Restriction Enzyme Digests of Rapidly Renaturing Fragments of Vaccinia Virus DNA

Copyright01976 American SocietyforMicrobiology Printed inU.S.A.

Restriction

Enzyme Digests

of

Rapidly Renaturing Fragments

of Vaccinia Virus DNA

FRANK M. DEFILIPPES

Laboratory of Biology of Viruses,NationalInstitute ofAllergyand InfectiousDiseases,

Bethesda,

Maryland

20014

Received for publication 17 July 1975

Vaccinia virus DNAfragments that have been denatured by alkali and then neutralized contain a fraction that rapidly reforms duplex structures. The

fraction isenriched byfractionating onhydroxyapatite columns andserves as a

substrate

fordigestion

by

tworestrictionendonucleases isolated from

Hemophi-lusparainfluenzae, Hpa I and HPaII.Thepatternsobtainedby gel

electrophore-sis of thedigested fragments show the presence of three major bands after Hpa I

digestion and four majorbandsafterHpa IIdigestion. TheDNAthat is isolated

from some of these bands quickly reforms duplex regions after alkaline

denaturation. The size of the DNA segments in the major bands has been

estimated tobein the rangeof0.44 x 106to 3.2 x 106daltons. Thefragments which rapidly reform duplex chains after denaturation are sensitive to single-strand-specific nucleases. These results are consistent with a model of vaccinia

virusDNA which has acovalent linkconnecting complementary chains.

The linear, double-stranded DNA molecule isolated from vaccinia virus weighs 122 x 106

daltons(8).Thecomplementarystrands of this

DNA donotseparateunder alkaline

denaturing

conditions, indicating that covalent bonds link thetwostrands (3). If this DNA is

sheared,

then the strandsof mostofthe

duplex

fragments will

separate after denaturation. The

fragments

which contain the covalent link connecting the

complementary

strands

(covalently-linked

com-plementary

[CLCI

fragments), however,

will rapidly reformduplex molecules

("snap-back")

when alkaline solutions are neutralized. The

CLC

fragments may be separated from single-stranded DNA

by

hydroxyapatite chromatogra-phy. Restriction endonuclease

digestion

ofthese CLC fragments

produces specific

segments of

the vaccinia DNA molecule. These segments

should be near the ends ofthe DNA since the covalent linksconnecting the strands have been locatedwithin 50nucleotidesofeach endofthe intact molecule (8).

One

model which has been proposed (8) for the structureofvaccinia DNA has thecomplementary chains joined

by

asmall

loopofsingle-stranded DNAateach end ofthe molecule.

Therestrictionendonucleasesfrom

Hemophi-lus

parainfluenzae

have been used to

digest

the

snap-back

fragments. Digestsof the entire

vac-cinia DNA molecule

by

theseenzymesproduce

alarge numberofsegments that are difficult to resolveby gel electrophoresis.Thisprecludesan

227

analysis of the entire vaccinia genome by

methods which have been usedto study smaller

DNA molecules isolated from simian virus 40

and otherviruses (4, 10, 14, 18). However, the presence of CLC fragments, which may be

generated by shearing theintactDNA, presents an opportunity to analyze a fraction of the

whole moleculeby restriction endonuclease

di-gestion.

MATERIALS AND METHODS

Virus. HeLa S3-1 cells, grown as suspension

cul-tures in Eagle minimal essential medium with 5%

horse serum, werekindly supplied by B. Moss and N.

Cooper.Vaccinia virus (strain WR) was grown in HeLa cells inmedium with 5% dialyzed horse serum

contain-ing2 or 4

MCi

of

['H]thymidine

per ml or 0.05gCiof

["4C thymidine perml(New England Nuclear Corp.). The virus was grown in phosphate-free Eagle medium forlabeling with either 1 or 5

sCi

of [2P

]orthophos-phate per ml (New England Nuclear). The procedure for virusinfectionhas been described previously (16). The virus was purified by the Joklik method (12), which wasmodifiedtoexclude sonic oscillation steps and the initial sedimentation through 36% sucrose. The crude cytoplasmic extract, obtained by Dounce homogenization of the cells followed by three low-speed centrifugations to remove nuclei, was incubated at 37 C for 5 h with 2x crystallized trypsin (Sigma ChemicalCo.) ataconcentration of1mg/ml. A band containing virus particles was isolated by

sedimenta-tion ofthedigested extract into a 25 to 40% sucrose gradient (12). After dilution of the band with an equal volume of 10 mM Tris-chloride, pH 9.0, the virus

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particles were pelleted by sedimentation at 21,000 rpm for 45min. The pellets were suspended in 10 mM Tris-chloride and repelleted through a 36% sucrose solution by sedimentation at21,000 rpm for60min. The pellet was suspended and stored in 10 mM Tris-chloride, pH 9.0.

DNA. Vaccinia DNA was prepared by digesting

virus particles with 100 ug of Proteinase K (EM Laboratories, Inc.) per mlat 37C for12h inabuffer containing 10 mM NaCi, 10mM sodium EDTA, 10

mM Tris-chloride, pH 8.3, and 1% sodium dodecyl sulfate(SDS). Solid sucrose was added to this digest togive a final concentration of 27% sucrose, andthe mixture wasextracted twice with phenol. The DNA was thendialyzed againstbuffer H (50 mMNaCl, 0.4 mMtrisodium EDTA and 10mM Tris-chloride, pH 7.8). 8H-labeled DNA had a specific activity of at

least 1.5 x 101 counts/min per

Ag

and "4C-labeled

DNAhad an activity of 1.9 x 104counts/minper

Ag.

32P-labeled DNAhad specific activities of 1.1 x 105 and 2.1 x 105counts/min per

jig.

Radioactivity was counted in aBeckman modelLS-250 liquid scintilla-tion counter inscintillation fluid containing toluene-Triton X-100-water (6:3:1) with 6.4% Spectrafluor

(Amersham/Searle). T4 and T7 bacteriophages were grown according to standard procedures (19) and the DNAwasisolatedby the method described by Bautz and Dunn for T4 DNA (2).

DNA solutions containing 1 M NaCl in buffer H werebubbled with nitrogenfor5 min at 0C, and the DNA, at a concentration of20

jg/ml,

wasshearedby 10 passages through a 9-cm, 20-gauge needle. The DNA was then dialyzed against buffer H for 12 h. DNA was denatured in solutions containing 0.2 N NaOH at 37C for 10 min. The solution was neutral-izedby addition of an equivalent amount ofHCland diluted into 5volumes of80mM sodium phosphate (pH 6.8)at 0Corintothesamebuffer containing50%

formamide.

Hydroxyapatite chromatography. Hydroxyapa-tite columns were prepared in disposable plastic

syringes that had a porous polyethylene support. In the mostbasic of several procedures, hydroxyapatite (Bio-Gel HTP, Bio-RadLaboratories)wassuspended

in80mM sodiumphosphate, pH 6.8, and poured into the column. The neutralized, alkali-treated DNA solutionwasapplied tothe column thatwaswashed with2column volumes of80mM sodiumphosphate. Single-stranded DNA was eluted with 8 column volumes of 0.18 M sodium phosphate, pH 6.8, and duplex DNA was eluted with4column volumesof0.4

M sodium phosphate, pH6.8.Trisodium EDTAwas

added toduplex DNA which had been eluted from the column to giveafinal concentrationof5mM, and the DNA was dialyzed against bufferH for 12 h. This DNAwas alkali denaturedasecondtimeand neutral-izedasbefore, except that the dilution buffer was 80

mMsodiumphosphate, 50%formamide, pH6.8.The

hydroxyapatite wassuspended in this bufferaswell,

and single-stranded DNA was eluted with 0.18 M

sodiumphosphatecontaining50%formamide.Duplex DNA was eluted as before using a buffer without formamide anddialyzed against bufferH.TheseDNA

fragments served assubstrate inthe restriction

en-zyme digestion, and they are referred to as CLC fragments.

Restriction enzymes. The two restriction endonu-cleases from H.parainfluenzae, Hpa I and Hpa II, were isolated by a modification of a previously pub-lished procedure for the isolation of Hpa I (6). The modifications included breaking the bacterial cells openinapress(Manton GaulinLaboratory Homoge-nizer) and precipitating the proteins in the crude extract with (NH4)2SO4 at 64% of saturation. The final step, after concentration onthe phosphocellulose column, was to separate the two enzymes by gel filtration on a Sephadex G-100column. The enzymes were stored and used aspreviously described (6). The digestion reaction was stopped by the addition of trisodium EDTA and SDS to final concentrations of 30 mM and 0.5% and enough sucrose was added to give a 15% solution.

Gelelectrophoresis.Agarose (1.6%) gels (11 by 0.5 cm; Seakem) were formed in plexiglass tubes. The gels were made up in buffer E (36 mM Tris, 30 mM NaH2PO4, 1 mM trisodium EDTA, pH 7.9). Electro-phoresis was in buffer E with 0.02% SDS and the samples were runfor10to 11 hat 22 V at 24 C. After electrophoresis of3H-or "4C-labeled DNAsegments,

thegelswere cuttransversely into 1-mm sections and

the DNAwas hydrolyzed by heating in 0.5 ml of 1 N HCl at 80 C for 5 h. The samples were counted as previously described (5). Gelscontaining 32P-labeled DNA segments were cut longitudinally, dried on Whatman 3MM paper, and placed in contact with

RP/R2X-ray film (Eastman Kodak Co.) according to standard procedures for obtaining radioautographs (7). In some cases gels containing 32P-labeled DNA segments were cut transversely as above. Each agarose slice was immersed in 5 ml of buffer H, and the radioactivity was determined by Cerenkov counting. The slices that contained the DNA segments were selected, the DNA was eluted by electrophoresis into dialysis sacs, and the samples were dialyzed against

buffer H. The DNA from the individual fragments

was then alkali denatured, neutralized, and passed throughsmall columns (0.2 ml) ofhydroxyapatiteto

separate single-stranded and double-stranded DNA.

Generallyless than5min wasinvolvedinneutralizing andapplying the entire DNA solution to the column.

Some gels were not cut but were immersed in a

solution of ethidium bromide (1 ,ug/ml) for 1 h and then examinedbyUVlight which showed the

fluores-centethidium-stained DNAbands (18).

The distance which the restriction enzyme

frag-ments migrated in the 1.6% agarose gels was

mea-sured and compared to the migration distance of

segments ofT7 DNAwhich had been produced by

digestion with Hpa I. M. Simon and F. W. Studier kindly providedestimates ofthemolecularweightsof

T7 DNA segments after Hpa I digestion (personal

communication), and these were used to determine the relative molecular sizes of the vaccinia DNA

segments. The logofthereciprocalofthe molecular

weightof avaccinia DNAfragmentwasassumedtobe related tothe distancemigratedinthe usual fashion (11).

Nuclease digestion. S. endonuclease from

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gillusoryzae waspurified bythe method ofVogt (22)

and his definition ofa unit ofenzyme activity was

used. TheDNAwasdigestedat37or55 C for30orfor 60minineither50or270mMNaCi, 1.5 mMZnSO4,

5%glycerol, and30mM sodiumacetate,pH 4.65,with 25Asgofsheared denatured calf thymus DNAperml,

and0.2or2U ofenzyme.Thereactionwasstopped by

additionofSDSto0.5%and EDTAto5mM,and the

sample waseither alkali denatured, neutralized, and

passed through hydroxyapatite or precipitated with

7% perchloric acidat0C.Precipitated sampleswere

collected on GF/C filters (Whatman) which were

driedand counted for radioactivity.

A crude preparation ofasingle-strand specific

en-donuclease associated withvaccinia virions was

pro-vided by P. Geshelin and K. Berns. The enzyme

digestionwasat37or55 C for 1 h in buffercontaining

2.5 mM trisodium EDTA, 0.1 M Tris-chloride, pH

7.8,and250Agofsterilegelatinperml.Usually0.1to 1.1 MgofDNAwasdigestedwith10to20Alofenzyme

preparation. The reaction was stopped by the

addi-tionofSDSto0.5% and the condition of the DNAwas

testedbythetechniques used afterS,digestion.

RESULTS

Hydroxyapatite fractionation of digested DNA. Shear breakage of vaccinia DNA, using the conditions described, produced fragments thatsedimented inaneutralsucrosegradientas abroad band whosepeak moved slightly faster

than simian virus 40 form III DNA (data not shown). This indicated that mostofthepieces

were lessthan 7% of the original length. These

fragments were denatured, neutralized, and

passed through a hydroxyapatite column to separate single-stranded from double-stranded DNA. Hydroxyapatite chromatography was at 24C rather than 60C (13) to reduce random

bond breakage, and some hydroxyapatite

frac-tionation was done inthe presence of

formam-ide. Goodman et al. (9) have shown that the fractionation of nucleic acids by hydroxyapatite chromatography at room temperature using buffers containing formamide is similarto the fractionation achieved at elevated tempera-tures.

When 0.1 ggorless ofshearedvaccinia DNA was denatured, neutralized, and diluted into

coldphosphate buffer containing 50% formam-ide and then loaded on hydroxyapatite, 12 to 15% of the DNA was recovered as duplex

material. If the sheared DNA had a uniform

molecular weight of 6 x 106 and was derived

from an intact molecule with a covalent link

connectingcomplementary strands ateach end ofthemolecule, then 10% of the DNA should be recovered asduplexmaterial after

hydroxyapa-tite fractionation. A typical result is shown in Fig. 1B. Less than5minwasneededto

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FIG. 1. Chromatography of [3H]thymidine

vac-cinia DNA on hydroxyapatite columnsat 24C with phosphate buffer, pH 6.8, containing50%formamide.

All samples in these figures were elated by step increasesin the molar concentrationof buffer, which

was applied as indicated by the arrows. (A) Native

DNA was shearedby 10passages througha22-gauge

needleand thenapplied and elated from the column.

(B) DNA was sheared and then alkali denatured,

neutralized, diluted into phosphate buffer,and

imme-diately appliedtothehydroxyapatite. (C-F) Sheared, double-stranded vaccinia DNA was digested witha

nuclease, alkali denatured, neutralized, diluted into phosphate buffer, and appliedtohydroxyapatite. (C) DNA was digested with S1 nuclease; (D) DNA was

digested with a combination ofrestriction

endonu-cleases, Hpa IandHpa II, isolatedfrom H. parain-fluenzae; (E)DNAwasdigested with HpaI; (F)DNA was digested with Hpa II. T4 DNA, labeled with

[14C]thymidine, was sheared and applied to each

hydroxyapatitecolumntoserve as adouble-stranded

DNA marker.

ize the solution and adsorb the DNA to the columnsothat renaturationwasminimal.If the

shearedDNAwasdigested withasingle restric-tion enzyme and denatured and neutralized,

then less duplex material was recovered from the hydroxyapatite. This indicated that the sheared fragments contained the proper

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[image:3.491.252.441.57.367.2]

quence for restriction enzyme digestion. After Hpa I digestion 4 to 7% of the DNA was recovered from the hydroxyapatite (Fig. 1E),

and after Hpa IIdigestion 3 to 5%ofthe DNA was recovered as double-stranded segments

(Fig. iF). Digestion of the DNA with both

restriction enzymes reduced the amount of

duplex DNA recoveredto 1 to 3% (Fig. iD). If

the DNAwasdigestedfor 60 min at 55 C with2

U of the

single-strand-specific

S1

nuclease,

in buffer with either 50 or 270 mM NaCl, and denatured and neutralized, then less than1%of

the fragmentswereretained by hydroxyapatite (Fig. 1C). In separate tests with native

"IC-labeled T7 DNA, 1 U of this S1 preparation nicked more than 90% of the molecules after

incubation for 60 min at 37 C in a reaction

mixture containing 270 mMNaCland 25

gg

of

denatured calf thymusDNA per ml (see

refer-ence 22). Thus it is assumed that the enzyme

nicks double-stranded portions of the CLC

fragments in addition to digesting single-stranded regions.

Isolation of CLC fragments. As stated above, about5%of the input DNA isrecovered

asCLCsegments after restriction enzyme

diges-tion. However these enzymes cleave vaccinia

DNA at many sites and produce so many segments that it is difficult to tell when the

reaction has gone to completion. Also, to pre-pare microgram quantities of CLC segments

routinely, relativelylarge amountsofrestriction enzyme mustbe used.To conservethesupplyof enzyme an alternative procedure was adopted. Sheared fragments were denatured, neutral-ized, and passed through hydroxyapatite. Du-plex fragmentswererecovered from the column, and the cycle was

repeated

usinga second

col-umn of

hydroxyapatite

with buffers

containing

50%formamideasdescribed above. The double-stranded fragments obtained from the second columnwereusedas asubstratefor arestriction

enzyme. About 3 to5% ofthe original sheared DNA was recovered with the elution of the

double-stranded fragmentsfromthe second

col-umn. The size distribution ofthese fragments afterelectrophoresis through a1.6% agarose gel isshowninFig. 2. The peak of thisdistribution

contains fragments which move more slowly

than the largest fragments obtained after

re-striction enzyme digestion. Figure 3 contains

electrophoretic profiles of restriction enzyme

digests of CLC fragments obtained as duplex DNA from the second hydroxyapatite column.

Thetop frameshowsthe pattern after digestion of

[3H

]thymidine

DNA fragmentsbyHpa I.The

middle frame is thepattern after digestion with

Hpa II and the bottom frame is the result

FRACTIONNUMBER

FIG. 2. Electrophoresis of sheared, duplex vaccinia DNA which is recovered from hydroxyapatite.

Sheared 3H-labeled DNA wasalkali-denatured, neu-tralized, passed through hydroxyapatite, and re-coveredasduplexDNA. Theprocedurewasrepeated, using bufferscontaining50% formamideasdescribed, and the duplex fraction from the hydroxyapatite,

called CLC fragments,wasdialyzedandanalyzed by electrophoresis through 1.6%agarosegels.In allgels

migration was toward the left (anode). Thegel was

sliced, digestedwith

HCl,

and countedasdescribed.

obtained after digestion with HpaIandHpa

II.

These types ofpatterns were consistently

pro-duced after

digestion

ofthree different

prepara-tionsof [3HHthymidine vaccinia DNA.TheHpa

I pattern always showed several large peaks

near the top of the gel (fractions 62-75), the largestofwhichalways movedmoreslowly than thematerialproviding the slowest moving peak

(fraction 65) found for the Hpa II digests. Two other peaks in the Hpa I pattern (fraction 46

and 36) show segments that migrate at faster

rates. However, in other gel patterns, the peak thatcorresponds totheonefoundatfraction36 is much less pronounced. The Hpa II digests always showed four major peaks which were

well separated in 1.6% agarose gels. The back-ground levelin some ofthese gelswashigh, and the definition of individual peaks was not as

good as that which would be found if the

original DNA substrate had beena

homogene-ous collection ofmolecules. Shearing the DNA

breakssomeofthepotentiallyuniform(specific size) segments which would be

produced

by

restriction enzyme digestion into fragments of random length.Afterdigestiononeend of these fragments isformedbyenzymatic cleavage and

the other end already existsdue to shear frag-mentation. Additional variation inthepatterns was probably due to radiation damage of the

DNA samples which had specific activities of

I

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0

x

0.

9 8 6 4 2

10 20 30 40 50 60 70 80 90 100

FRACTIONNUMBER

FIG. 3. Electrophoresis ofDNAsegmentsproduced by restriction enzyme digests of 3H-labeled CLC

fragments. (A) The fragments were digested with enzyme Hpa Ias described and then analyzed by

electrophoresis through 1.6%oagarosegels. (B)

Diges-tion withenzymeHpaII.(C) Digestionwithenzymes

Hpa I and HpaII.ThegelsweretreatedasinFig.2.

105counts/minperjgormore.Thetwo denatu-ration steps whichwereused toselect theCLC fragments allowed separation ofpartsofnicked fragments of DNA. Since single-stranded tails have little effect on the affinity of duplex

regions for hydroxyapatite (20), CLC segments with tails of differentlengths will be retained by the column and they will migrate through agarose gels atdifferentrates. The susceptibil-ity of 3H-labeled DNAtoradiation damage has

been cited(19). Radioautographs of electropho-retic separations of digests of "P-labeled DNA fragments provided better profiles. Figure 4 shows thatthe patterns obtained afterdigestion of 32P-labeled DNA fragments by Hpa I and

Hpa II agree with the major features of the profiles shown in Fig. 3, but that it is easier to

visualize the major bands. The Hpa I pattern has the three majorbands at thetop of the gel and one more easily distinguishable band,

la-beled E in Fig. 4A, as well as several minor bands. Bands A, B, C, and E in Fig. 4A correspondto thepeaks atfractions 74, 66, 62, and 46 in Fig. 3A. Bands D and F are always much less intense than bandE in the

radioauto-grams and I regard them as minor bands. The

Hpa IIprofile shows that the peak representing thelargest DNAsegmentsinFig. 3B, atfraction

65, can be resolved into 4 bands, designated

a-ay. This profile is very reproducible andthe

four bands are always present. It is not known

whetheranyofthese bands ariseasdegradation

products from any of the other bands. The patternremainsconsistent when thesame

sam-ple of 32P-labeled fragments is digested by Hpa II upto a week after the original digestion and

electrophoresis. Another one ofthe four peaks

shown inFig. 3B, atfraction 43, corresponds to twobandsdesignated cl andc2inFig. 4B. This

is slightly more variable than the presence of

the fourbandsnearerthetopofthe gel. Bands b and dcorrespondtothe peaks foundatfractions 50 and 30 inFig. 3B. Thesetypesofpatternsare

alsorepeated when CLC fragmentsareprepared

fromunlabeled DNA and the digestedsegments

areseparated by electrophoresis. Ethidium

bro-mide stained gels are shown in Fig. 5. They

confirm the patterns shown for previous gels. Relatively large amounts of vaccinia DNA (50 to100

Ag)

havetobepreparedtopurifyenough CLC fragments for digestion when the ei-thidium staining procedure is used. The back-ground staining due to the presence ofbroken

fragments of DNA limits the sensitivity of this procedure. The right hand gel in Fig. 5 was

prepared from 32P-labeled fragments which

were stored for 10 days at 0C. The pattern, although recognizable, has deteriorated because ofradiationdamage.

Fractionation of DNA from gel slices. It

wasgenerallymostusefultoprepare32P-labeled

CLCfragments. After digestion and electropho-resis the gel can be sliced and the radioactive

profile is determined by counting the slices in

dilute buffer. DNA was recovered from the

individualslices by electrophoresis and discrete

segments could be tested for the CLC property

by alkali denaturation, neutralization, and hy-C

17,

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Ir

a

₂

a--4

9.

a-

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4_ b

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A

B

FIG. 4. Electrophoresis of32P-labeledDNAsegmentsproduced by HpaIdigestion of CLC fragments.The DNAwasdigestedandanalyzed byelectrophoresis. Thegelwasthenslicedlongitudinallyintotwoslices,anda

radioautographwasprepared bytheprocedure ofFairbanksetal. (7). Electrophoretic migration is towardsthe

bottom (anode). The bands of the radioautogram have been designated by large letters. Gel segments

corresponding tothese bandsinsimilargelswereexcisedand the '2P-labeled DNA segmentswereelated by electrophoresisasdescribed.(B)Electrophoresis of "2P-labeled DNAsegmentsproduced by Hpa IIdigestionof

CLCfragments. Theprocedureisthatdescribed in (A). The fourcloselyspaced DNA bandsnearthe top ofthe

gelhavebeendesignateda,-a4,witha,beingtheband nearest the cathode.

droxyapatite fractionation. When this was

done, however, no one gel bandwas associated

with segments which gave 100% ofthe counts

eluting from hydroxyapatite as duplex DNA.

Thiswasexpectedhowever becauseofradiation

damage and the presence of random sheared fragments. In addition, the peaks obtained by slicing the gel were probably not pure, so that

individual segments were contaminated by

other segments. Despite the difficulties in-volved itwas found thatbands Aand B in the

Hpa I digest shown in Fig. 4 contained a

substantial fraction ofDNAwhichappearedto besnap-back DNA. Bandsaand d intheHpa II

digestof Fig. 4 alsoappeared tocontain snap-backDNA. The relative amountof DNAwhich

A

B

C

* D

J. VIROL.

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[image:6.491.58.449.65.494.2]

FIG. 5. Electrophoresis of unlabeled DNA

seg-mentsafter digestion of CLC fragmentswith restric-tion enzymes. Thegels were stained with ethidium

bromideasdescribed. ThegelontheleftshowsHpaI

digestion products. The middle gel shows Hpa II

digestion products.ThegelontherightshowsHpaII

digestion products of "2P-labeled CLC fragments which had been stored at 0C for 10days prior to

digestion. Electrophoretic migration is toward the

bottom (anode).

is retained by hydroxyapatite after removing single-strand chains is given in Table 1. An

attempt was made to split the a band of the

Hpa II digest byusingalonger time for

electro-phoresis and dividing the increased number of discs cut from across this band into two frac-tions. Both fractions showed some segments eluting as duplex segments. After digestion of 'C-labeled CLCfragments the four bands in a

could beresolvedinethidium-stainedgels when viewed underUVlight. However, previous

expe-rience with other DNA samples has shown that this exposure to UV light (mineralight short

wave, model R-51) degrades the DNA and it is

also knowntocausecross-linking (15). For these

reasons no attempt was made to use this tech-nique toisolate theindividualbands, which are

shown in Fig. 5.

Denaturation of DNA segments produced

by a restriction enzyme. Another approach, used initially, wasto digest the CLCfragments

with a restriction enzyme and then alkali dena-ture the entire reaction mix and apply it

di-rectly to agel. Those unique segments produced

by therestriction enzyme which do not contain the covalent link connecting complementary

strands remain single-stranded after

denatura-tion. Any random size duplex fragments

with-out the covalent link also remain single

stranded after denaturation. In addition the unique segments which contain the covalent linkmayby nicked, andpart of thechain which isbrokenatthe nick willbecome single stranded. Figure 6B shows that denaturation produces single-stranded DNA, which migrates more

quickly than duplex DNA (Fig. 6A). Some of

this isdue to the migration ofdegraded single strands, although intact single-stranded DNA moves fasterthan double-stranded DNA ofthe

same length in these gels. Those restriction enzyme segments which retain the covalent linkage should move as double-stranded

mole-culesaftertheyenterthegel and migrateinthe pH7.9buffer. The lowerpanel shows the result

of denaturation, neutralization, and passage

throughhydroxyapatite. The duplexDNA from

the

hydroxyapatite

gives the electrophoretic

profile shown in Fig. 6C. Most ofthe smaller

(single-strand)

DNA is removed by this

proce-dure, leaving only the DNA initially seennear

the top of the gel, with a shoulder on the low-molecular-weight side. Definition of indi-vidual peaks inpanel C isreduced by the high background. This indicates the presence of some degraded CLC segments, which are only partial

duplexes

that have been retained

by

the hydroxyapatite column. If the CLC segments are in the fractions withpeaks at 80 and 74in

panel

A, then

they

would

provide

a source of

partially

degraded

segmentswhichare

responsi-ble for the background in panel C. This is not

surprising since these experiments were

per-formed with [3H

]thymidine-labeled

DNA

frag-ments which are particularly susceptible to radiationdamage. Figure 7 shows the same type

experiment except that [3H

Ithymidine-labeled

fragments were digested with Hpa II. The

middle panel shows that denaturation leaves one high-molecular-weight segment which movesataboutthe same rateasthemajor peak

in panel A. The bottom panel demonstrates

again that hydroxyapatite treatment removes

thesingle strands, leavingthe major peak found

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TABLE 1. Fraction of recovered CLC segments retained by hydroxyapatitea

Total counts 0.4M phosphate counts/0.08 M + 0.18 Mphosphate counts Sample

Expt 1 Expt 2 Expt 3 Expt1 Expt 2 Expt3

Hpa I

A 1,399 883 1.02 0.71

B 1,366 621 0.62 0.51

C 1,160 471 0.18 0.14

E 509 231 0.16 0.09

Hpa II

a,-a4 2,481 2,326 0.92 0.74

b 1,148 863 0.13 0.14

c-cC2 900 527 0.09 0.15

d 999 850 1.55 0.65

a,-a2 933 0.71

a3-a4 666 0.77

a32P-labeled CLC segments, produced by digestion with restriction enzymes Hpa I and Hpa II, were

separatedby electrophoresis throughagarosegels. Gelsweresliced into 1-mmdisks,and theradioactivitywas measuredby the Cerenkov method. Selected gel fractions, corresponding to those bands which are labeledin

Fig. 4A andB, were placed in electrophoresis buffer and the DNA segmentswererecovered byelectrophoresis intodialysis sacs. The DNA was denaturedin0.3NNaOHat 37C for10minand then neutralized withHCl anddiluted with bufferto afinal concentration of80mM sodiumphosphate, pH6.8,with 50% formamide. The

sampleswereloadedon0.25ml ofhydroxyapatite columns and eluted with 0.18 M phosphate-50% formamide followed by0.4Mphosphate-50% formamidebuffer, pH 6.8, at 24C.Radioactivitywascounted in the triton fluorgiveninthetext.Thespecific activityofthe DNA inexperiment1 was 1.1 x 101counts/minperggand in experiments2and3 was 2.1 x 101counts/minper fig. Adigest of this latter DNAby Hpa II was separatedby electrophoresisand the mainabandwascut intopiecesthoughttocorrespondapproximatelytoa,plusa,and

a,plus a4.

nearthe top of thegel and a smaller peak on the

low-molecular-weight end. The data presented in Fig. 6 and 7 suggest that the larger DNA segments containspecieswhich haveacovalent link connecting complementary chains. The HpaIIdigest also showsa peak

(about

fraction

29, Fig. 7 panel C) with DNA which runs

slightly faster than thesegments producing the peak at fraction 34 in panel A. When native

C-labeled T7 DNA was denatured and

neu-tralized,

it was eluted from hydroxyapatite in

the 0.18 M phosphate buffer. Since single

chains of T7 DNA are longer than thesheared vaccinia DNA strands, it is clear that the

hy-droxyapatiteis not retaininglong single chains

ofvaccinia DNA.

Size of the DNA segments. The size of the enzyme segments derived from the CLC frag-ments has beenestimated by comparison with

an Hpa I digest of T7 DNA which was run

simultaneously, usingidentical conditions.The

molecular weights of the T7 fragments have been determined by M. Simon and F. W. Studier (personal communication) who kindly provided their data. This indicated that bands A, B, C, D, E, and F of the Hpa I digest shown

in Fig. 4A had DNA segments with molecular weights which wereabout 3.2 x

106,

2.2 x 106,

1.9 x 106, 1.35 x 106, 0.9 x 106 and 0.56 x 106.

The Hpa II digestof Fig. 4B has bands a,b, c, and dwithDNAwith molecularweights of 2.1 x 106. Although these sizes are onlyestimates, it seemscertain

that

the relative sizes of themajor segments are

reliable

sincethe patterns are

re-producible despite the high background levels.

Thus fragment A ofthe Hpa I digest is larger

thanfragment A of the Hpa II digest. It is not

likely that the presence of a small

single-stranded end region would cause abnormal

migration of a segment which ispredominantly

duplex.

Digestion of CLC fragments

by

single-strand-specific nucleases.A

single-strand loop

in a CLC fragment should be digested by single-strand-specific nucleases. Very small

loops would be resistant to such nucleases but

they should expand with increasing tempera-tureandbemoresusceptibletodigestion. Since both the

S,

(1) and the vaccinia nuclease

(17)

are active at 60C, they have been used by

others to determine the nature of the covalent linksconnectingcomplementarychains (8, 21).

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[image:8.491.59.456.81.262.2]

0

x 10

_M 5

3

2

1

0

20 40 60 80 100

FRACTION NUMBER

FIG. 6. Electrophoresis of denatured 3H-labeled

DNA segmentsproduced by HpaIdigestion of CLC fragments. (A) Electrophoresis of native digestion products. (B) A 50-,ul digestion reaction was

termi-nated bythe additionofEDTA andSarkosyltofinal

concentrations of30mMand 0.5%, and then NaOH

was added to 0.2 N. After 10 min at 37C the

0

x

CL

y

[image:9.491.40.439.55.588.2]

FRACTION NUMBER

FIG. 7. Electrophoresis of denatured 'H-labeled

DNAsegmentsproduced by HpaIIdigestion ofCLC

fragments. (A) Electrophoresis of native digestion products. (B)A50-,gldigestionwas treatedasinFig.

6B. (C)A 50-uldigestwastreatedasinFig. 6C.

denatured digest wasapplied directly toan agarose gelfor electrophoresis. (C)Theprocedureused in(B) wasfollowed but the alkalinedigestwas neutralized andpassed through hydroxyapatite. TheduplexDNA retained by thehydroxyapatite was dialyzedagainst buffer H and analyzed by electrophoresis. All gels

were cutandanalyzed as described in the legendto

Fig.2. 17,

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[image:9.491.47.237.68.599.2]

Breaking the covalent link converts the duplex to a denaturable form which is eluted from

hydroxyapatite by 0.18 M sodium phosphate. Results ofthe hydroxyapatiteassay aregivenin

Table 2 as the relative amount of DNAwhich

remains double stranded after enzyme treat-ment and denaturation. As anticipated, en-zymaticdigestion eliminates duplex DNA and

is more effective at temperatures above 37C. Thisagreeswith theresultreportedby Geshelin andBerns (8), who used the vacciniaenzyme to

digest native DNA to produce molecules with

openends.Theiranalysis of their sedimentation

data indicated that there were no

single-strandedregions in nativevaccinia DNA except at theends of the molecule. TheSlendonuclease

fromA.oryzaealsodigestsCLCfragments when

[image:10.491.53.249.312.382.2]

tested by the same criterion used for the vac-cinia nuclease. The digestion buffer contained

TABLE 2. Effect of nucleases on spontaneous

renaturationofCLC fragments

Enzyme Time Temp DNArecoveredin

Enzyme (min) (C) 0.4 M phosphate(%)

S 1 30 37 32.0

S1 30 55 4.9

Vaccinia 60 37 77.3

Vaccinia 60 55 28.5

a

0.27

,g

of

[14C

]thymidine-labeled

CLC fragments

(19,000 counts/min per ,ug) were incubated at the temperature and timeindicated inareaction volume of0.12ml in buffersdescribedinthe text, with and without enzyme. The

S1

reactionbuffer contained 270 mMNaCiand 0.2 U(22) of enzyme and the vaccinia nucleasecontained 10 Ill of a crude preparation. Each reactionwas terminatedby addition of SDS and then

chilling the tube. Afteradjusting the Si tubes to 5

mM EDTA all samples were denatured in 0.3 N NaOH at 37C for 10 min. The sampleswere neutral-ized with HCl and diluted with buffer to a final concentration of80mM sodiumphosphate, pH6.8, in 0.81ml.Thesampleswereloadedon0.25-ml

hydrox-yapatite columns and eluted by 0.18 M phosphate

followed by 0.4 M phosphate buffer, pH 6.8.

Radio-activefractionswerecountedinthe triton fluor. The

results aregiven as the ratio of the number ofcounts

elutedin0.4Mphosphateintheenzyme-treated

sam-ple to the number of counts in the 0.4 M

phos-phate fractioninthe controlsample without enzyme, expressedaspercentage.84% ofthe totalcounts inthe controlsample elutedinthe0.4Mphosphatefraction. Denatured[3HIthymidine-labeledHeLacell DNAwas 96% acid soluble after digestion with

S,.

The same

DNA was 70% acid soluble aftertreatment with the

vaccinia nuclease (see text for details). 97% of the radioactivity of these controls was eluted from

hy-droxyapatite columnsby0.18 Mphosphatebuffer be-foreenzyme treatment andall the radioactivitywas

eluted by this buffer aftertreatment.

270mM NaClsince Vogt (22) has reported that

the specificity of purified

S,

preparations for

single-stranded DNA is greatly increased at

high salt concentrations. In 270 mM NaCl at 55C, theamountof enzymeusedtodigest

CLC

fragments inthe reaction given in Table2will

degrade over 95% of sheared, denatured, 3H-labeled HeLa cell DNA(2.5

,ug)

toacid-soluble materialin 30min.With thesameconditionsat 37C the S enzyme willdigest 76%ofthisDNA to acid-soluble material. The amount of

vac-cinianuclease usedintheexperiment forTable

2, in the Tris-EDTA buffer, causes 70% ofthe denatured HeLa cell DNA (1.1 ,g) to become

acid soluble in 60 min at 55 C. At 37 C 58% of

this DNA becomes acid soluble after60 min of digestion.

One would expect these enzymes to digest single-strand tails on degraded, hook-shaped CLC fragments. If the covalent link is at the

extremeendofthefragment, then it ispossible that digestion of the tail might leave a very

small hairpinstructure that hasasmall, intact

single-stranded loop. This loop would resist

digestion because it was extremely small,

per-haps consisting of one or twounpaired

nucleo-tides. The small duplex fragment which

re-mained would not be retained by hydroxyapa-tite. Wilson and Thomas (20) estimate that

duplex

chains withamolecularweight less than

18,000 will be eluted from hydroxyapatite by 0.14M phosphate buffer. It mightappearthen that digestion had split the link connecting strands when the actual resultwasthe produc-tion ofaveryshortduplex CLC fragment.

How-ever, S,

digestion

of CLC fragments reduces theamountofduplexDNAretainedby hydrox-yapatite to a

background

level of about 5%.

This implies that most ofthe CLC fragments

would have the unusual hooked-shaped struc-ture described above, which is unlikely. The most plausible explanation oftheresultsofthe

S,

digestion

is that this amount of enzyme is

splitting all the single-stranded regions,

includ-ing those that contain the covalent link.

Eighty-nine percent of the CLC fragments

(not denatured) remain acid insoluble after

S,

treatment. If these fragments, inbuffer H, are

heldat 100Cfor 10min and then cooled to 0C anddigestedwith 0.2 U ofS,at 55 Cfor 30min, then 39% of the fragments remain acid

insolu-ble, although this amount of enzyme will de-grade over 95% of five times this amount of

single-stranded DNA toacid-soluble material.

DISCUSSION

Hydroxyapatite

chromatography

has been

used to isolate a rapidly renaturing fraction of

J.- VIROL.

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thesheared vaccinia DNAmolecule. Restriction

enzyme digests of this fraction yield a set of DNA segments which have been separated by

gel electrophoresis. The main features of the

electrophoretic profiles are reproducible and

characteristic of the enzymeusedfordigestion, indicating that the original substrate contained

aset offragments that had a specific nucleotide sequence. It is assumed that the ends of this DNA molecule which have a covalent link con-nectingcomplementary chains (8)arethesource ofthe specific fragments. Berns andSilverman

(3) noted the possibility that such cross-linked DNA was biologically inactive, and that infec-tious virus containeda small percentage of the

DNA which was free of links. IfCLCDNA has thesameduplex basesequence asthepresumed

free DNA plus a small number ofbases in the links ateach endofthemolecule, then

restric-tion enzyme digests of either DNA should produce nearly identical sets of segments. If

single-stranded loops link theendsthey mustbe less than 50 nucleotides long, since electron micrographs of intact vaccinia DNA do not

revealany single-stranded regions (K. I. Berns, personal communication).

The DNA segments which are produced

by

restriction enzyme cleavage are those which include and are adjacent to the covalently linked region. If the target sequence for the

enzyme is located near the link, then the

segment containing the link could migrate

through the gel during electrophoresis and be lost, as is the case with any small hairpin

structure. Conversely, ifthetarget sequence is

located at a great distancefrom the link, then

the

duplex

chain would be broken at some

random position

between

the link and thetarget sequence

during

the shearing process. Since shearing reduces the molecular size below 8 x 106daltons these fragments wouldnotbe large

enough to migrate as a single band at some

slow, limitingratethrough theagarosegel (11). Also, theelectrophoretic profiles obtained with 1.6% agarose gels show the same pattern as

those obtained with 1% agarose gels (not

shown), where larger DNA segments are more

easily separated. These arguments, plus the

continuous natureoftheelectrophoretic distri-bution ofnondigested sheared fragments (Fig.

2), indicate that the largest DNA segments

produced by restriction enzyme digestion are

specific segments. Random sheared fragments

which have not been removed by the second

hydroxyapatite step do contaminate these gels

but theyarespread through the gel.

The most abundant restriction enzyme seg-ments should be those which contain the link,

and theabundanceofadjacent segments should

decrease with their distance from the link. I

expected two bands in each gel to contain the linked end segments and this seems to besofor the Hpa I digest. The HpaII digest, however, has two regions ofthe gel with a total of five

bands whichcontainsnap-backDNAsegments.

The fourbands

(a-a,)

ofFig. 4could be several forms or variations of one or more linked

seg-ments.Breakingthe covalentlinkmightcause a

conformational change that alters themigration rate. Radioautogramsofgelsafter electrophore-sis of alkali-denatured 32P-labeled DNA

seg-ments produced by Hpa II digestion show a

single band in a region corresponding to the a region. These gels have a considerably higher background level than do gels of

double-stranded DNA so it is harder to distinguish multiple peaks. The possibility remains that

some ofthe a segments are adjacent to linked

end segments and migrate at nearly the same rate. Ifthearegioncontainsbothend segments

then band d ofFig. 4B may be either aspecific degradation product ofthe a segmentsor may come from the interior region ofthe molecule. Band d isalways present as asharp peakafter

electrophoresis of Hpa IIdigests andcontains 20 to 25% of the counts in band a, so it does not seem tobegenerated from banda.One possible

structure however is a predominantly single-stranded molecule which is

generated by

a

breaknearthe covalent linkinthe endsegment onthe strandcomplementarytothelong single strand. This

hooked-shaped

structure would have enough double-stranded length to allow retentionby hydroxyapatite but would migrate as asingle chain.

Vaccinia doesnothave small repetitive DNA

sequenceswhich wouldrapidly reanneal due to

theirhigh concentration (3). Since thereare no

fixed internal cross-links (8), duplex DNA

formedby rapid reannealing would havetoarise

from single chains folding backon themselves. This would be the case if single chains

con-tained inverted repetitions. Ofcourse, the ends of the vaccinia DNA molecule could be de-scribed as a single chain with a turn-around. However there may be other, internal regions, where the duplex DNA is palindromic and consists ofsingle chains which can

self-associ-ate. Single strands ofeukaryotic DNA which

form duplex molecules with a small

single-stranded hairpinturnhavebeen described(21).

My hydroxyapatite procedure would retain

partial duplex structures which have large or

smallsingle-stranded loops(20). Iftheloopwas very large then this fragment would probably

nothaveenough duplex structure to contain the

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restriction enzyme target sequence. If these putative hairpins contain long duplex regions and a moderately large single-stranded loop, then the loop should be easily digested by the

single-strand-specific

nuclease from vaccinia.

Theevidence presented here agrees with earlier results (8) and indicates that the single-stranded turn-around region is small since it is

not readily digested by the vaccinia nuclease. Finally, if the internal hairpins contain long duplex regions and a small turn-around, then there is very little physical difference between them and the ends of vaccinia DNA. These

internal structures would also define specific regions of thevaccinia genome.

The model forvaccinia DNA (8) allows more

than one link at each end of the molecule, but I

can see no reason for having a single-strand hairpin and aninternal cross-link next to each other. A clover leaf arrangement at the end would have several

turn-arounds

butthere is no indication of extensive clover leafbranches in

electron micrographsofvaccinia DNA. The procedurepresented inthis paper shows how restriction enzymes may be usedto cleave limited regions of large viral DNAs. CLC seg-ments maybeobtained

by

digestionofvaccinia

DNAwith different restriction enzymes. These

segments can be

redigested

with the H.

parainfluenzae

enzymestogivenew

electropho-reticpatternsthatcan be compared with those presented here. This

technique

will

identify

new

segmentsthatareassociated withCLC

regions.

Although no genetic functions may be associ-ated with the CLC

fragments,

they

mayserveas

referencesequencesthat will

help

toestablisha

physical map of vaccinia DNA.

ACKNOWLEDGMENTS

This work was initiated after discussions with B. Moss who provided manyhelpfulsuggestionsduringthisstudyaswell asviral stocksand cells. I also thank NormanCooperforhis assistance and M.Simon and F. W. Studier for their dataon restrictionenzymedigestsofT7DNA. I also appreciate the gift ofvaccinia nuclease from P.Geshelin and K. Berns.

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