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Vaccinia virus encodes a polypeptide with DNA ligase activity

Citation for published version:

Kerr, SM & Smith, GL 1989, 'Vaccinia virus encodes a polypeptide with DNA ligase activity', Nucleic Acids

Research, vol. 17, no. 22, pp. 9039-50. https://doi.org/10.1093/nar/17.22.9039

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10.1093/nar/17.22.9039

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Vaccinia virus encodes a polypeptide with DNA ligase activity

Shona M.Kerr and Geoffrey L.Smith*

Department of Pathology, University ofCambridge, TennisCourt Road, CambridgeCB2 lQP, UK

Received August25, 1989; Revised and Accepted September 25, 1989

ABSTRACT

Vaccinia virus geneSalF 15R potentially encodes a polypeptide of 63 kD which shares30% amino acid identity with S. pombeand S. cerevisiae DNA ligases. DNA ligase proteins can be identified

by incubation with _-(Q2P)ATP, resulting in theformationof a covalent DNA ligase-AMP adduct, anintermediate intheenzymereaction. Anovel radio-labelled polypeptide of approximately61 kD appearsin extracts from vaccinia virus infectedcells afterincubation with _-(32P)ATP.This protein

ispresentthroughoutinfectionand is a DNA ligase as theradioactivity is discharged in the presence of either DNA substrate orpyrophosphate. DNAligaseassays show an increase in enzyme activity incell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion

proteinof,B-galactosidase and aportion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts ofvaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNAligase-AMPadduct, thusprovingthat the observed DNAligase is encoded by SalF 15R.

INTRODUCTION

Vaccinia virus, the prototypical member of the poxvirus family, is a large DNA-containing

virus which replicatesinthe cytoplasm of the host cell. The lineardouble-strandedgenome ofapproximately 185,000 base pairs has the potential to encode at least 200 proteins

(reviewedin 1). Althoughthenucleus isrequired for formation of maturevirus particles,

vaccinia virus DNA replication can occur in enucleated cells (reviewed in 2). The

cytoplasmic siteofreplication requiresthat vaccinia virus encodes many enzymes andprotein

factors necessary for DNAsynthesis,thusprovidingaunique system for molecular analysis ofthe functionsassociated with virusreplicationinamammalian cell. Identification and

analysis of such virus-encoded proteins may also provide valuable information on the

enzymology ofreplication ofcellular DNA.

Agrowing numberof virus genesexpressing proteinsinvolved in DNAreplicationhave

beenmappedonthe vacciniavirus genome andsequenced. ThiscomprisesDNApolymerase (3),DNAtopoisomerase (4)andaDNA-dependentATPase(5). The enzymesofprecursor

biosynthetic pathways, thymidine kinase (6,7) thymidylate kinase (8) and the large and small subunits of ribonucleotide reductase(9,10)have also beencharacterisedinthisway. In addition, a genehas been identified inour laboratory which has extensive sequence

homologytoyeast DNAligases (11). Anincreasein DNAligase activityhas beenreported

invaccinia virus infected cells (12,13), althoughgeneticevidence of virusoriginwasnot

obtained. Otherreplicativeenzymes for whichanincreaseinbiochemicalactivityhas been

reported include single-strand specific nucleases (14) and nicking-joining enzyme (15).

DNAligasesjoin strandbreaks in DNAbycatalysing theformation ofphosphodiester bonds, requiring a divalent metal ion and either ATP or NADas cofactor. Studies on

conditional lethal mutants of yeast, E. coli and

bacteriophage

T4 have established the involvementofDNAligaseinthereplication, repairandrecombination ofDNA(reviewed

in 16). The basicmechanism by whichDNAligase sealsaDNAstrandbreak isthesame

in mammalian cells as in bacteria. A DNA ligase-AMP covalent complex is formed,

followed by transfer of theAMPmoietytothe 5' terminus of the chain

break,

togenerate

a covaent DNA-AMP reaction intermediate prior to closure of the strand interruption.

A substantial amountofbiochemical information on mammalian DNA

ligases

has been

obtained (reviewed in 17). Two

immunologically

unrelated

proteins

with DNA ligase

activity, DNAligases I andII, have been

described,

and may be

responsible

for DNA

replication and repair, respectively. The molecular defectin thehuman genetic disease

Bloom's syndrome istheresult of adeficiency in DNA ligase I (18,19). However, the cloning andsequencingofaDNA

ligase

genefroma

higher

eukaryote

havenotyetbeen reported.

The SalI FfragmentofVacciniavirusDNAwassequenced andanopen

reading

frame, SalF 15R,withthepotentialtoencodea

polypeptide

of63kDwasidentified. Computer-assisted database searches revealed extensive amino acidsequence

homology

withtheDNA

ligase genes of the yeasts S. cerevisiae and S. pombe

(11).

Here, biochemical and

immunological analyses ofDNAligaseinvaccinia virus-infectedcellsare

presented.

These

experiments demonstrate that the

polypeptide

encoded

by

SalF 15R isa DNA

ligase,

a

resultwhichmayleadto abetterunderstandingofthe mechanismsof vacciniavirusDNA

replication and recombination. MATERIALS ANDMETHODS Cells and Virus

Africangreen moneykidney (CV-1) cellsand human TK- 143cellsweremaintainedin

Glasgow modified Eagle's medium (GMEM) containing 10% foetal bovineserum.Vaccinia virus strain WR was grown in BHK cells andtitrated on CV-1 cells (20).

Chromatography

andBuffers

DE52 ion-exchange cellulose (Whatman) was prepared according to themanufacturers

instructions. P11 phosphocellulose (Whatman)wasprepared asdescribed (21). All sodium

chloridebuffers were at pH 7.5 and contained 50 mM

Tris-HCl,

1 mM EDTA and 10 mM 2-mercaptoethanol.

Extraction

ofDNA ligase

All operations werecarried out at4°C. CV-1 cells in monolayer culture, 1 x107 cells

per sample either mock infected or infected with 100

p.f.u./cell

vaccinia virus, were

collectedby scrapingwith a rubberpoliceman and

centrifugation,

thenwashed in phosphate-freecell culturemedium. Cells wereharvestedat thetimespost-infection (p.i.) indicated

in the presence or absence of cytosine arabinoside (40

itg/ml).

The

cell

pellet was

resuspended in 1 ml ice-coldextraction buffer(0.1 M NaCl, 50 mM

Tris-HCl,

pH 7.5,

10mM2-mercapto-ethanol, 1 mMEDTA, 23

,ug/ml

aprotinin, 0.5

Itg/ml

each ofpepstatin,

leupeptinand chymostatin) and disrupted in a glass hand homogeniser, asdescribed in

(18).Insomeexperiments, DNAligase was partially purified by a two column procedure.

TheDouncehomogenatewascentrifugedtoremove cell debris then the

supernatant

made

0.4 M NaCl and passedthrough a DE52 column equilibrated with 0.4 M NaCl buffer.

Fractionscontainingprotein (measured by the Bradford assay (22)) were pooled and dialysed

against 50 mMNaCl buffer. Thedialysed sampleswereloadedontoaphosphocellulose column, equilibratedwith50 mM NaCl buffer. The column waswashedwiththree column

1 2

3 4 5

-200

-

92.5

-

69

-46

-30

Fig. 1: Identification of vaccinia virusDNAligase protein. Crudeextracts werepreparedfrom mockinfected

orvaccinia virus infected(100pfu/cell) CVI cellsby Douncehomogenisationin100mMNaClbufferasdescribed inMaterials and Methods. Vaccinia virus infectedearly (lane 2),late(lane 3)ormock infected(lane 1) CVl cellextractsandpurifiedcalfthymusDNAligaseI(agiftfrom T. Lindahl) (lane5)wereincubated with

a-(32P)ATP(Methods). Reactions were terminatedby trichloroacetic acid andcovalently labelled polypeptides

analysed by SDS PAGE ona 12.5% gel.

volumes ofthesamebuffer, thenenzymeactivityelutedwith 1 MNaCl buffer. The resulting fractionsweredialysed against 50mM NaClbuffer containing 50% glyceroland stored at -20°C

prior

toassaysofDNAligaseactivity, formation ofDNA

ligase-AMP

adducts,

andimmune-precipitation. DNAligasewasalsoanalysedincrudeextractswithout column fractionation. Inthiscase,cellswereharvestedasabove and afterDouncehomogenisation

andcentrifugationat 10Kfor 20 minutesat4C the

supernatant

andpellet fractionswere

assayed directly.

AssayofDNALigase Activity

DNAligase was assayedusinga polydA. oligo

5'-(32P)-dT

substratebyamodification

of theprotocoldescribed in(23). Incubationswere at

16WC

for15 minutes and the reaction wasstoppedand substrate denaturedbyheatingto

90WC

for 10 minutes. The

samples

were

extracted with phenol:chloroform,

precipitated

in ethanol then

resuspended

in water.

Formamide dye mix was added and the samples electrophoresed on a 12%

acrylamide

Formation

of

DNA ligase-AMP adduct

DNA

ligase

was radioactively labelled by incubating cell extracts with cx-(2P) ATP followed by TCA precipitation essentially as described in (25), except that incubations

were atroomtemperature for30 minutes. DNA ligase-AMP adductswere visualisedby

12.5% SDS

polyacrylamide

gel electrophoresis and autoradiography ofthe dried gel.

Antisera and Immune-Precipitations

A

plasmid, pSK12, expressing

a(3-galactosidase/SalF 15R fusionproteinwasconstructed

by cloning

the 550 nucleotide

BglII

fragmentfrom SalF 15R DNA intotheBamHIsite of

pEX3 (26).

The

BglIl

sitesare atpositions 516 and1066 in thefragmentofSalFpresented

in

(11).

Oninduction of bacterial

culures

containing pSK12, a

(3-galactosidase

fusion protein

ofthe predicted Mr was detected by SDS-PAGE and staining with Coomassie brilliant blue. After

gel purification

and

electroelution,

250 ,galiquots of thisprotein were used

to immunise a rabbit

resulting

in the

production

of pEX LIG antiserum.

TK- cells infected at 30 p.f.u./cell with either wild-type vaccinia virus, or mock

infected,

were incubated in methionine-free medium from 1 to 1.5h post infection, then radiolabelledby incubation in100

MCi/ml

of

(35S)-methionine

in MEM

witiout

methionine for 2.5h. Immuneprecipitation was asdescribed in (27). The cells were lysed in RIPA

buffer

(50

mMTris-HCl pH7.2, 150mMNaCl,

1%

sodium deoxycholate, 0.1% SDS, 1% Triton

X-l00) plus

5

,ug/nml

DNase and 1 mMphenylmethylsuphonyl fluoride for 15 minonice. Lysateswereclarified by centrifugation at 10,000 r.p.m. for 15 min at 4°C.

Supernatants

wereincubated with rabbitserafor30 minatroomtemperature and immune

complexes precipitated by

incubation with 50

gig

ofa 50% suspension of Protein

A-Sepharose

for 2h at4°C. The beadswere washed twice with NP40 buffer and once with 2Murea, 0.4MLiCl,10 mMTris-HClpH8.0, then SDS sample buffer added and proteins

analysed by

SDS-PAGE.

In some experiments, unlabelled cell lysate fractions containing DNA ligase were incubated with

aI-Q2P)

ATP toforma DNAligase-AMP adduct then immune-precipitated

and

analysed

as described above.

RESULTS

Identification

of

the Vaccinia Virus DNALigase Protein

Afeature of the reactionmechanism of allDNAligases is the intermediate formation of

astableDNA

ligase-AMP

adduct. Anassaymaking use of this feature has been developed

by

whichDNA

ligase polypeptides

canbeidentified by incubation withCa-C2P) ATP(25). Thetype ofcovalentlinkage formed is rare among other proteins, therefore the assay is

suitable for use on relatively crude preparations and was used to determine whether a

vaccinia virus DNA ligase could be detected.

Extracts from vaccinia virus infected cells contain a novel radio-labelled polypeptide

ofmolecularweight approximately 61 kD after incubation with

a-g2P)

ATP (Figure 1). Thisactivity isdetectable inboth crude and

partially

purified extracts, at early (lane 2)

andlate(lane

3)

times

postinfection.The size

estimated

bySDS-PAGE is in good agreement

withthatpredicted from the amino acid composition of SalF 15R, 63 kD (11), which would

be consistent with a lack ofextensive post-translational modification. The extent of

incorporation

ofradioactivity is much greater than that in mock infected cells, in which

only

afaint bandofapproximately 46kDisvisible

(ane

1). This polypeptide is also present

atreduced

intensity

in extracts fromvaccinia virus infected cells. The 130 kD mammalian

1

2

3

4

5

6

200--'

92.5

.

69--

46--

30-Fig. 2: The 61 kD polypeptide isa DNAligase. A DNA ligase preparation partially purified from vaccinia virus infected cells late(15h)post infectionwaslabelledwithca_(2P)ATP(lane 3). PreparationsofcalfthymusDNA

ligase (a gift fromT. Lindahl, ICRF)(lane 1) and bacteriophage T4DNAligase (NewEngland Biolabs)(lane 2) were labelled in parallel. The vaccinia sample was divided into four equal parts. One part was analysed without further manipulation (lane 3) and the remainder centrifuged throughacolumnto removeunincorporatedATP

asdescribed in (25) except thatSephadex-G25 was used. The excluded volumewasdivided into threeequal parts and incubatedat37°C for 30 minutes witheithernoaddition(lane 4), coldpoly(dA):oligo (dT) DNA

ligase substrate (lane 5)or100ltMsodiumpyrophosphate (lane 6). The productswereanalysed as in Figure 1.

containno polypeptidewhich co-migrateswith the61 kDband in infected cell extracts,

suggestingtheappearanceofthisproteinisaconsequenceofinfectionwithvacciniavirus. The 61 kD polypeptide has the properties expected ofa DNA ligase (Figure 2). A

phosphocellulose column fraction derived from extracts of vaccinia virus infected cells

lateininfectionwas incubatedwith

a_C2P)

ATPandthenexcessATP wasremovedusing

SephadexG-25.The excludedproteinwasincubated with eithernoaddition(lane 4), DNA

ligase substrate (lane5)orsodiumpyrophosphate (lane6). ThepresenceofDNAsubstrate

allows theligase reactionto proceedto completion, withadisappearance of

(32P)-AMP

fromtheenzyme.Conversely, highconcentrations ofpyrophosphatedrive theequilibrium

back towards free enzymeand ATP, again with aconsequent discharge ofradioactivity

from thepolypeptide (Figure 2). This resultstronglysuggeststhat the61 kD

polypeptide

1

234

_5678910

-- -t

-_'.W,.. U,_114W"...lsm...-... ...r

a.

Fig. 3: DNAligaseactivityinvaccinia virus infected cells. CrudeextractsfromCV-lcells infected with vaccinia

virusearly(3h), late (17h) post infectionormockinfectedwereassayed forDNA ligase activity (Methods).

The30mer(32p)oligo dT:poly dA substrate is shown in lane 1 andcorrespondstothemonomer n = 1. Four

units(lane 2), 0.4 units (lane3)and 0.04 units(lane 4)ofbacteriophageT4DNAligase (NewEngland Biolabs)

DNA Ligase Activity in Vaccinia VirusInfected Cells

An assay whichmeasuresligation of 30 mer (32P)-dT oligodeoxynucleotides annealed to

polydA wasused todetermine whetheranincreasein DNA ligase activity could be detected uponvacciniavirusinfection. Activity is represented by the appearance oflabelled products

correspondingto two ligatedmolecules of the dT oligonucleotide (n =2), trimers of dT (n=3) andfurther higher oligomers. The assay of bacteriophage T4 DNA ligase provides a standard for this activity. An increase in DNA ligase activity above the basal level

measurable in mock infected cells is observed after vaccinia virus infection (Figure 3). The ligaseactivity early in infection(lanes5 and 8) is only slightlygreater than the cellular

activity in mock infected cells (lanes 6 and 9), but by late in infection (lanes 7 and 10)

the activityissubstantiallyhigher. Extractspreparedinthislowsaltextraction buffer(100

mMNaCl, Methods) haveanappreciable portion of thetotal DNAligase activitylocated in thepellet fraction aftercentrifugation(lanes8-10) compared to the supernatant (lanes 5-7) but anincrease inthesaltconcentrationto 1 Mshifts themajorityof the total DNA ligase activity into the soluble fraction (data not shown).

Immunological Analysis ofVaccinia Virus DNA Ligase

Approximately one third (183 amino acids) of theproteinencoded by SalF 15R was cloned intothebacterialexpression vector pEX3 (Methods). A rabbitpolyclonal antiserum(pEX LIG) wasraised against the resulting

,3-galactosidase/SalF

15R fusion protein. The pEX

LIGantiserumimmune-precipitatestwoviruspolypeptidesfrom extractsofcellslabelled

with

(35S)-methionine

1.5-4h post infection (Figure 4A, lane 2). The upper band, of

molecularweightapproximately 61 kD on SDS-PAGE, is more intensethanthe lower,

ofapproximately 54 kD. No protein is recognised in mock infected cells (lane 1). The

portion of the gene inserted intothepEX LIGconstruct doesnot includethe regionsof strongestaminoacidsequencehomology withyeast DNAligases, therefore cross-reaction with mammalian ligases might not be expected. Both polypeptides areearly virus gene products as treatment ofthecells with cytosine arabinoside,aninhibitor ofDNAreplication,

doesnotaffecttheirexpression (data notshown). Denovosynthesis ofboth

proteins

can

be detected early in infection by pulse labelling with

(35S)

methionine 1.5-2.5h post

infection followedbyimmune-precipitation, butonlyslightlyreducedlevelsareobserved

as late as 7-8hp.i. (data not shown).

Thelargerofthetwopolypeptides detected by

immune-precipitation

with thepEXLIG

antiserumco-migrates with theDNAligase-AMP adductfromvacciniavirus infectedcells

onSDS-PAGE(Figure 4B). The

marginal

differencein size between the

(32P)

and

(35S)-labelled polypeptides which may be detected on

electrophoresis

to achieve maximum

resolution ispossibly duetotheadditionof the AMPmoiety inthe

(32P)-labelled protein.

Theidentity of the lowerband is not clear. Pulse-chase

experiments

have not indicated

aprecursor-product

relationship (data

not

shown).

Itis

possible

that the 54 kD

polypeptide

representsthetranslationproduct of the minormRNAof SalF

15R,

detected

by

SI

mapping

(11),

butthis is unlikely dueto the relative ratios of the 54 and 61 kD

proteins

versus

the shorter and full

length

RNAs.

SalF15R Encodes the Vaccinia Virus DNA Ligase

The DNA ligase would be likely to be an essential gene if it was involved in DNA

replication, inwhichcaseit wouldnotbe

possible

toselect recombinantvirus

containing

fractionsfromearly,mock and latesamplesrespectivelyafter Douncehomogenisationandcentrifugationat1OK for 20minutes.Lanes8,9 and10arethepelletfractions fromearly,mock and latesamples.Anautoradiograph

A

1

2

3

B

1

2

3

-200 -92 -69 -46 -30 ..1.t.

Fig.4: A. Immune-precipitationof(35S)-methionine labelledpolypeptidesfromvaccinia virus infected cells.

TK- cellsinfected with vaccinia virus(30pfu/cell)ormockinfectedwerelabelled with(35S)-methionine1.5-4h postinfection. Cellextractswerepreparedandimmune-precipitatedwithpEX LIGantiserum(Methods).Lane

Irepresentsuninfected cells and lane 2 vaccinia virus infected cells. Molecularweightmarkersareshownto

therightof thegel. B. Co-migrationof(35S)-methionineand a-_(32P)-ATPlabelled proteins.A(32P)-labelled

DNAligase-AMP adductfrom vacciniavirus infected cells(lane1)and cellextractslabelled with(35S)-methionine 2.5-6hp.i. from either vaccinia virus infected(lane2)ormockinfected cells(lane 3), immune-precipitated

with pEXLIG antiserumasdescribed in PartA, wereelectrophoresed througha12.5% polyacrylamide gel.

aspecificdeletion ofthisgene.Analternative approachtoprovethatSalF 15Rgeneproduct wasresponsible for the increased DNA ligaseactivity in vaccinia virus infected cellswas

thereforechosen. This madeuseofthepEXLIGantiserum, raised against SalF15Rencoded

protein, inimmune-precipitation experiments against the radiolabelledDNAligase-AMP

adduct. ThepEXLIG antiserumcanefficiently precipitate the(32P)-labelled DNA ligase

proteininextractsfromvaccinia virus infectedcells(Figure 5, lane 5), whereas pre-immune serumfromthe samerabbit(lane4), oranon-specific immune serumraised againstan

unrelated pEX fusionprotein (lane 6), do not recognise the 61 kD polypeptide (Figure 5). Controlexperiments indicate that neither purified calf thymus(lane 1)norbacteriophage

T4 DNAligase (lane2)canbeimmune-precipitated by thepEXLIG antiserum (datanot

shown).Theimmune-precipitationof the novel DNAligase-AMPadductby theantiserum

-9g2.

..69

92.5-

69-

46-

30-Fig.5: Immune-precipitation of labelled vaccinia virus DNA ligase. Calf thymus DNA ligase (lane 1), T4 DNA ligase (lane 2) and aphosphocellulose column fraction from vaccinia virus infected cells (lane 3) were incubated

witha-(32P)-ATP(Methods).Each sample was divided into four equal parts and either analysed directly by TCA precipitation and SDS-PAGE(lanes1,2 and 3) or, in the case of extract from vaccinia virus infected cells, immune-precipitated with either pre-immune serum (lane 4), pEX LIG serum (lane 5) or a non-specific pEX immune

serum(lane 6), followed by SDS-PAGE.

raisedagainst SalF 15Rencodedproteinclearly demonstrates thatthisvaccinia virusgene

encodes the observed DNA ligase activity.

DISCUSSION

Avacciniavirusgeneencodingapredictedpolypeptideof63 kDwhich shares 30% amino acidsequenceidentitywith S. cerevisiae and S.pombeyeastDNAligaseshas beenidentified

(11). Inthispaperthe protein encoded by this genehas been detected and showntohave

the biochemical characteristics of a DNA ligase. The extensive amino acid sequence

homologytoyeastDNAligases,increase inDNAligaseactivity in vaccinia virus infected

cells and appearanceofanovel DNAligase-AMPadduct ofapproximately thepredicted

molecular weight all strongly suggest that SalF 15R encodes this enzyme.

Immune-precipitation of theDNAligase-AMP adduct by antiserum raised against SalF 15Rencoded protein proves thatthis is indeed the case.

The ability to label the vaccinia virus DNA ligase by incubation with

ca_(32P)

ATP

shows that incommonwitheukaryoticDNAligasesandbacteriophageDNAligases, ATP

typhimurium

are

NAD-requiring.

The

lysine

residue whichforms the covalent enzyme-AMP

linkage

is

likely

to be amino acidnumber

382,

by alignment with thatpreviously

identifiedin yeast DNA

ligases (11).

Itwillbeof interesttodetermine whether the properties of vaccinia virus DNA

ligase correspond

morecloselytothe type IortypeH mammalian enzymes. The type I enzymecan

catalyse

blunt-endjoiningofDNA,increases inrapidly

dividing

cells

during

rat liver

regeneration

and is the major ligase of DNA replication

(reviewed

in

28).

DNA

ligase

II is involved in DNA

repair

processes, andunlike type I can use

hybrid

DNA:RNA molecules as

substrate,

allowing the development of

type-specific

assays

(23).

It will be

possible

to assessthe biochemical properties of vaccinia

virus DNA

ligase,

such as pH and temperature

optima

and salt requirements, after

purification

assisted

by high

level

expression of

thegene in a suitable vector system.

A substantial increase in DNA

ligase

activity, measured by joining synthetic dT

oligonucleotides

annealedto

poly dA,

is observed after vaccinia virus infection. Thisresult is consistent with the increase in DNA

ligase

activity reportedtwentyyears ago(12). In that

study,

upto a 13-fold increasewasmeasuredearlyininfectionand was notinhibited

by cytosine arabinoside,

aninhibitorof DNA

replication

andtherefore lategeneexpression. The level of

activity

late in infectionwas not reported. Sambrookand Shatkin (12) did

notdetect

activity

in

purified

virions after detergent solubilisation, aresult alsoobtained

in

(13).

The moresensitiveassay for DNAligase-AMP adduct formationdescribed here

was also unable todetect DNA ligase activity packaged within the virion core (data not

shown).

DNA

ligase

istheproductofanearlygene, therefore the activitywill be present

prior

totheonsetof DNAreplication. However, theenzymeactivity increases with time,

asthere isagreaterlevelof enzyme

activity

lateininfectionthan early. Thisis somewhat

surprising

in view ofthe smallamount of SalF

15R-specific

RNAdetectable late (lOh) postinfection

(1

1)

and may be due in parttoprotein synthesised earlyininfectionremaining stable.

The identification ofageneencodingDNAligaseprovesthat theincreasein DNA ligase

activity

is of viral

origin.

This need nothavebeenthecase, asvacciniavirus replication

models

existforwhichaDNA

ligase

isnot

required. Furthermore,

evenif such anactivity wasnecessary, it is

possible

thatrather than encoding itsownDNAligase, vaccinia virus could causethe transport ofcellularDNA ligase from the nucleus, or induce a cellular DNA

ligase

gene. There wouldbesomeprecedentforthis, asthe large subunit of RNA

polymerase (29)

andacellularlamin(30) arebothrecruited to the cytoplasm upon vaccinia virusinfection. Cellular DNAligaseIis known to be susceptible to proteolysis, possibly

dueto adomain structure with protease-sensitive hinge regions. The intact mammalian

enzymehasamolecularweightof200kD,which is rapidly cleaved to a 130 kD fragment andmore

slowly

toan85-90 kD active fragment(17; see also Figure 2, lane 1). DNA

ligase

H has a molecular weight ofapproximately 80 kD. A mammalian DNA ligase

fragment

of 61 kD has not, however, been reported.

Although

much progress has been made in unravelling the overall features of the mechanism of vaccinia virus DNA replication (reviewed in 31) several possible models exist. After

infection,

nicks are introducednear one or both of the ends of the genome and DNA

replication

startsvia self-priming by the 3' ends generated by the nicks. This initial

self-priming

alone, followedby a strand displacement mechanism, could account

for DNA

synthesis

along the entire genome leading to the generation of concatameric

structures. DNA

ligase

wouldnotberequired, as a nicking-joining (NJ) enzyme capable offirst

introducing

and then sealing nicksatthe ends of the genome has been described

(15) and would be capable of resolving concatameric molecules into monomers. This

enzyme, which is probably encoded by vaccinia virus although the gene has not been

identified, is distinct from the DNA ligase described here on the basis of its smaller

molecular size (50 kD processed to 44 kD), ATP independence and concerted mode of

action, cleavingsuperhelicalDNAsite-specifically followed by joining toformcross-links. Furthermore, nicks introduced by DNase I treatment, equivalent to thepoly(dA):oligo(dT)

DNAligase substrate, are not joined by NJ enzyme (32). NJ enzyme is also distinct from the vaccinia virus type IDNA topoisomerase, which has anicking-closing activity (33).

Asecondpossible modelisthatafterself-primingatthe genomeends, DNAreplication

proceedsbi-directionallyvialeadingandlagging strandsynthesis, with formation of RNA

primers andOkazaki-type fragments of DNA. Concatamers couldagain be formed, and

some evidence for this mechanism does exist (e.g. 34,35). Acentral requirementwould

be for a DNAligase to join the short DNAfragmentsonthelaggingstrand. Theconstruction

ofconditional viral mutantsinthe SalF 15Rgene willallow these models and the various

functions of the DNA ligase in vaccinia virus DNA replication and recombinationto be

investigated.

ACKNOWLEDGEMENTS

Wethank Dr. ThomasLindahl (ICRF, London)andmembers of hislaboratoryforproviding

purified calfthymus DNA ligase I and for helpful discussions on methods of extraction

andassay of DNAligases. Wealso thank Anita Hancock and Pam Woodward for

typing

and Paco Rodriguez for critical reading ofthe manuscript. This workwas

supported by

grant G8425735CA from the Medical Research Council and GLS is a Lister

Institute-Jenner Research Fellow.

*To whomcorrespondenceshould be addressed atSir WilliamDunn School ofPathology, University of

Oxford,South Parks Road, OxfordOXI 3RE,UK

REFERENCES

1. Moss B. (1985)In B.N.Fields,D.M.Knipe,J.L.Melnick,R.M.Chanock,B.R.Roizman andR.E.Shope

(eds.), Virology. RavenPress, NewYork, pp. 685 -704.

2. Moyer, R.W. (1987) VirusRes. 8, 173-191.

3. Earl, P.L., Jones, E.V. andMoss, B. (1986) Proc. Natl. Acad. Sci. USA, 83, 3659-3663.

4. Shuman, S. andMoss, B. (1987) Proc. Natl. Acad. Sci. USA, 84, 7478-7482.

5. Rodriguez, J.F., Kahn, J.S. andEsteban, M. (1986) Proc. Natl. Acad. Sci. USA, 83, 9566-9570. 6. Weir, J.P., Bajszar, G.and Moss, B. (1982) Proc. Natl. Acad. Sci. USA, 79, 1210-1214. 7. Hruby, D.E. and Ball, L.A. (1982)J. Virol., 43,403-409.

8. Smith, G.L., deCarlos, A. and Chan, Y.S. Nucleic Acids Res. Inpress.

9. Slabaugh, M., Roseman, N., Davis, R. and Mathews,C. (1988) J. Virol. 62, 519-527.

10. Tengelsen, L.A., Slabaugh, M.B., Bibler, J.K. andHruby, D.E. (1988) Virology, 164, 121-131.

11. Smith, G.L., Chan, Y.S. andKerr, S.M.,Nucleic Acids Res. Inpress.

12. Sambrook,J. and Shatkin, A.J. (1969)J. Virol. 4, 719-726.

13. Spadari, S. (1976)Nucleic Acids Res. 3,2155-2167.

14. Pogo, B.G.T. andDales, S. (1969)Proc. Natl. Acad. Sci. USA, 63, 820-827. 15. Reddy, M.K. andBauer,W.R. (1989)J. Biol. Chem. 264,443-449.

16. Engler,M.J.andRichardson,C.C.(1982)In P.D.Boyer(ed.) 7heEnzymes.Vol.XV,pp.3-29.(Academic

PressInc., NewYork).

17. Lindahl, T.,Willis, A.E.,Lasko,D.D. andTomkinson, A. (1989). InM.Lambert and J. Laval (eds.),

DNARepair Mechanisms andTheirBiological ImplicationsinManmalianCells. Plenum Press. In Press. 18. Willis, A.E. and Lindahl,T. (1987) Nature, 325, 355-357.

20. Mackett, M.,Smith, G.L. and Moss, B. (1985) In D.M. Glover (ed.),DNACloning:APracticalApproach. Oxford andWashington DC,IRLPress.

21. Greene, P.J., Heynecker, H.L., Bouvar, F.,Rodriguez, R.L., Betlach, M.C., Covarrublas, A.A., Backman, K.,Russell, D.J., Tart,R. and Boyer, H.W. (1978) Nucleic AcidsRes., 5, 2373-2380.

22. Bradford, M.M. (1976)Anal. Biochem. 72, 248-254.

23. Arrand, J.E.,Willis, A.E., Goldsmith, I. and Lindahl, T. (1986)J. Biol. Chem. 261, 9079-9082. 24. Bankier, A.T., Western, K.M. and Barrell, B.G. (1987)In R. Wu(ed.), Methods inEnzynology, 155,

51-93. AcademicPress,London.

25. Banks, G.R. and Barker, D.G. (1985) Biochim. Biophys. Acta., 826, 180-185.

26. Stanley, K.K. andLuzio, J.P. (1984) EMBO J. 3, 1429-1434. 27. Sullivan, V. and Smith, G.L. (1987) J. gen. Virol. 68, 2587-2598.

28. Soderhall, S. and Lindahl, T. (1976) FEBS Lett., 67, 1-8.

29. Morrison, D.K. and Moyer, R.W. (1986) Cell, 44, 587-596.

30. Bloom,D.C., Massung, R., Savage, L., Morrison, D.K. and Moyer, R.W. (1989) Virology, 169, 115-126.

31. Moss,B., Winters, E. and Jones,E.V. (1983)Mechanisms of DNA ReplicationandRecombination. Alan

R. Liss,Inc., NewYork, pp.449-461.

32. Lakritz,N., Foglesong, P.D., Reddy, M., Baum, S., Hurwitz, J. and Bauer, W.R.(1985) J. Virol. 53,

935-943.

33. Shaffer, R. andTraktman,P. (1987) J. Biol. C/tem. 262, 9309-9315. 34. Esteban, M.,Flores, L.andHolowczak, J. (1977) Virology, 83, 467-473. 35. Pogo, B.G.T. and O'Shea, M.T. (1978) Virology, 84, 1-8.

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