Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells.

Copyright © 1988,AmericanSocietyforMicrobiology

Identification and Characterization of

a

DNA Primase

Activity

Present in Herpes

Simplex

Virus Type 1-Infected HeLa Cells

ANDREW M. HOLMES,* STEVEN M. WIETSTOCK, ANDWILLIAM T. RUYECHAN

Department ofBiochemistry, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road,

Bethesda,

Maryland

208144799 Received 31 July 1987/Accepted 1 December 1987

A novel DNA primaseactivity has beenidentified inHeLa cells infected withherpessimplex virus type 1 (HSV-1). Suchanactivity hasnot been detectedinmock-infected cells. Theprimase activitycoeluted witha

portion of HSV-1 DNA polymerase from single-strandedDNAagarosecolumnsloaded withhigh-saltextracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primasewasrelativelybroad andpeakedat8.2 to8.7pH units. Incontrast,thepH optimum of the HeLaDNAprimasewasverysharp and fell between pH

7.9and 8.2. Second, freshlyisolated HSV DNAprimasewasless salt sensitive than the HeLaprimaseandwas

elutedfrom single-strandedDNAagaroseathighersaltconcentrations than the hostprimase. Third,antibodies raisedagainstindividualpeptidesof the calfthymusDNApolymerase:primase complexcross-reacted with the HeLa primase but didnot react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associatedwith the HSV polymerase, but after storage at4°C for several weeks, the DNA primase separatedfrom the viral DNA polymerase. Separation or decouplingcould also be achieved by gel

filtration of the HSV polymerase:primase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereasfree HeLaDNA primase hadan apparent molecular size of

approxi-mately 110 kilodaltons. On the basis of these data, we believe that the novel DNA primase activity in HSV-infected cellsmaybe virus coded and that thisenzymerepresentsa newand important function involved

in thereplication ofHSV DNA.

The isolation ofa virus encoded DNA polymerase from

herpes simplextype1(HSV-1)-infected cells (21, 22, 35)was

an important first step toward understanding viral DNA

replication. The DNA polymerases isolated from HSV-1-and HSV-2-infected cells show very similarproperties (26)

whichservetodistinguish the viralenzymefrom mammalian

DNApolymerases. These properties include salt optimum, inhibition by polyamines (such as spermine), inhibition by phosphonoacetate and phosphonoacetate analogs, column chromatographic behavior, sedimentation rate on sucrose

gradients,andinhibitionby 9-(2-hydroxyethoxymethyl)GTP and 9-(1,3-dihydroxy-2-propoxymethyl)GTP (10, 11, 26).

The presence of a 3' -> 5' exonuclease activity (9, 26, 35)

also serves to distinguish HSV DNA polymerase from mammalian DNA polymerases ox, 3,and-y.The size of HSV

DNApolymerase wasoriginallyestimatedtobe 180

kilodal-tons (kDa) (35), but apolypeptide with a molecular size of approximately 140 kDa has nowbeenassigned tothe DNA polymerase activity (26), and thegene for this polypeptide has been cloned and sequenced (28).

Althoughthe propertiesof HSV DNApolymerase distin-guishit from mammalian DNApolymerases, oneimportant

similarity exists between HSV DNA polymerase and

mam-malian DNA polymerase a., the enzyme believed to be

responsible forthebulkof the cellular DNA replication (see reference 16 for review). Thatpropertyis the abilitytouse a

short piece of RNAas aprimer for DNA synthesis (35). A

DNAprimasecanbecopurified from eucaryotic cells in tight association with DNA polymerase a (6, 14, 20, 34). This

primasecansynthesize oligoribonucleotides of 8to 10bases inlength, whichthe DNApolymerase canthenusein DNA

synthesis (7, 17, 32). Nascent DNA chains extracted from

*Corresponding author.

mammalian cells contain oligoribonucleotides of this size at

their 5' ends (29, 33).

Mature HSV DNA molecules contain alkali-labile sites (12, 13, 31, 36). Thesemaybe duetotheuseof RNA primers

for DNA replication, some of which may have persisted during maturation of the DNA, although alternative expla-nations for alkali lability and the presence of RNA-like material inmatureviral DNAarepossible (3,25, 38). Newly synthesized HSV DNA has been demonstrated to contain RNAcovalentlylinkedtoDNA(2, 15, 24) whichis removed

over a period of time (24). These RNA segments are esti-matedtobe 35 nucleotides inlengthandarelinkedtoDNA averaging 36 x 103 nucleotides in length (24). This would indicate amajordifference between the waythe virususes

RNA toprimeDNA synthesis and thewayeucaryoticcells replicate. The lengthof the DNA attachedtoRNAprimers in the eucaryotic cell is usually 200 nucleotidesor less (8). These differences may reflect the involvement of virus-codedproteins in the synthesis of the RNAprimers aswell

as inthesynthesis of the viral DNA.

We report hereonthe identification and characterization ofanenzymewhichcanbeclassifiedas aDNAprimasefrom HSV-1-infected HeLa cells and whoseenzymaticand

struc-tural properties distinguish it from the DNA primases iso-lated from uninfectedHeLacells.

MATERIALS AND METHODS

Chemicals and reagents. Calfthymus DNA and unlabeled deoxynucleoside and nucleoside triphosphates were ob-tained from Sigma Chemical Co. [8-3H]dATP, [methyl-3H]TTP,and [a-32P]ATPwere obtained fromNewEngland Nuclear Corp. Escherichia coli DNA polymerase I was

obtained from New England BioLabs, Inc. Sepharose 6B

was obtained from Pharmacia Fine Chemicals.

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stranded DNA agarose was obtained from Bethesda Re-search Laboratories. Tissue culture media were obtained from GIBCO Laboratories.

Poly(dT)3500

was prepared by using terminal deoxynucleotidyltransferase as described elsewhere (5). Calf thymus DNA polymerase:primase com-plex and free calf thymus DNA primase were prepared as described previously (6). Antibodies were prepared by im-munizing individual rabbits with individual peptides from the calf thymus DNApolymerase:primase complex as described elsewhere (18). They were further purified by chromatogra-phy on protein A-Sepharose and then affinity purified by chromatography on a calf thymus DNApolymerase:primase complex Sepharose 4B column. All otherchemicals were of reagent grade.

Cells and viruses. HeLa S-600cells were grownin suspen-sion culture in suspensuspen-sion-modified Eagle medium to a concentration of 1.6 x 106/ml. Infection was with HSV-1 (mP) grown in Vero cells at a multiplicity of 20 PFU per cell as described by Derse et al. (10). Cells were harvested 8 h after infection, the time viral DNA replication reached its maximum under these conditions. The cells were harvested by centrifugation and stored at -70°C or used immediately for the isolation of HSV DNApolymerase.

Isolation of HSV DNA polymerase:primase. The isolation procedure was based on a procedure described by Powell and Purifoy (27) and Ruyechan and Weir (30). The cells were suspended in extraction buffer (20 mM Tris [pH 7.5], 1.7 M KCI, 5 mM EDTA, 1 mM dithiothreitol [DTT], 0.2% Noni-det P-40, 10% glycerol), sonicated three times for 30 s in a Heat Systems Ultrasonics sonicator, andcentrifuged at 500 x g, and the supernatant was subjected to a 10% polyethyl-ene glycol phase separation. The precipitated material, in-cluding nucleic acids, was spun down. The supernatant was dialyzed against 0.15 M KCI in the buffer described above and chromatographed on a single-stranded DNA agarose column. Enzyme were eluted with a linear 0.15 to 1.0 MKCI gradient. All procedures were carried out at 4°Cin asolution containing 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mMDTT, and 10% (vol/vol) glycerol. Amodified procedure ofAllenet al. (1) has also been used to isolate DNA polymerase from HSV-1-infected HeLa cells. In thisprocedure, the cells were lysed in hypotonic buffer, brought to 0.4 M potassium phosphate, as described above, and passed through DEAE-Tris-acryl. The unbound material was dialyzed against a solution containing 20 mMpotassium phosphate (pH7.5), 1 mM DTT, 20% (vol/vol) glycerol, and 0.2% Nonidet P-40 and rechromatographed on DEAE-Tris-acryl. HSV DNA polymerase bound to the column was eluted with a linear

KCI

gradient (0 to 0.5 M). Fractions containing DNA poly-merase:primase assayable at 0.2 M salt were pooled, dia-lyzed against the above buffer, and chromatographed on a single-stranded DNAagarose column underidenticalelution conditions.

In some instances, the DNA polymerase:primase was concentrated by adsorption and batch elution with 0.5 M KCI from a 1-ml single-stranded DNA agarose column and stored at 4°C.

Enzymeassays. DNA polymerase activities were assayed by using three different protocols. DNApolymerase eluted from single-stranded DNA agarose columns to which high-salt extracts had been applied werecarried out in asolution containing 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 8.0), 8 mM MgCl,, 300

pg

of DNase I-treated DNA per ml, 0.2 mM each dATP, dCTP, and dGTP, 0.04 mM [3H]TTP at 372 dpm/pmol of 2 mM 2-mercaptoethanol, and 200 mM KCI. Aliquots (10 LI) of

individual column fractions were added to 40 ,ul of the reaction mixture andincubatedat

35°C

for 20 min.Aliquots (40

IL)

were

spotted

on

glass

fibersquaresand

placed

in5% trichloroaceticacid-1% sodium

pyrophosphate.

The squares werewashedtwice with1 NHCl andoncewith95%

ethanol,

airdried, and counted in a

liquid

scintillation counter. One unit ofDNA

polymerase

activity

in this assayis defined as

the

incorporation

of 1

pmol

of

[3H]TMP

into insoluble material in 1 minat

35°C.

DNA

polymerase

isolated

by

the modified

procedure

ofAllenetal.

(1)

was

assayed

in 40 mM phosphatewithorwithout200 mMKCl.All otherconditions wereexactlyasdescribedabove.Aftertheinitialisolation of HSV DNA

polymerase

activity

by

these two

methods,

all additional

polymerase

assays werecarried outin a solution

containing

40 mM

potassium

phosphate

(pH 7.0),

8 mM

MgCl2,

100 ,ugof bovineserumalbumin

(BSA)

per

ml,

1 mM DTT, 100 ,ugofDNAase I-treated DNA per

ml,

0.1 mMeach dATP,

dCTP,

and

dGTP,

and 0.1 mM

[3H]TTP

at 40to 50

cpm/pmol,

with or without 0.15 M NaCl.

Assays

were

incubatedat

35°C,

andat thetimes

indicated,

samples

were

spotted

on

glass

fibersquaresand

placed

in the5% trichloro-acetic acid-1% sodium

pyrophosphate.

One unit of DNA polymerase

activity

isdefined asthe

incorporation

of1nmol of

[3H]TMP

into acid-insoluble material in 60 minat

35°C

in this assay.

DNA

primase

was

assayed

directly

ina solution

contain-ing 50 mM Tris

(pH

8.0),

8 mM

MgCl2,

100 ,ugofBSA per ml, 1 mM

DTT,

10

,uM

poly(dT)3,500,

and 0.1 mM

[a-32P]ATP

(100to 200

cpm/pmol). Assays

were done at

35°C,

and

aliquots

were

spotted

onWhatmanDE-81 papers

previ-ously

spotted

withan

equal

volumeof5mMunlabeledATP. This

technique

reducescontamination

by

the

[32P]ATP.

The papers were washed four times with 0.2 M

dipotassium

hydrogen

phosphate

andtwice with

glass-distilled

waterand counted in

glass-distilled

water in a

liquid

scintillation

counterset onthe

3H-labeled

channel.Thecounts

remaining

onthe paper

give

a direct measurement of

oligoribonucleo-tide

synthesis.

One unit ofenzyme

activity

is definedasthe

incorporation

of 1 nmol of

[32P]AMP

into

oligoribonucleo-tide in 60 minat

35°C.

DNA

primase

was

routinely

assayed

by

anindirect assay. Theassayconditionswere asdescribed

above,

except

that 2 mMunlabeledATP

replaced

the 0.1mM

[at-32P]ATP

and 0.1 mM

[3H]dATP

at40to50

cpm/pmol

and 20 UofE.

coli

DNA

polymerase

I per ml was

usually

present.

Assays

were

carriedout at

35°C,

and

aliquots

were spottedon

glass

fiber

paper and

processed

as for DNA

polymerase

assays. This

coupled

assay

procedure

measures the extension of

primers

synthesized

by

the DNA

primase,

and the rate of

incorpo-rationof

[3H]dAMP

intoacid-insoluble

product

is

dependent

on the rate of

primer

synthesis.

In the absence ofATP or

DNA

primase,

no

incorporation

of

[3H]dAMP

is observed. This isan

amplification

of DNA

primase

activity.

One unitof enzyme

activity

in this instance is defined as the

incorpora-tionof 1 nmol of

[3H]dAMP

into

acid-insoluble

material in 60 min at

35°C.

Exonuclease

activity

was measured in a

solu-tion

containing

50 mM Tris

(pH 8.0),

8 mM

MgCI2,

1 mM DTT, 100,ug of BSA per

ml,

and 10,ug of

single-stranded

"4C-labeled

T7 DNA at 30 x 103

cpm/,ug.

Assays

were

incubatedat

35°C,

and at the times

indicated,

aliquots

were

placedon

glass

fiber squares and the squareswere

processed

as for DNA

polymerase

assays.

Sepharose 6B

chromatography.

Sepharose

6B

chromatog-raphy

was carried out on an

85-by-1.4-cm

column in a

solution

containing

0.25 M

NaCl,

50 mM Tris

(pH

8.0),

100 ,ugofBSA per

ml,

1 mM

DTT,

and 20%

(vol/vol)

glycerol.

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Fractions (50 drops, or 1.7 ml, each) were

column was calibrated with calf thymus

catalase, rabbitIgG,E. coliDNApolymeraseI,

cytochrome c, and DTT.

Antibody-binding experiments. Antibody-binding

mentswerecarriedoutasfollows. Atotal p.l

enzyme preparation and 20,ul ofrabbit

(IgG) raised against a specific calf thymus

peptide (68 kDa,55kDa,or48kDa)inTBS

Tris, 0.15M NaCl, 2 mg of BSA per ml)

incubated for 18 h at 4°C. Samples RI

removed, and to each sample was added plI 50%

(vol/vol) protein A-Sepharose in TBS

continued for an additional 18 h. The

spundown inan Eppendorfcentrifuge, 5-pLI

the resulting supernatants were assayed

polymerase activity asdescribed above.

RESULTS Association of DNA primase with HSV

Whenextractsof HSV-1-infectedHeLa

graphed on single-stranded DNA agarose,

polymerase activity eluted between 0.3

With the indirect,orcoupled,DNAprimase

DNAprimase activity was detected toward

HSV DNA polymerase peak (Fig. 1).

primase assay also detected someDNA

this area of the elution profile (data

difficult to quantitate the DNA primase

fractionsby thedirectprocedure, since

[32_p]AMP incorporation, it was necessary

assaytime far beyondthatshown inFig.

of high levelsofexonuclease activitydestroyed

atlater times. Thedirectassaywasalso

presence ofaDNA-dependentATPase

H activity inthisareaoftheelutionprofile

The direct DNAprimaseassaycontained

whereas the indirect, or coupled, assay

ATP, helping to negate the effects of

ATPase. Further,thecoupledDNAprimase

theDNAprimaseactivity tosuch anextent

and the amount ofthe DNA agarose

500

=9 C1

C2

=c

E

400

300

200

100

0l

0 5 10 15

FRACTION NUMBER

20 25

FIG. 1. Chromatography ofHSV DNA

stranded DNAagarose. HSV DNA polymerase

infectedcells by themodifiedPowelland

AssaysweredoneasdescribedinMaterials

O,

DNA polymerase in the presence 0,

primase in the coupled assay in the coli

polymeraseI ; A,exonuclease;

assay could be reduced so that neither

ATPase affectedthe assay. Theincorporation [3H]dAMP

shown in Fig. 1 was taken from time courses

individual fractions in which the incorporation

linearwith time. The resultshowninFig. 1

position of elution ofthe DNAprimase was

the contaminating enzyme activities. That

primase elution position was toward the

polymerase elution position. The fractions

primase activity were pooled, andthepooled

referred to as HSV DNA primase.

Uninfected HeLacells were subjected tothe

tion and chromatography procedures.

activity present in controlextracts did not

agarose column. This DNA polymerase itedby 0.15 M NaCI intheassay and could

in the absence ofsalt.This salt-inhibited

was also present in the DNA agarose column

of the infected cell extracts. The properties

polymerase are consistent with those of

polymeraseao. On application of the KCl

control (uninfected) DNA agarose column,

of DNA primase began to elute from

continued to elute uptoabout 0.4 MKCl (data shown).

This material, comprising less than 10%

eluting from the infected cell DNA agarose

pooled forenzymecomparisonpurposesand

control DNAprimase. This relatively small

primase activity identified in uninfected suggest that the much greater amount

observed in this areaof the elution profile

infected cells were subjected to the same

consequenceoftheviralinfection, although

that it is necessarily virally encoded.

By using the modified procedure of

isolate DNA polymerase from infected

tion was achieved between the HeLaDNA

the HSV DNA polymerase by the KCI

second DEAB-Tris-acryl column. The

polymerase was pooled, dialyzed, and

stranded DNA agarose as indicated in

ods. The elution profile from this DNA

shownin Fig.2. Thesalt-sensitiveHeLa ot

eluted between 0.1 and0.15 MKCI. Thesalt-activated

DNApolymerase again elutedbetween KCI.

Two peaks of DNA primase activity

major peak wasassociatedwith the HeLa

cx and the minor peak eluted at the rear

polymerasee

peak. The first peak of DNA

waspooled forenzyme comparison studies

as the HeLa DNA primase.

When this second purification procedure

uninfected HeLa cells, a salt-sensitive

meraseactivity was observed againeluting

0.15 M KCI. Salt-activated DNA polymerase not observed in this elution profile (data

peak of DNA primase activity was observed,

elution positionasthe DNApolymerase,

of DNA primase smeared off the column

concentration ofabout0.4M KCI. Again,

10% of the amount of enzyme activity

infected cell extracts were subjected RNApolymerase wasnotdetected with

procedure.

Initial characterization of the HSV

relationships betweenthedifferentDNA

100 80

40

1-20 ... .20 25

0

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[image:3.612.68.304.505.648.2]

20 0 23a1

la.2ChoaorpyofHVDAplmeaeo aige

meras in~the prsnc f0.MNCl ,DN5oymrsih

z

CD~~I

~100

501 .

EI

50 -25

0 10 20 30 40

FRACTION NUMBER

FIG. 2. Chromatography of HSV DNA polymerase on single-stranded DNAagarose. HSV DNApolymerase was isolatedfrom infectedcellsby themodified procedure of Allenet al. (1). Assays

were doneas described in Materials and Methods. 0, DNA

poly-meraseinthepresenceof 0.2MNaCl; 0, DNApolymerasein the absence of NaCl; A, DNA primase in the coupled assay in the

presenceofE. coliDNApolymerase I; .--, KCIgradient.

infected and uninfected cell extracts were

explored.

The effect of increasingconcentrations ofNaCl on the HSV and HeLa DNA primases was determined by using the coupled assayin the presenceofE. coli DNA polymerase I(Fig. 3). It is important to note that these primase samples also containedHSV and HeLa DNA polymerases, respectively. Freshly prepared HSV primase was less sensitive than the HeLa DNAprimase, whichshowed the same saltsensitivity as the DNA primase activity of the calf thymus DNA polymerase:primase complex. E. coli DNA polymerase I wasactive throughout therangeof salt concentrations used. SinceHSV DNApolymerase isasalt-activated enzyme(26, 35), however, the relative salt resistance of the HSV DNA primasecould have been due to the HSV polymerase begin-ning to function at high salt concentrations to efficiently elongate a reduced number of primers synthesized by a salt-sensitive DNAprimase. Accordingly,acomparisonwas madeofDNAprimase activities in the presence and absence of0.15 M NaCl and with and without the additionofE.coli

125

100

75

5'

25

0

I

<:0~~~~~~~~~~~

0 .025 .05 .075 .1 .125 .15

[NaCq

(M)

FIG. 3. Effects ofincreasing NaCl concentration on DNA pri-mase activity. DNA primase was assayed in the coupled assay

systemin thepresenceofE.coli DNApolymeraseI.0,HSV DNA

primase; A, HeLa DNA primase; 0, DNA primase of the calf

thymusDNApolymerase:primase complex.

TABLE 1. Effects of the additionof 0.15M NaClonthe activitiesof HeLa DNA primase and freshly

prepared HSVDNAprimase

Picomoles of[3HldAMP incorporated

per5 ,ul per60min

WithE.coli DNA WithoutE.coli polymeraseI DNApolymeraseI

No 0.15 M No 0.15 M

NaCl NaCl NaCl NaCl

HeLa 55 1 25 0

Freshly prepared HSV 180 72 105 33

DNA polymerase (Table 1). Inthe absence of bothE. coli DNA polymerase I and NaCI, the elongation of primers synthesizedby both the HeLa andHSV DNAprimases by the respective DNA polymerases was relatively efficient comparedwith theelongationof theprimersin the presence of E. coli DNA polymerase I (Table 1). At 0.15 M NaCl, there was no incorporation of [3H]dAMP with the

14eLa

DNA primase either in the presence or absence ofE. coli DNApolymeraseI, whereas therewasstillincorporationin both instanceswith theHSV DNA primase. Table 2 shows the activities of the DNA polymerase at the two salt con-centrations.

These results were obtained by using freshly prepared HSV DNA primase. The salt profile shown in Fig. 3 was obtainedwith HSV DNAprimasewhich had been isolateda few days before. HSV DNA primase stored at 4°C for severalweekswasalmostas salt sensitive as the HeLa and calfDNA primases were

(Table

3). Furthermore, although both the DNA polymerase and DNA primase activities of this stored enzymesamplehadnotdeclinedsignificantly (the DNA primaseactivity beingmeasuredin the presenceofE. coli DNA polymerase I by the indirect assay), the HSV DNA polymerase could now not elongate the primers pro-ducedbythe DNAprimaseineither the presenceorabsence of0. 15 MNaCl, indicatingamajor changein therelationship ofthe DNA polymerase and DNA primase in this sample. This change could be the decoupling of the two enzyme activities or the loss ofanother factor which mediates the two activities. This change in the salt sensitivity and the inability of the HSV DNA polymerase to function in the coupledDNAprimaseassayhasbeenobservedonlong-term storageofthe enzyme at

4°C

orafterdialysis ofthe enzyme against50% (vol/vol) glycerol and storage of the enzyme at -20°C. Thus, these dataindicate that the relative insensitiv-ityto saltdisplayed by freshlypreparedHSVDNAprimase is not an intrinsic property of the primase but may also indicate its participationinacomplex. The uncoupledHSV DNApolymeraseis stillcapableofincorporating[3H]dAMP into product by using a poly(dT) template when given an

TABLE 2. Effects oftheaddition of0.15 MNaClonthe DNA

polymerase activities of HeLaDNAprimaseand

freshly preparedHSV DNAprimase

Picomolesof[3H]dTMP

incorporatedper 5p.l

Originof DNApolymerase per 60min NoNaCl 0.15 M

NoNaCi NaCI

HeLaDNAprimase 73 10

Freshlyprepared HSV DNAprimase 92 720

21-t=

210

p

C.3

dc -e

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[image:5.612.318.556.66.215.2]

TABLE 3. Effects ofaddition of 0.15 M NaCl on the activities

of freshlyprepared HSV DNA primase and concentrated HSV DNA primasestored at 4°C for 35 days

% Activity

WithE.coliDNA WithoutE. coli HSVDNAprimase polymeraseI DNApQlymeraseI

No 0.15M No 0.15 M

NaCl NaCl NaCl NaCl

Freshlyprepared looa 64 76 60

Concentrated and stored 100b 3 5 3

aAnactivity of 100% for freshly prepared HSVDNAprimasewas590pmol of[3]dAMPincorporatedper 20[LIper 60min.

bAnactivity of100%forconcentrated stored HSVDNAprimasewas152

pmol of[3H]dAMPincorporatedper 1RI1per 60min.

oligo(A) initiator (data not shown), indicating that this loss of coupling ofDNA polymerase andprimase activities is not duetothe DNA polymerase losing the capability of using an oligoriboadenylate initiator.

The pHoptima of both freshlyprepared and stored HSV DNAprimase were the same, pH 8.2 to 8.7 (Fig. 4A and B). Those of the HeLa DNA primase and the DNA primaseof the calfthymus DNApolymerase:primasecomplex andfree DNAprimasefrom calf thymus were all pH 7.9 to 8.2(Fig. 5A and B). The control DNA primase (the DNA primase fromthe DNA agarose column chromatography of extracts of uninfected HeLacells) did not show a defined pH opti-mum over the pH range studied but rather indicated a gradualincrease in activitywith increasing pH (Fig. 4B).

[image:5.612.59.296.96.193.2] [image:5.612.320.556.446.609.2]

Size oftheHSV DNA primase. Theresults of the chroma-tography of a stored (uncoupled) sample of HSV DNA primaseon acalibrated Sepharose 6B columnareshown in Fig. 6. The HSV DNA polymerase activity eluted in advance of beef liver catalase with an apparent molecular size in excess of250kDa, compared withthemolecularsizeof140 kDa obtainedfromsedimentation studiesandpredictedfrom the DNA sequence (28). Allen et al. (1) have previously made a similar observation with the equine herpesvirus DNA polymerase. This discrepancy in the molecular size

estimnates

is explicable on the basis that the molecule is asymmetric in shape (19).

TheHSV DNAprimaseactivityeluted between the posi-tions of elution of ovalbumin and cytochrome c with an

150 125

i 100

Z 75

X50

2 5

oc 25

0

7.5 8 8.5 9 9.5 7.5

[image:5.612.63.301.522.672.2]

pH

FIG. 4. The pH optima of HSV DN.

wasassayed in the coupledassaysystem DNA polymerase I in constant-molarit Stored HSVDNAprimase (O); freshlypr

(0); control DNA primase (A).

57@= _ Z75G

F - 500A

150

.100

2L 2501EC

50-0F

0

7.5 8 8.5 9 9.5 7.5 8 8.5 9 9.5

pH

FIG. 5. The pH optima of mammalian DNA primases. DNA primasewasassayed in the coupledassayinthepresenceof E.coli DNA polymerase I in constant-molarity Tris buffers. Symbols: HeLa DNA primase (O); calf thymus DNA polymerase primase complex (0); free calf thymus DNA primase (A).

apparent molecular size of approximately 40 kDa (Fig. 6). The material eluting between fractions 40 and 50 was not present in chromatography of other samples ofHSV DNA primase on the Sepharose 6B column and may represent aggregated material. Most of the DNA primase activity of freshlypreparedHSVDNAprimasealsoelutedfrom

Seph-arose6Bwith anapparent molecular sizeof 40 kDa. These dataindicatedthat(i) either theprimase activitywas

uncou-pled fromthe HSV DNA polymerase activityon long-term storageoruncouplingoccurred duetodilutionduring chro-matographyor(ii)itwasnotpartofatight complextobegin with. Most of the HeLa DNA primase eluted at the same

volume as E. coli DNA polymerase I, with an apparent molecular size of 110 kDa (Fig. 6). This result seems to

indicate that most of this DNA primase is also not tightly

1 234 5

111 1

125

LU

I--:f g

40

Is

E

30 b

co

20

IC

in

6 7

100-

75-50[

251

30 40 50 60 FRACTIONNUMBER

70 80

a FIG. 6. ChromatographyofHSV DNAprimaseonan

86-by-1.4-cm Sepharose 6B column. Stored HSV DNA primase was

chro-matographedon Sepharose6Basdescribed in Materials and Meth-ods. Assays were done as described in Materials and Methods.

8 8.5 9 9.5 Symbols: l, DNApolymerase activity in the presence of 0.15 M

NaCl;0,DNAprimase activityin thecoupledassayin thepresence

of E.coliDNApolymeraseI.The numbers refertostandardsrunon

A primase. DNA primase thecolumn under thesameconditions:1, Calf thymus DNA; 2,beef

inthepresenceofE. coli liver catalase; 3, rabbit IgG; 4, E. coli DNA polymerase I; 5,

ty Tris buffers. Symbols: ovalbumin; 6,cytochromec;7,DTT.Thearrowmarksthepeakof

epared HSVDNAprimase elutionof thefree HeLaDNAprimase rununder thesame

condi-tions.

A A~~~~~~~

L

i

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associated with DNA polymerase under these elution con-ditions. It is important to note that free calf thymus DNA primase elutes at the same position and sediments with the sameS value as E. coli DNA polymeraseI, indicating that it is a globular molecule with an approximate molecular size of 110 kDa (18). This does not necessarily indicate that the apparent molecular size of 40 kDa observed for the HSV DNAprimase is the true molecular size of this enzyme (see Discussion).

Reactivity with anti-calf DNA primase IgGs. Rabbit IgGs raised against individual peptides of the calf thymus DNA polymerase:primase complex and previously used to assign functions to these individual peptides (18) were used in antibody-binding experiments to ascertain the relationship of the HSV, HeLa, and calf DNA primases. Previously, by using both an immunoblotting procedure and enzyme neu-tralization, peptides of 55 and 48 kDa had been assigned to thefreeDNAprimase ofcalf thymus and peptides of 68 kDa, 55kDa, and 48 kDa had been assigned to the DNA primase of the calf thymus DNA polymerase:primase complex, with the 48-kDa peptide being derived, perhaps proteolytically, from the 68-kDa peptide (18). The results of the antibody-binding experimentsarepresented in Table 4. All threeIgG samples reacted with the HeLa DNA primase, but none of themreacted with the HSV DNAprimase. Threetimes the amount of calf thymus DNA primase showed the same reactivity with these antibodies as the HeLa enzyme did. Thus, although cross-reactivity of these antibodies with the HeLa DNA primase was not 100%, the results show a significant difference between the HeLa and HSV DNA primases. Immunoblots with the anti-calf DNA primase antibodiesshowedcross-reactivitybetween theseantibodies and the HeLa DNA primase and indicated that the HeLa DNA primase has a peptide composition similar to that of the calf DNA primase. No cross-reactivity with the HSV DNA primase wasobserved (datanot shown).

DISCUSSION

[image:6.612.60.299.603.680.2]

A new DNA primase activity has been identified in ex-tractsderivedfrom HSV-1-infectedHeLacells. Thisactivity appearstobecoupled with the HSV DNApolymerasewhen freshly isolated, but it appears to decouple from the poly-merase activity upon long storage and is separable by gel filtration chromatography. Severalpropertiesof thisprimase activity distinguish it from the host HeLa cellprimase. First, freshly prepared HSV primase retains approximately 50% activityatsaltconcentrationsupto125to150mM,although this salt resistance is lost when the primase is uncoupled

TABLE 4. Percentage of DNAprimase activity remaining in solution after treatment withanti-calfpeptide IgGsandafter binding of the antibody-antigen complextoprotein

A-Sepharose"

%Activitywith antibodiesagainst Enzyme the following calf peptides:

68 kDa 55kDa 48kDa

CalfthymusDNApolymerase: 41 74 25

primase complex

HeLa DNAprimase 54 73 31

HSV DNAprimase 96 96 118

a An activityof100%wasthatobtained aftertreatmentof enzymes with controlIgGand was34, 13,and 14pmolof[3H]dAMPincorporationper 5 ,ul per 60minforthecalfthymusDNApolymerase:primase complex,the HeLa DNA primase, and HSV DNA primase, respectively, with E. coli DNA

polymerase I presentin the assays.

from the polymerase. In contrast, HeLa primase is ex-tremely salt sensitive both when it is coupled to the host polymeraseand when it is free. Second,thepHoptimumfor the HSV primase is higher and much broader than that determined for the HeLa primase. Third, the apparent molecularweight of the HSVprimaseas determined by gel filtration isapproximatelyone third that determinedforthe HeLaprimase. Fourth, antibodiesraisedagainstsubunits of the calf thymus DNA primase complex which bind to the HeLaprimaseactivitydonotshow anycross-reactivitywith theHSVprimase.

The question then arises as to the origin of this primase activity. Severalpossibilitiesmust beconsidered. First, the putativeHSV

primase

couldsimplybe HeLaprimasewhich hasbecome associated with the HSVDNA

polymerase

and is used in viral DNAreplication. Such anassociationcould alter the pH optimum and salt

sensitivity

of the HeLa primase. Thefact that thepHof

uncoupled

HSV

primase

is the same as that offreshly prepared (coupled) HSV DNA primase would argue that this

activity

is not the HeLa primase.Thisfact,inconjunctionwith thedifferentapparent molecularweightsdetermined for thetwo

primase

activities by gel

filtration,

appears to eliminate this

possibility.

Sec-ond,

the HSV

primase

could be a modified

(proteolytic

fragment) ofthe HeLa

primase.

Inthis case, the salt and

pH

optima

of the enzyme could

again

be altered

significantly

and would not necessarily change upon decoupling ofthe pri-mase from thepolymerase. Theevidencewe have

gathered

argues

against

this

possibility.

The

cross-reactivity

of anti-bodies to the

peptides

of the calf DNA

primase

with the HeLa DNA

primase

and the elution

position

ofthe HeLa primasefromSepharose6B suggestastructural

similarity

of the DNA

primases

from these two sources. The free calf DNA

primase

has beendemonstratedtocontain

peptides

of 55 and 48 kDa

(18).

If the size of the HSV DNA

primase

is indeed 40 kDa, it is

possible

that it is derived from the

catalytic

subunit of the HeLa

primase.

In this case, how-ever,itwould be

expected

thatsome

epitopes

recognized by

these

polyclonal

antibodies raised

against

the calf DNA

primase

would be retained and that some

reactivity

with these antibodies would have been observed with the HSV primase. No such

reactivity

was observed.

Thus,

while we

cannot

absolutely

ruleoutthissecond

possibility,

webelieve itto be

unlikely

on the basisof the above lineof

reasoning.

A third

possible

source of the HSV

primase

could be mitochondrial DNA

primase,

which is solubilized and be-comes

fortuitously

associated with HSV DNA

polymerase

during

the extraction

procedure.

This

possibility

also does not seem

likely.

TheratlivermitochondrialDNA

polymer-ase hasa

pH

optimum

of

9.3,

with very little

activity

below pH8.5(23).Further,the humanmitochondrialDNA

primase

hasbeen showntobe boundto

RNA,

which,

when

removed,

leaves an enzyme that sedimentsatthe same rate astheE. coli

polymerase

I

large

fragment (37).

If the

protein

is

spherical,

it would

correspond

to a molecular size ofabout 70kDa. In the

light

of these

considerations,

the control DNA

primase

we have observed may represent a mixture of mitochondrialDNAprimaseandHeLa DNA

primase

onthe basis of the lack of a

pH

optimum

(Fig. 4B)

and a small amount of

reactivity

with the antibodies raise'd

against

the calfDNA

primase peptides (data

not

shown).

Thefinal

possibility

tobeconsidered is that the HSV DNA primase

activity

is in fact a virus-coded enzyme. The

cou-pling

of the

primase

withtheviralDNA

polymerase

in fresh extracts,the

unique pH

profile

of its

activity,

and its lack of

cross-reactivity

with antibodies

against

the calf

primase

all

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argue that this may be the case. Challberg (4; personal communication)hasrecentlydemonstrated that thereare at least seven openreadingframes presentonthe HSV genome whose products are essential for the replication of HSV DNA.Thegeneproducts potentiallyencodedbythese open reading frames range in size from 140 to 51 kDa. The two largestopenreadingframescorrespondto the genesforthe viral DNA polymerase and the HSV major DNA-binding

protein,

ICP8. The geneproducts from theother five open reading framesarealllargerthan the apparentmolecularsize of40kDa which wehavedetermined forthe HSV

primase.

Thiswouldimplythat if the HSVprimase isencodedbyone ofthese openreading frames,theactivitywhichwedetected represents a posttranslationally modified protein. Whether this modification is required for the activity of the HSV primaseorrepresents nonspecific proteolytic degradationof theprimase during extraction and partial purification ofthe enzyme is notknown.

Furtherexperiments needto be carriedout to purify the HSV DNA primase to homogeneity, to characterize its enzymatic activity in depth, and to demonstrate that the primase can synthesize RNA primers of the size seen in newly replicating HSV DNA, both in the presence and absence of HSV DNA polymerase and deoxynucleoside triphosphates. These last experiments would demonstrate theeffectsof the DNApolymeraseonprimer synthesisand theuseofsuchprimers bythe DNApolymerase. However, suchproductcharacterization is meaninglesswith the HSV DNAprimase at its present level ofpurification because of the presence of other activities, such as RNase H, which affect the size of the product or the ability of the DNA polymerase to use these products. Additional work also needstobe donetodefinitively show that the DNAprimase is partofanHSV DNApolymerase:primasecomplex andto discover other proteins which may be involved in holding enzymatically active peptides together or in coupling the DNA primase activity with the DNA polymerase activity. Theseexperiments areunder way.

ACKNOWLEDGMENTS

The expertassistance of Steven C.Maresinthepreparationand

chromatography of cell extracts is gratefully acknowledged. We

thankF. J. Bollum, L.M. S. Chang,and M.Challberg for helpful discussionsduringthecourseof this work.

This workwassupported by Public Health ServicegrantA122468 fromtheNationalInstitute forAllergy and InfectiousDiseases (to W.T.R.)and UniformedServices Universityof the Health Sciences grant R07156(toA.M.H.).

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