama in Birmingham, Birmingham, Alabama 35294*

Received for publication 29 September 1975

Superhelical simian virus 40(SV40) DNA Iwasreactedwith N-cyclohexyl-N'-8-(4-methylmorpholinium)ethylcarbodiimide (CMC), and the location ofCMC

sites was mapped using the Hin D restriction endonuclease. The use of 14C0

labeledCMC allowsaquantitativeanalysisof thebindingto therespective Hin Drestriction endonuclease fragments. The percentage ofreactivity was 6.54%

for fragment A, 3.87% for fragment B, and 2.74% for fragment G. No CMC

radioactivity was detected in other fragments. This reactivity is in agreement

with the evaluation of binding by buoyantdensity measurements. The above

fragmentsalso contain the sitessusceptibleto

S,

endonucleaseaction.Thisadds

further support to the view that superhelical DNA can contain regions of

localized interrupted secondary structure which may be capable of forming

intrastrand hairpin structures if sequence relationships are favorable. The

possible structure-function relationships for this model arediscussed with the

emphasisontranscription.

In the previous paper (15) we showed that

superhelical simian virus 40(SV40)DNA I can

be modified with

N-cyclohexyl-N'-j8-(4-methyl-morpholinium)ethylcarbodiimide (CMG). CMC

reacts preferentially with the imino sites of

unpaired thymineand guanine residues toform

a stable covalent product in the neutral pH

range (19, 20). Consequently, one can map the

locationsofCMConSV40DNA I. Thisinvolves

locating those Hin D restriction endonuclease

fragmentscontaining CMC. A comparison can

then be madebetween the CMCreactive sites

and those sites that are cleaved by

single-strand-specific S, endonucleaseaction.

If a correspondence exists between the re-gions ofreactivity of CMC,

single-strand-spe-cificendonucleases (3, 15),andgene 32 protein

(21, 22), this would addfurther support to the

view that superhelical DNA can contain

re-gions of localized interrupted secondary

struc-ture which may be capable of forming

intras-trand hairpin structures. An examination of

possible structure-function relationships for the

model proposed arediscussedinthe contextof

recentstudies, particularly transcription data.

MATERIALS AND METHODS

Cell culture, SV40, and DNA preparation. Full details for the above procedures have been described

IThis research wasconducted while on sabbatical leave

fromSyracuse University, Syracuse, N.Y. 13210.

previously (15).

Enzymes. The endonuclease R-Hin D was

puri-fiedby the method of Smith and Wilcox (25) andwas

storedat -20Cin0.2 MKCI-0.01 MTris(pH

7.5)-50%glycerol at 6 U/ml.

Chemicals.Cold CMCwasordered fromthe

Ald-rich Chemical Co., Milwaukee, Wis.; 14C-labeled

CMC was prepared by the New England Nuclear

Corp. (Boston, Mass.)custom synthesis laboratory

using1-cyclohexyl-3-[2-morpholino-(4)-ethyl]

carbo-diimide thatwasprepared byusfollowing previous

procedures (19). This intermediate carbodiimide

wasconverted to CMC by reaction with 14C-labeled

CH3Iinthe NewEnglandNuclearCorp.laboratory.

Electrophoresis. All of the electrophoretic runs

werecarried outinanECvertical-gel

electrophore-sis apparatus (model E-C 470). Samples (0.05 ml)

were applied to slab gels (17 by 12 by 0.30 cm)

consisting of 5%polyacrylamide and 0.5% agarose.

Electrophoresis was carried out for16h at20Cat 50

V ina buffer consisting of 40 mM

Tris-hydrochlo-ride, 20 mM sodium acetate, and 1 mM EDTA

ad-justed to pH 7.2 with acetic acid. To quantitate

radiolabeled bands, the gels were frozen and sliced

into 1-mm segments.Each segment was dissolvedin

0.2ml of30% H202 and counted with Triton X-100

toluene scintillation fluid.

Analysis of the binding of "4C-labeled CMC to

SV40 DNA. A defined amountofSV40 DNA I was

placedinthegel slab afterHin D digestion. Since

the weight fraction of eachfragmentisknown, the

calculation ofbindingwasperformed, e.g.,for

frag-mentA,asfollows. The Afragment is 22.5%of the

SV40 genome and consequently the gel contained

211

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212 CHEN ET AL.

2.25

jLgg

of A. Since only T and G are capable of

reacting we have 1.125,igor 0.00338 ,umol of

possi-ble bases as potential covalent binding sites. The

amount of"4C-labeledCMC bound afterbackground

subtraction was 292 counts/min, which was

con-verted into disintegrations per minute per

micro-mole using the specific activity for CMC, 2 mCi/

mmol, and the determined counting efficiency of

30%. In the case of the Afragment, 0.000212Mmol of

CMC wasbound, giving a reactivity of 6.54%.

Simi-lar calculations were performed for the B and G

fragments.

RESULTS

Electrophoretic analysis of a Hin D digest

ofSV40DNA Imodified with unlabeled CMC.

The first experiment utilized 3H-labeled SV40

DNA Itreated with unlabeled CMC (15). The

Hin Ddigestionwascarriedout inthe presence

of untreated "4C-labeled SV40 DNA after

re-moval of excess CMC. Figure 1shows that the

20[ | IX

16

o12

-a.

0U

X

8~

electrophoretic mobilities of the Aand B

frag-ments are clearly slowed by onefraction.There

is an indication that the G fragment is also

displaced by the CMC reaction. To confirm

these assignments, it was necessary to obtain

labeled CMC. This was provided to us as

14C-labeled CMC by custom synthesis from the

NewEngland Nuclear Corp. (see Materials and

Methods).

Electrophoretic analysis of theHinDdigest

of 3H-labeled SV40 DNA Imodified with

14C-labeledCMC. Thereactionof"4C-labeledCMC

was carried out under conditions similar to

those that wereused for the reaction between

cold reagent and 3H-labeled SV40 DNA. After theexcess reagent wasremoved, the 3H-labeled

DNA,with covalently bound"4C-labeledCMC,

was digested in the presence of 32P-labeled

SV40 DNA. The results of thistriple-label

ex-perimentareshowninFig.2. Itcanreadilybe

X | | W l l 50

40

30;

0~

a.-U

20 u

-0 20 40 60 80 100 1

-FRACTION NUMBER

FIG. 1. 3H-labeledSV40 DNA I (specific activity, 5300 counts/min per

pg)

wasreacted with an800-fold

excessof cold carbodiimide (moles per mole of nucleotide) in a buffer containing 0.01 MNa2B4O7, 0.1 M

Na2SO4,0.25M NaCl, pH 8.0. The excess carbodiimide was removed by dialysis against the same buffer. The

carbodiimide-reacted 3H-labeled(0) SV40 DNA Iwasdialyzed against6.6mMTris, 50 mMNaCl(pH 7.4)

before being cleaved with endonuclease Hin D. "4C-labeled(+)SV40 DNA I markerdigest(specific activity

16,000 counts/min perMg)wascleavedsimultaneouslyinthesameHin Dbefore electrophoresisin 5%

acryl-amide-0.5% agarosein anECvertical-gel electrophoresisapparatusfor16h. Gelswerethenslicedinto 1-mm

segmentsand dissolvedin 0.3ml 30%H202 overnightat65C. Ten millilitersofscintillationfluidwasadded

and counted.

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. I i

0K

0. u

xI

6T

S-2

4x

25

20

Is

0

m

10 gona

5~ 5

10

FRACTION NUMBER

FIG. 2. The experimentwasessentially thesame asinFig.1,except'4C-labeled carbodiimide(A) (2mCi/

mmol) wasreactedwith3H-labeled (l) SV40DNAI.32P-labeled SV40DNAI(0) markerwascleavedinthe

sameHinDdigestwith 14C-labeledCMC-modified3H-labeledSV40DNA.

seen that no 14C counts are observed (closed

triangles)inanyofthefragments, exceptA,B,

and G. It is also apparentthat the 14C counts

follow the 3H counts in those fragments that

reactwith CMC. Itcanbe concluded fromthis

experiment thatA,B,and Garetheonly Hin D

fragments thatreact with CMC. It ispossible

fromthespecificactivityof14C-labeled CMCto

calculate theamountofbindinginthe

respec-tivefragments.The totalreactivitycanbe

com-paredtothebindingcalculated from the buoy-antshift measured in theprecedingpaper (15).

Binding analysis of[14C]CMCtotheHin D

A,B,and GfragmentsofSV40DNA. Basedon

thespecificactivity of"4C-labeled CMCand the

amountofSV40 DNA used intheexperiment,

we cancalculate theamount ofbindingtothe

respectivefragments. Theresultsarepresented

in Table 1. The tabulated reactivity for each

fragmentcanbe readilyconverted intoatotal

valueforthe entire SV40genomeby

multiply-ing the percentage of reactivity by the

frac-tional molecular weightofeach fragment and summing the total. This value is compared to

thevalue obtained from thepreviousstudy (15)

inwhichweemployedthebuoyant densityshift

produced by CMCand the partial specific

vol-TABLE 1. Percentage of reactivity of CMC to respective Hin D fragments and the number of CMC

molecules bound perfragment

HinD fragment No. of

and relative mol Reactivity with "'C-la- CMC wt(%ofSV40 beled CMC(%) mol

DNA) bound

A 22.5 6.54 80

B 15.0 3.87 31

G 7.0 2.74 10

Totals in terms of 2.24% of SV40 DNA 121 base pairs

Analysis bybuoyant 2.0%ofSV40 DNA 108 density shift and

densityof CMC from previous study(15)

ume of the reagent. The reaction conditions

were very similar except for the different

sourcesofCMC. Itcanbeseenthat the results

are in good agreementusingtwo independent

methods.

DISCUSSION

The results of the CMC bindinganalysis

us-ing theHin D restrictionendonuclease can be

best examined in thecontextofpreviousresults

on themapping of the single-strand-specific S,

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214 CHEN ET AL.

endonuclease (3),gene 32 protein, andthe

cur-rentresults on thespecificity of theinitiationof

transcription of Escherichia coli polymerase

(14, 16,30).

Figure 3 summarizes thedata in theHin D

fragment map (6). It is apparent thatthere is

excellent agreement between the binding of

CMC tofragmentsA,B, andGand thesites cut

byS1 endonuclease onSV40 DNA I.

Inthe light ofthe aboveresults, we can

con-sider the possible structure-function

relation-ships for the sensitive regions ofsuperhelical

SV40DNA.The firstquestionthatisof

particu-larsignificance is thestructure ofthe regions containing unpaired bases in superhelical

DNA. The previous two papers (15, 29) have

presented evidencethatcanbeinterpreted with

themodelthatsupercoilingproducesregionsof

interruptedsecondarystructure. One can

envi-sion that different single-strand regions,

pro-duced by superhelical torsional forces, could

migrateuntiltheyreachedregionswhere

suffi-cientintrastrandcomplementarity, i.e., forma-tion ofhairpin structures atthese sites, would

lower the free energy and localize the

inter-rupted duplex structure. This localization of

interrupted secondary structure is dependent

onsequencerelationships. Thus, SV40 and

pol-yoma superhelicalDNAsappearto have a

lim-itednumberoflocalizedsitesfor

S,

cleavage(3,

8) whereas 4X-RF is cleaved by Neurospora

crassa endonucleaseinanonpreferential man-ner (K. Bartok and D. T. Denhardt, personal communication).

We suggest that some superhelical DNAs will contain localized hairpin regions which 0

.8 D .2

-~~~~~~~~~

CMIC

BOUND

.7

\t.5 3/ E. coli RNA

Polymerase

Transcription

Early in vivo

Transcription

FIG. 3. Map of theHin DfragmentsofSV40DNA with the location of the following sites: dark regions

represent the area sensitive to the single-strand endonuclease S1 (3); the Hin D fragments binding the indicated number of CMC molecules determined in this study. Gene 32 protein binding sites (22, 23) are represented by arrows inside the circle. Three pppAp initiation sites determined according to the methods ofP.

Lebowitz and Zainet al. (15, 17, 30; manuscript in preparation) used by E. coli polymerase (see text) are

shown by the outside arrows with the direction of transcription. Early in vivo transcription is shown for

comparison (13).

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may show fluctuations from the most stable

state (hairpin configurations). This introduces

amoredynamic view which allows for the

in-fluence of environmental factors. Beard et al.

(3)did observe that

S,

cuttingshifted

preferen-tiallyto 0.45and 0.55 sites upon an increase in

NaCl concentration from 75 to 250 mM. The

additional consideration ofsequence

relation-ships suggest that some superhelical DNAs

could have many transient intrastrand

hair-pins or single-strand sites and, consequently, many sitesforsingle-strand nuclease attack. A

testfor thisview would be the precise locations

of CMC in order to sequence the regions

re-acted. Suchastudy is inprogess.

Sincegene 32proteinbinds cooperativelyto a

single-strand DNA (26), it is striking that it

binds only at one site, at 0.46, which

corre-sponds to one of the three sites at which

S,

cleaves SV40 DNA, whereas itfailstobind to

the B and G sites which are susceptible to

S,

cleavage. Inaddition, another gene 32protein

sitehas beendiscovered that doesnot mapwith

S,

orCMCsites(22).It has beenpointedout(26)

thatthefreeenergyof binding ofaproteinto a

single strand ofDNA

(AGbind),

in aregion

nor-mally double stranded under the prevailing

en-vironmentalconditions,must atleast offset the

free energy favoring the native over the

ran-dom coil conformation at that locus (AGCOflf).

These energetic considerations are applicable

tothemeltingoutof A-T-richregionsinduplex

DNA, aswell asthe meltingoutof short

hair-pinregions.Bothevents areneeded during

rep-lication(13, 26).

Inthecaseof SV40DNAI,itwould be

neces-sarythat

AGCOff

orhairpins

<AGbhld

inorderto

melt out the proposed hairpin structures. The

inabilitytosatisfy favorableenergeticsforgene

32proteinmelting of the0.55 site in Aand the

sites in B and G fragments suggests stable

hairpinsitesatthese locationsinSV40 DNA I.

Other structural featuresmay play an

impor-tantrole. The cooperativity ofhairpin melting

might bepartially controlled byprotein-protein

interactions,asboundgene 32proteinsinteract

favorably with oneanother (26). Steric factors

insupercoiled DNA could affect the binding of

gene 32protein.

A variety of in vitro transcription studies

have beenperformed on different superhelical

DNAs(4, 5, 11, 14, 17, 18, 23, 24, 27, 28, 30, 31).

Hayashi andHayashi(10)showed that UX-RFI

ismorerapidly transcribedthan

4X-RF

II. This

kindof resulthasbeenobtained for

superheli-cal X (4) and PM2 DNA (23, 24, 27, 31). Itwas

proposed that supercoiling increased

nonspe-cific initiation(4, 27),sinceanincrease in

bind-ing is morefavorable inordertolower the free

energy by a coupled unwinding ofthe duplex

structure and supercoils. However, ifhairpin

regions exist,then binding ofRNApolymerase

could occur at these sites. Thiswould present

easily recognizable, specific regions for the

en-zyme aswell as promote more favorable

ener-geticsofbinding.

A number ofstudies (11, 14, 17, 18, 24) sug-gest that the above model may reflect the

en-hanced transcription seen for superhelical

DNA. Richardson (24) found that PM2 DNA I

forms 16 stable binary complexes with E. coli

RNApolymerase, whereasonly 2 are foundfor

closed, nonsupercoiledPM2 DNA. The reaction

of PM2 DNA Imodified with CMC,atabinding

ratio of0.01, eliminates 95% of the

transcrip-tion capacity relative to untreated form I (M.

Flashner and J. Lebowitz, unpublished data).

This hasalso been found for SV40 DNA I (P.

Haleand J. Lebowitz, unpublished data).

Recent studies(14, 16, 30) of thetranscription

of SV40 DNA with E. coli RNA polymerase

show that therearethreeprincipal y-[32P]ATP

initiation fragments: one, designated

A,,,

cop-ied fromthe Hin D-G fragment, and the other

twodesignated

A,

and

A,,,,

each copiedfromtwo

separatesitesinthe Hin-A fragment(16, 30; P.

Lebowitz, unpublished data). It was also

re-ported thattwoadditionalpppAp andalimited

number ofpppGp sites (14) exist.

InFigure 3weshow the three pppAp

initia-tionsites onthe SV40map. Itcanbeseenthat

initiation occurs at 0.52and0.44 intheA

frag-ment,andat 0.17 inthe G fragment. The

direc-tionofE.coli RNApolymerasetranscription is

showninFig. 3,anditiscompletely

asymmet-ric (28). The strand copied corresponds to that

copied early in lytic infection and has been

designatedthe (-) orearly strand (12). An ex-aminationofFig. 3 shows anextremely

inter-esting structural feature: there is a potential

hairpin site beforeeach pppAp initiation site.

Consequently, hairpin regions in these cases

would promote considerable specificity in the

interactionofE.colipolymerase with

superhel-ical SV40DNA. The formation of RNA

polym-erase promoter complexes would be achieved

morereadilyathairpinsites, sincetheenergy

neededtoopentheDNAforaccess tothe

tem-plate bases should be less than that forafully

paired structure (5).Hence SV40 DNAIcould,

inthepppApinitiationsitesshown, bypassthe

activation step of opening an intact duplex

strand. This would allow for more initiation

events, which would explain the enhanced

transcription ofsupercoiledDNA.The

specific-ity of the RNApolymeraseinteractionwouldbe

dependentonwhetheropenregionsorhairpins

canbefixedonthe molecule. Further

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216 CHEN ET AL.

ingbythe formationofaDNA-RNA hybrid (14)

would be assisted by the free energy decrease

caused by acoupled unwinding of duplex and

superhelicalturns.

Recently, a series ofpapers was published

on the in vitro transcription of SV40 DNA I,

relative to the linear form, usingcalf thymus

and rat liver RNA polymerases (11, 17, 18).

These results are very similar to the

OX-RF

and PM2dataandshowenhanced transcription

for SV40 DNA I. They conclude that unpaired

basesareresponsible for theenhancedratedue

to an increased binding affinity for the RNA

polymeraseatunpairedsites(11).In contrastto

E. coli polymerase, the mammalian RNA

po-lymerasestestedtranscribed SV40 DNA I

sym-metrically. The incorporation of -y-[32P]ATP

and y-[32PIGTP suggests multiple initiation

sites,although thenumberand locationare not

known (17). It is important to note that an

initiationcomplexof theBRNApolymerase of calfthymuswith SV40 DNA I apparently pro-tectsagainstS1 cleavage (11).

An in vivocomparisonof SV40 mRNAinthe

nucleus and thecytoplasmsuggeststhat RNA

synthesis occurssymmetrically and that

anti-message isdegraded priortoitstransport tothe

cytoplasm(1,12).Thisproposalagreeswiththe

symmetrical synthesis observed in vitro for

mammalian RNA polymerases. The

hybridiza-tionpatternof cytoplasmic mRNA withHin D

fragments reveals that early message starts

almost attheend of the A fragment (12) (Fig.

3). It is difficult at this time to reach

conclu-sionssolely from thehybridizationdata. First,

wedonotknow thedetails of processing of viral

mRNA and second, weknow verylittle about

the transcription of SV40 DNA complexes with

histones(7, 9).It isentirely possible that

struc-turalvariationwill beproduced by the

forma-tionof nucleosomes (7, 9). For example, astudy

of the mapping of the SV40-specific sequences

transcribed in vitro from chromatin of

SV40-transformed SV 3T3 cells shows that the early

regions A throughB (Fig. 3) were transcribed

fiveto ten timesmore frequently than the

re-maining regions (2). In contrast, transcription

ofpurifiedSV3T3 DNAbyE. colipolymerase

produced equal frequencies of transcription fromall regionsof theintegratedDNA (2).

Thisbriefsurveyofaspectsoftranscription of

SV40clearly suggests thatfurther

experimen-tal exploration is required in order to

under-stand the structural relationship involved in

transcriptional events for SV40 DNA I. We,

alongwith other (3, 10, 11, 17, 18),suggestthat

there maybe an important role played by

re-gionsofinterruptedsecondary structure

form-ingpossiblelocalized hairpinsinthe

transcrip-tionalprocess.

ACKNOWLEDGMENTS

J. L. wassupported by Public HealthService Research Career AwardK04-CA00141-03 (formerly5K04-CA07514) from theNational Cancer Institute throughout theconduct and preparation ofthisstudy.J. L.thanksSyracuse Uni-versity for thenecessarysabbaticalleave to carry out the reportedresearch.

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