Processive Replication of Single-stranded DNA Templates by the Herpes Simplex Virus-induced DNA Polymerase*

THE JOURNAL OF BOLOGCAL CHEMSTRY 0 987 by The American Society of Biological Chemists, nc. Vol. 6,. 9, ssue of March 5, pp. 45-459,987 Printed in U.S.A. Processive Replication of Single-stranded DNA Templates by the Herpes Simplex Virus-induced DNA Polymerase* Michael E. O DonnellS, Per Eliass, and. R. Lehman (Received for publication, September, 986) From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 The DNA polymerase encoded by herpes simplex virus consists of a single polypeptide of M, 36,000 that has both DNA polymerase and 3 +5 exonuclease activities; it lacks a 5 +3 exonuclease. The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular singlestranded DNA (turnover number -0.5 nucleotide). Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (& e nm). The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number -4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the singlestranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 0-30 min required to replicate the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles. ATP (or datp) is not required to initiate or sustain highly processive synthesis. The 3 +5 exonuclease associated with the herpes DNA polymerase binds a 3 terminus tightly (K, < 50 nm) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki-4 h ~ ) suggesting, close communication between the polymerase and exonuclease sites. The replication of a duplex DNA chromosome requires the concerted action of several proteins that are thought to assem- Biochemical Co. Phosphonoacetic acid was obtained from Sigma. Bio-Gel A-.5m and protein molecular weight markers were obtained ble into a multiprotein complex (). To understand the mo- from Bio-Rad. Plastic-backed polyethyleneimine-cellulose sheets lecular mechanism by which a eukaryotic chromosome is (Polygram MN300) were obtained from Brinkmann nstruments; replicated, we have chosen to study herpes simplex virus Centricon 0 was from Amicon. (HSV-l). The HSV- genome which is a linear duplex ap- Bufiers-Buffer A was 0 mm Tris-C (ph 7.5), 6 mm MgClz, 4% proximately 50 kb in length encodes many of the enzymes required for its replication, including a DNA polymerase and glycerol, 0. mm EDTA, 40 pg/ml bovine serum albumin, 5 mm * This research was supported by Grant GM0696 from the National nstitutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 8 U.S.C. Section 734 solely to indicate this fact. $ Fellow of the Helen Hay Whitney Foundation. Present address: Dept. of Microbiology, Cornel University Medical School, 300 York Ave., New York, NY 00. 5 Supported by a fellowship from the Knut and Alice Wallenberg Foundation, Sweden. The abbreviations used are: HSV, herpes simplex virus; Hepes, 4-(-hydroxyethyl)-l-piperazineethanesulfonic acid; dntps, deoxyribonucleoside triphosphates; MMP-PNP, -deoxy-5 -adenyl imidodiphosphate; $X, bacteriophage 4x74; ssdna, single-stranded DNA; RF, closed circular duplex DNA; RF, circular duplex DNA with a nick in one strand; kb, kilobases; SDS, sodium dodecyl sulfate; SSB, E. coli single-stranded DNA-binding protein; CP8, infected cell protein 8. 45 a single-stranded DNA-binding protein (). Partial purification of the herpes DNA polymerase has shown it to be an approximately 50-kDa polypeptide (3) in good agreement with the molecular mass of 36 kda predicted from the nucleotide sequence of the gene (4, 5). As a first step in our analysis of HSV- DNA replication, we have purified the herpes-induced DNA polymerase to homogeneity and exam- ined the dynamics of its replication of ssdna templates. A second viral encoded protein known to be essential for HSV- DNA replication, CP8 (6-8), binds ssdna tightly and cooperatively (9) and is therefore analogous to the prokaryotic single-stranded DNA-binding proteins typified by Escherichia coli SSB and T4 gene 3 protein (). The interaction of CP8 with the DNA polymerase in the presence of single- and double-stranded DNA templates is the subject of the accompanying paper (0). EXPERMENTAL PROCEDURES Materials-Unlabeled and labeled nucleotides were purchased from Pharmacia P-L Biochemicals and Amersham Corp., respectively. damp-pnp was a gift from Dr. B. Alberts (University of California, San Francisco). $X and Ml3Goril viral DNAs were prepared as described (); all viral DNA concentrations are expressed as DNA molecules and were calculated using an AZW of as equivalent to 36 pg/ml. (da)lm and (dt)7 were purchased from Pharmacia P-L Biochemicals. Calf thymus DNA, purchased from Sigma, was activated as described (). SSB (4 X lo4 units/mg) (3) was a gift from Dr. D. Soltis (this department). DNA polymerase holoenzyme fraction V (7 X lo6 units/mg) was prepared as described (4). E. coli DNA ligase was prepared as described (5). T4 DNA polymerase and T4 polynucleotide kinase were obtained from the United States dithiothreitol. Buffer B was 0 mm Hepes/Na+ (ph 7.5), 0.5 mm dithiothreitol, 0.5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0% (w/v) glycerol. Buffer C was 50 mm Tris-C (ph 7.5), 50 mm (NH,),SO,, 0.5 mm dithiothreitol, 0. mm EDTA. Cells and Viruses RA305 (6), a thymidine kinase-deficient mutant of HSV- [F], was used to infect roller-bottle cultures of Vero cells using a multiplicity of infection of 5-0 plaque-forming units/ cell. Purification of HSV- DNA Polymeruse-The herpes-induced DNA polymerase was purified by a modification of the method of Powell and Purifoy (3). The steps in the purification up to chromatography on phosphocellulose have been described (7). Briefly, nuclei were prepared from 50 roller bottles of infected cells (3 g, wet weight) and extracted with.7 M NaC. The DNA was removed by ultracentrifugation at 00,000 X g, and the supernatant (00 ml) was dialyzed for 6 h against two changes ( liters each) of buffer B and loaded onto a phosphocellulose column (6-ml bed volume) equilibrated with buffer B. The phosphocellulose column was eluted with a linear gradient from 0. to 0.6 M NaCl in a total volume of 00 ml of buffer B. Herpes DNA polymerase activity eluted at approximately 0.3 M

TABLE Purification of HSV- DNA polymerase from HSV--infected Vero rdls Fraction Total Total Specific Overall Purifiprotein activitf activity yield cation units x unitslmg mg 0" x 0-6 7% -fold a. Cytoplasm.9 67 0.79 b. Nuclear extract Before dialysis 3 3.0 0.5 8 After dialysis 60 4.0 3 88 6b. Phosphocellulose 7.9 4.8 6 30 6. DNA-cellulose 0.30 3.7 30 3 36 V. Glycerol gradient 0..6 00 6 583 One unit is eaual to pmol of DNA synthesis in 0 min at 37 "C. Relative to the combined fractions a and b (before dialysis). NaC. Polymerase fractions were pooled, dialyzed, and applied to a 5-ml ssdna-cellulose column (prepared as described in Ref. 8) equilibrated with buffer B containing 30% (w/v) glycerol. The column was eluted with a linear gradient from 0. to.0 M NaCl in a total volume of 00 ml of buffer B containing 30% (w/v) glycerol. DNA polymerase eluted at approximately 0.3 M NaCl. Active fractions were pooled, dialyzed against buffer C containing 5% (w/v) glycerol, and concentrated by ultrafiltration in a centrifuge (Centricon 0) to 0.75 mg/ml in a total volume of 400 pl. The concentrated DNA polymerase fraction was divided into two equal portions, and each was loaded onto a 0.6-ml linear 0-30% gradient of glycerol in buffer C. The glycerol gradients were centrifuged at 40,000 rpm in a Beckman SW4.Ti rotor for 40 h at 4 "C. After centrifugation, 0.-ml fractions were collected from the bottom of the tubes. The purification is summarized in Table. Fractions at each step of purification were also analyzed by electrophoresis in a 7.5% SDS-polyacrylamide gel, and proteins were visualized by staining with Coomassie Blue (9). Protein concentration was measured by a modification of the method of Bradford (see Ref. 0) using bovine serum albumin as a reference. The concentration of active DNA polymerase molecules was deter- mined from the amount of SSB-coated singly primed circular @X ssdna that was replicated by a given amount of polymerase in 40 min at 30 "C (assuming one polymerase molecule/primer terminus). The number of circles replicated was calculated from the total amount of DNA synthesis, the specific activity of nucleotides, and the size of the primed ssdna circle () (i.e. moles of nucleotide polymerized per 5386). Templates-The sequence and synthesis of the deoxyoligonucleotide 5-mer primers are described in a previous report (). n most of these studies, the @X ssdna was primed with primer ; in one experiment, the ssdna was primed with primer 4 (see text). The M3Goril ssdna was primed using primer 5. Hybridization of synthetic primers (. p~ 5-mer) to viral ssdna (60 nm circles) was in 0 mm Tris-C, 0.3 M NaCl, and 0.03 M sodium citrate (final ph 8.5). The hydridization mixture was heated briefly to 00 "C, allowed to cool to room temperature over 0 min, and incubated a further h at 30 "C. Primed @X ssdna labeled at the 5' terminus of the synthetic DNA primer was made by labeling the primer with T4 polynucleotide kinase and [T-~~PATP, and then hybridizing the labeled primer to @X ssdna, followed by filtration on Bio-Gel A-.5m in 0 mm Tris- C (ph 7.5), 0.5 mm EDTA, 00 mm NaCl to remove excess labeled ATP and primer. (da)000.(dt)7 was made by incubating (da)lwo (0.5 mg/ml) and (dt),, (5 pg/ml) in 0 mm Tris-HC, 0.3 M NaC, and 0.03 M sodium citrate (final ph 8.5) at 30 "C for h. Synthetic 3' end-labeled hairpin templates (synthetic 57-mers) with either a paired 3'-deoxyadenylate or unpaired 3"deoxythyrnidylate were generously provided by Hisaji Maki (this department).' Measurement of DNA Synthesis-For the assay of DNA polymerase activity during purification, reaction mixtures (5 pl) contained. pg of activated DNA, 0.5 mm ATP, 60 p~ each datp, dctp, and dgtp, 5 pm [3H]dTTP (40 Ci/mmol), and 50 mm (NH,),SO, in buffer A contalnlng 3 mm MgCl,. The reaction was initiated by adding pl of enzyme fraction containing up to 30 units of DNA polymerase. ncubation was for 0 min at 37 "C. Reactions were quenched with ml of cold 0% trichloroacetic acid, 0. M pyrophos- H. Maki and A. Kornberg, manuscript in preparation. HSV- DNA Polymerase 453 phate. ncorporation of labeled nucleotide into acid-insoluble material was measured as described (). Specific details of DNA synthesis on singly primed @X ssdna are given in the figure legends. Reactions were incubated at 30 "C, and samples taken at the times indicated were quenched by adding them to an equal volume of % SDS, 40 mm EDTA. The samples, after quenching, were usually divided into two parts. One-half was analyzed for DNA synthesis by measuring the total nucleotide incorporated into acid-insoluble material as described (). (The values for DNA synthesis refer to the entire reaction mixture). The other half was used for the analysis of the replication products by electrophoresis in neutral 0.8% agarose gels as described (). For autoradiography, dried gels were exposed to Kodak XAR-5 x-ray film. Measurement of3"hj' Exonuclease Actiuity-Synthetic DNA hairpin templates containing either paired or unpaired 3"labeled termini were used as substrates for measurement of 3'+5' exonuclease activity. Reaction mixtures (.5 pl) contained 3. pmol of hairpin template, 83 fmol of DNA polymerase, and 60 mm NaCl in buffer A. When present, dntps were 60 p~ each. ncubation was at 30 "C. Samples ( pl) were quenched instantly upon being spotted onto polyethyleneimine-cellulose strips containing ATP and ADP as carrier and developed in M formic acid and 0.5 M LiCl. Positions of unused substrate (origin) and dtmp or damp (near the solvent front) were identified by autoradiography and cut out, and their radioactivity was determined by scintillation counting. RESULTS HSV- DNA Polymerase s Stimulated by E. coli SSB- The HSV- DNA polymerase was only minimally active with circular 4X ssdna (5.4 kb) primed with a synthetic 5-mer (Fig. ). Activity was, however, stimulated more than 0-fold upon coating the primed ssdna with E. coli SSB (Fig. ). The herpes DNA polymerase was also stimulated (.5-5-fold) by 50 mm NaCl (data not shown), the extent of stimulation depending upon the relative amounts of DNA polymerase and DNA in the assay. The influence of ionic strength on polymerase activity when the herpes DNA polymerase was in molar excess over singly primed ssdna circles is shown in Fig.. DNA polymerase activity was stimulated %fold by 50 mm NaCl at early times 0 0 30 40 50 60 MNUTES FG.. Stimulation of HSV- DNA polymerase by E. coli SSB. Herpes DNA polymerase (3 fmol) was incubated with 78 ng of singly primed 4X ssdna (45 fmol as circles) in buffer A (68 pl) containing 60 p~ each dctp and dgtp. The reaction mixture was incubated 3 min and then replication was initiated by addition of 7 plof.5 mm datp and 0.5 mm [a-3p]d'm'p (400 cpm/pmol). Samples (0 pl) were removed at the times indicated, quenched, and analyzed for DNA synthesis as described under "Experimental Procedures." A, no additions; 0, ssdna was coated with 0.78 pg of SSB.

HSV- DNA Polymerase 454 of SSB, theherpes DNA polymerase was maximally active on SSB-coated ssdna at anionic strength of approximately 70 mm (data notshown). HSV- DNA Polymerase s Highly Processiue-DNA synthesis by the herpes DNA polymerase withssb-coated 5 ssdna as template reached a plateau value after 0-30 min even though all of the available DNA had notbeen replicated -(Fig. ). Moreover, the extent of DNA synthesis was proportional to the amount of DNA polymerase added (data not - 0 shown). The limited DNA synthesis suggests a highly procesy sive mode of nucleotide polymerization wherein each polymx erase molecule completely extends a DNA primer around the 5> 5 ssdna circle without dissociation and is slow in cycling to a another primed template. z n Analysis of replication products from singly primed SSB0 coated ssdna by native agarose gel electrophoresis supports the highly processive mode of nucleotide polymerization (Fig. 3B). During the time in which full-length products (RF ) 5 were formed, most of the primed ssdna remained unchanged (detected by UV-induced ethidium bromide fluorescence). The 0 min required for the complete replication of a 4X ssdna molecule (5.4 kb) yields an average turnover number 5 0 5 of 4.5 nucleotides/s/polymerase molecule. The lack of signifmnutes icant radioactivity in theregion of the gel between the primed FG.. nfluence of ionic strength on replication of singly ssdna and RF product after 30 min indicates that cycling primed &X ssdna. Reaction mixtures (5 pl) contained 04 fmol of singly primed @XssDNA (as circles), 500 fmol of herpes DNA of the polymerase to otherprimed ssdna molecules is slow. polymerase, 0.5 mm ATP, 60 p~ dctp, dgtp, and datp, and 0 That the remaining primed circles were effective templates was demonstrated by their replication upon further addition p~ [m3*p]dttp(3000 cpm/pmol) in buffer A. Samples (0 pl) were removed at the indicated times, quenched, and analyzed for DNA of DNA polymerase (not shown). synthesis as described under Experimental Procedures. The conthe herpes DNA polymerase was also highly processive in centration of NaCl in individual reactions was: A, 0; 0, 3 0 mm; A, 60 the absence of SSB. An agarose gel analysis of the replication mm; 0,90 mm; 0, 0 mm; 0, 5 0 mm. products formed with singly primed 4X ssdna showed that A ) NO ADDTON B) SSB ADDED most of the DNA templates had not reacted; nevertheless, discrete, partially replicated species and some fully replicated MN: 0 0 40 60 MN: 3 6 0 0 30 RF molecules were evident (Fig. 3A). The processivity of the HSV- DNA polymerase was demcrfll a-rf onstrated in a second type of experiment diagramed in Fig. 4A. The DNA polymerase was preincubated with an 8-fold excess of singly primed +X ssdna coated with SSB so that each polymerase molecule was bound to a primer terminus. dctp and dgtp (the 3 terminal nucleotides of the primer and the first4 nucleotides needed for synthesis) were present during the preincubation to protect theprimer from removal of the terminalnucleotides by the 3 +5 exonuclease activity of the polymerase (see below). After a short preincubation -%DNA period, an excess (%fold over 4X ssdna circles) of challenge cssdna DNA, i.e. singly primed,m3goril ssdna (8.6 kb), was FG. 3. Time course of replication of singlyprimed &X added. After further preincubation for either 5 s or 3 min, ssdna analyzed by neutral agarose gel electrophoresis. Au- replication was initiated by the addition of datp and[ c Y - ~ ~ P ] toradiogramsof 0.8%neutral agarose gel electrophoresis of replication dttp; after6 min, further incorporation of radioactivity was reactions of Fig. in the absence of SSB ( A )and replication reactions prevented by addition of excess unlabeled dttp. ncubation of Fig. in the presence of SSB (B). Electrophoresis and autoradiog- for an additional 40 min ensured complete replication of raphy were as described under Experimental Procedures. The arrows mark the,position of RF DNA and singly primed ssdna templates towhich a polymerase molecule was bound at the time unlabeled dttp was added. Analysis of the replication standards detected by UV-induced ethidium bromide fluorescence. products by agarose gel electrophoresis is shown is Fig. 4B. in the reaction (-3 min). However, at later times (after 5 Essentially allthe label was incorporated into the4x ssdna min), thepolymerase activity was maximal at 60-90 mm NaCl template following the 5-s preincubation period. Hence, the and then decreased progressively as the salt concentration herpes DNA polymerase remained bound to the 4X DNA was increased. Analysis of the products of replication by during synthesis.after a 3-min preincubation period, most of neutral agarose gel electrophoresis showed that most of the the radioactivity was associated with the 4X DNA, showing only minimal transfer of the herpes polymerase to the chalprimers were extended to a few uniquepositions on the circular template (data notshown, but see Fig. 3A). A differ- lenging M3Goril DNA before initiation of synthesis.a ent setof polymerase pause or termination sites was observed controlreaction in which the polymerase was added to a with another 5-mer (primer 4 in Ref. ) which hybridizes mixture of $X and M3GorilssDNAs showed about twice as to 4X ssdna at a site.9 kb distant from the primer 5- much incorporation of labeled nucleotide into the M3Goril $X ssdna (Fig. 4B, third l a n e ), consistent mer. n contrast to thestimulation of activity in the absence ssdna as into the 30 v) v) 0 NaCl

HSV- DNA Polymerase A) 455 [3u-P]dTTP DNA '$xl 74 dctp G o r i ldgtp M3 CHALLENGE SSDNA -- M 3 G o r ir l Fll- 0x74 R F FG.4. Processive DNA replication by herpes DNA polymerase in presenceof challengingtemplate. A, scheme of challenge experiment. Herpes DNA polymerase (7 fmol) was preincubated for 3 min (30 "C) with 0 fmol of singly primed 4X ssdna (as circles) coated with SSB (. pg) in 5 p of buffer A containing 60 p~ each dctp and dgtp.after the preincubation, the reaction was mixed with a solution (5 pl) containing 40 fmol of singly primed M3Goril ssdna (as circles) and 6.9 pg of SSB in buffer A containing60 p~ each dgtp anddctp. After further incubation for either 5 s or 3 min, pl of a solution containing.5 mm datp and 0.5 mm [m3*p] dttp (375 cpm/pmol) was added. After 6 min, excess unlabeled dttp was added ( mm final concentration), and the incubation was continued for 40 min before quenching with an equal volume of %SDS and 40 mm EDTA. B, autoradiogram of 0.8% neutral agarose gel electrophoresis of replication reactions. Addition of datp and [ c Y - ~ ~ P ~was T T Peither 5 s (first lune) or 3 min (second l a n e ) after addition of Ml3Goril DNA. The third lune (marked C ) is a control reaction where herpes DNA polymerase was added to a mixture of the @X(0 fmol) and Ml3Goril (40 fmol) ssdnas. The tick marks correspond to theposition of DNA standards detected by UVinduced ethidium bromide fluorescence. with the molar excess of M3Goril over 4X ssdna circles. The concentrations of primed DNA circles and HSV- DNA polymerase in the experiments of Figs. 3 and 4 provide an upper limit of nm for the equilibrium dissociation constant (&) for polymerase binding to a primer-template. Unlike E. coli DNA polymerase holoenzyme, whose tight binding to DNA and highly processive DNA synthesis requires ATP (or datp) hydrolysis, there was no effect of ATP or damp-pnp on processive DNA synthesis by the HSV- DNA polymerase (data notshown). HSV- DNA Polymerase Completely Replicates Primed 4X ssdna Circles-To determine the extent to which the circular DNA template is replicated by the herpes DNA polymerase, the products of replication of singly primed 4X ssdna were treated with E. coli DNA ligase, which requires directly apposing 3'-hydroxyl and 5'-phosphoryl termini toform a phosphodiester bond (3). n the experiment shown in Fig. 5, a portion of the reaction mixture was removed after 0 min, and E. coli DNA ligase and NAD+ were added. Samples were removed after an additional 0, 0, and 30 min of incubation and analyzed by electrophoresis in an agarose gel containing ethidium bromide (Fig. 5B, eighth to tenth lanes). Approximately 70% of the RF products of polymerase action were sealed by the DNA ligase within 30 min and asa consequence co-migrated with a closed circular duplex DNA marker. t therefore appears that synthesis by the herpes DNA polymerase proceeds directly to the 5' terminus of the primer, leaving a nick that canbe sealed by DNA ligase. HSV- DNA Polymerase Lacks 5 ' 4 ' Exonuclease Activity-The experiment used to test for 5'+3' exonuclease activity associated with the herpes DNA polymerase is diagramed in Fig. 5A. A -fold molar excess of polymerase was added to SSB-coated 4X ssdna primed with a synthetic 5mer labeled with 3Pa t its 5' terminus. Samplesof the reaction were removed a t intervals up to 30 min, the time required to replicate the entire circular template. f the polymerase has significant 5'43' exonuclease activity, it should remove the 3P-labeled 5"terminal nucleotide of the primer upon complete replication of the template. As shown in Fig. 5B, the 5'terminal nucleotide persisted throughout the 30-min period of replication. Quantitation of radioactivity of excised gel slices showed that after30 min, the 3Pat theposition of the completed RF circles was approximately 75% that of the primer before the beginning of replication (0 min); 9%of the remaining 3Pwas present in the smear below the RF products. The reaction of Fig. 5 was initiated by adding DNA polymerase to a solution containing the primed DNA and all four dntps; hence, the replication intermediates appeared as a smear. A discreteband of replication intermediates (as infig. 3) is observed only when the polymerase is preincubated for a brief period with the primed DNA and synchronous replication is initiated by addition of the dntps. Herpes DNA Polymerase and 3 ' 4 ' Exonuclease Are Present within the Same Polypeptide-The HSV- DNA polymerase and its associated 3'45' exonuclease co-sediment perfectly in a 0-30% glycerol gradient at theposition of a 58kDa marker protein (Fig. 6A), consistent with previous reports (4, 5). Moreover, as shown in Fig. 6B, the herpes DNA polymerase is nearly homogeneous (>go%) as judged by Coomassie Blue staining of the gradient fractions following SDS-polyacrylamide gel electrophoresis. The DNA polymerase and 3'+5' exonuclease active sites would therefore appear to reside within the same polypeptide chain. The herpes polymerase showed no detectable primase activity (data not shown). 3 ' 4 ' Exonuclease Has Proofreading Activity-An earlier report demonstrated that the 3'+5' exonuclease associated with the herpes DNA polymerase has proofreading activity (6). We have examined the proofreading capacity of the 3'+

HSV- DNA Polymerase 456 A) 5 LABELLED DNA 5-MER V RFl DNA LGASE RF * B) MNUTES: 0 3 6 0 5 030 0 0 30 =-RFl --RFl (Table )is considered more fully in the accompanying report (0). During DNA synthesis with the homopolymer template, (da)looo. (dt),,, a significantamount of dtmp was generated, approximately 0% of the level incorporated into the homopolymer (compare Fig. 7, B and C). However, in the absence of aprimer-template, dtmp was not produced (data not shown). The dtmpis most likely formed upon hydrolysis of paired 3 termini during DNA synthesis as observed previously for the herpes polymerase (5) and for other DNA polymerases that contain3 +5 exonuclease activity (). Phosphonoacetic Acid nhibits DNA Polymerase and 3 4 Exonuclease to Equal Extents duringdna Synthesis-Phosphonoacetic acid inhibits both the polymerase and 3 +5 exonuclease activities of the herpes DNA polymerase with a K j value of approximately 4 p M (Fig. 8, A and B ). n the absence of dntps, theki for phosphonoacetic acid inhibition of the 3 +5 exonuclease on the hairpin template with either paired or unpaired 3 termini is 50 p~ (Table ), the same as the K, for hydrolysis of both paired and unpaired 3 -terminal nucleotides (see above). DSCUSSON Despite its slow rate of DNA synthesis, the HSV--induced +ssdna DNA polymerase is strikingly processive. Once bound to its primer-template, the herpes polymerase does not dissociate FG.5. Complete replication of primed &X ssdna circles by herpes DNA polymerase which lacks 5 43 exonuclease ac- during the approximately 0 min required to fully replicate a tivity. A, scheme for detecting 5 +3 exonuclease activity activity 5.4-kb +X ssdna circle. This highly processive mode of and complete replication of ssdna. The 5 end-labeled 5-mer primer nucleotide polymerization may be ofimportance in replicating annealed to @XssDNA was prepared as described under Experimen- the 50-kb viral chromosome and, even more important, in tal Procedures. A slight excess of herpes DNA polymerase (000 the synthesis of multiple copies of the genome in rolling circle fmol) was added to initiate replication of the singly primed @XssDNA DNA replication (). Although it is not known whether Oka(.3 pg, 750 fmol as circles) in 50 pl of buffer A containing 3 pg of SSB, 0.5 mm ATP, 60 p~ dctp, dgtp, and datp, and0 p~ [3H] zaki fragments are intermediates in replication of the herpes removed and chromosome, the high processivity, replication to a nick sealdttp (500 cpm/pmol). After 0 min, 50plwas incubated with NAD+ (0. mm final concentration) and 0.3 pg of E. able by DNA ligase, and lack of 5 +3 exonuclease activity coli DNA ligase.samples (.5 pl) were removed at thetimes indicated are clearly desirable features in thesynthesis of discontinuous and quenched with SDS/EDTA; the DNA products were analyzed by DNA fragments. neutral agarose gel electrophoresis; and DNA synthesis was quantithe rateof fork movement during replication of pseudoratated as described under Experimental Procedures.?,autoradibies virus, a member of the herpes virus family, is approxiogram of 0.8% neutral agarose gel electrophoresis of the products of replication. Eight to tenth lunes, the time noted is after the addition mately 50 nucleotides/s at 37 C (6),similar to that of of ligase. Arrows mark the positions of RF, RF, and ssdna eukaryotic chromosomes (7, 8). One might therefore anticstandards detected by UV-induced ethidium bromide (EtBr)fluores- ipate a turnover number of at least 30-40 nucleotides/s for cence. the herpes DNA polymerase at 30 C. The apparentturnover number of 0.5 nucleotide/s with singly primed 4X ssdna 5 exonuclease using defined synthetic DNA hairpin tem- circles is far too low to sustainproductive herpes virus infecplates (57-mers) having either a paired or unpaired labeled 3 tion (assuming 0,000 copies/cell in 0 h). However, the 0terminus (diagramed in Fig. 7A). n the presence of the 4 fold stimulation of the herpes DNA polymerase upon coating dntps, the 3 +5 exonuclease completely removed the un- the ssdna with E. coli SSB approaches the ratein uiuo. HSV may therefore encode a functional analogue of E. coli SSB. paired3 -terminal nucleotide (Fig. 7B).ncontrast,the Like E. coli SSB, the herpes-induced CP8 binds tightly and paired 3 terminus was stable to the 3 45 exonuclease in the presence of dntps (Fig. 7B), due presumably to theaddition cooperatively to ssdna (9), is essential for ongoing DNA of nucleotides to the paired 3 terminus by the polymerase. replication (8), and is present a t stoichiometric levels (9). However, despite its similarity to the E. coli SSB, binding of The 3 +5 exonuclease hydrolyzed an unpaired 3 -terminal nucleotide at justover twice the rate atwhich the paired 3 - CP8 to ssdna completely inhibits the replication of singly terminal nucleotide was hydrolyzed (Fig. 7C). The rates of primed +X ssdna by herpes DNA polymerase (0). n conhydrolysis of paired and unpaired 3 -terminal nucleotides did trast, CP8markedly stimulates synthesis by the polymerase not change over the range p M to 50 nm template, setting an on duplex DNA templates (0). E. coli DNA polymerase holoenzyme hydrolyzes the P,rupper limit of the K, for hydrolysis at 50 nm (Table ). Since substrate was present at saturating concentrations, apparent phosphodiester bond of ATP (or datp) to initiate highly turnover numbers for removal of paired and unpaired 3 - processive DNA synthesis (). n contrast, the monomeric terminal nucleotides could be calculated from the observed herpes DNA polymerase does not require ATP or datp. hydrolysis of the rates of hydrolysis (Table ). Use of Mn+ inplace of Mg+ Thus, a complex subunitstructureand hadno effect on the rate of hydrolysis of the paired 3 terminal phosphate of ATP or datp are not essential for terminus but stimulatedremoval of the unpaired 3 terminus highly processive DNA synthesis. The response of the herpes DNA polymerase to ionic.5-fold (Table ). The effect of the herpes-encoded ssdnabinding protein,cp8,on the 3 +5 exonuclease activity strength is complex. At a molar excess of DNA polymerase to

HSV- DNA Polvmerase 457 B) - FG. 6. Glycerol gradient sedimentation analysis of herpes DNA polymerase. A, glycerol gradient sedimentation of herpes DNA polymerase is described under Experimental Procedures. DNA polymerase (Pol, 0) was assayed using activated calf thymus DNA, and the 3 +5 exonuclease (Exo, A) was assayed using the synthetic 57mer hairpin DNA with a labeled unpaired 3 terminus as described under Experimental Procedures. Position of protein standards in a parallel glycerol gradient are marked by brackets. B, SDSpolyacrylamide gel electrophoresis of glycerol gradient peak fractions. The tick marks correspond tothe migration of molecular mass standards in the same gel. 40 kda5836 E c + H 00 FRACTON NUMBER 0.. -.- i 5 3 4 ~-e,.... 44 7 -, stds kda -6-9 80-66 y,, 60 ee 5 40 Y Q: - Q - 45 o 30 0 0 FRACTON NUMBER TABLE Steady state kinetic parameters for hydrolysis of pairedand unpaired 3 termini by 3 +5 exonuclease of HSV- DNA polymerase 3 termini A 3 PARED 7 A 8 3 UNPARED Unpaired Paired * Turnover number (nucleotides/s) 30 C, no addition 0.6 0.066 37 C, no addition 0.3 0.073 30 C, CP8 added 0.046 0.78 30 C, Mn+ 0.066 K, 3 termini ( p ~ ) C0.05 <0.05 Ki PAA (pm) 50 7 B) a 0.4 50 PAA, phosphonoacetic acid. primer termini, the polymerase is stimulated at early times of synthesis by an ionic strength of 00 mm; however, it is inhibited at later times under theseconditions. This behavior may be explained by salt stimulation of a rate-limiting step 4 3 in polymerase catalysis while enzyme is bound to DNA, which is offset by an increase in the stability of DNA sequencespecific pause sites at salt concentrations above 00 mm (e.g. hairpin structures). However, at substoichiometric ratios of polymerase to primer termini, polymerase activity is stimulated up to anionic strength of 50 mm. Under these conditions, the polymerase can extend a primer to a sequencespecific pause site and then dissociate from the terminus to gain access to anunused primer terminus.maximum activity of herpes DNA polymerase onssb-coated singly primed ssdna circles at moderate ionic strength (60-70 mm) may result from the combined effect of SSB-induced removal of sequence-specific barriers and anintrinsic stimulation of nucleotide polymerization at thisionic strength. The exceptionally low K,,, value of the 3 4 exonuclease for a primer terminus may underlie the only -fold difference in rates of removal of paired and unpaired 3 termini. The 3 4 3 +5 exonuclease of the DNA polymerase isolated from MNUTES HSV--infected cells has been reported to remove a 3 -unfg. 7. Comparison of paired and unpaired 3 termini as paired terminus six times faster than a 3 -paired terminus in substrates for the 3 +5 exonuclease of herpes DNA polym. ~ basis for the discrepancy between erase. A, diagram of synthetic DNA hairpin substrates labeled at the the absence of ~ N T P sthe that result and the one presented here is unclear, but may 3 terminus with either a paired deoxyadenylate or unpaired deoxyand/or source of thymidylate residue; B, plots of 3 +5 exonuclease activity on the reside in the differentassayconditions 3 -paired and -unpaired hairpin templatesin thepresence of dntps; enzyme. C, plots of3 +5 exonuclease activity on 3 paired and -unpaired hairpin templates in the absence of dntps. Reactions were as described under Experimental Procedures. J. Abbotts, Y. Nishiyama, S. Yoshida, and L. A. h e b, unpublished observations.

458 HS V- DNA Polymerase FG. 8. Effect of phosphonoacetic acid on herpes DNA polymerase and 3 45 exonuclease during DNA synthesis. The reaction mixture (50 pl) contained.5 pg of (da)lm.(dt)l, (: molar ratio), 40 fmol of DNA polymerase, 60 mm NaCl, and 0 p~ [3H]dTTP (8.000 cpm/pmol) in buffer A. After 3 min at 30 C, 0-pl samples were placed in separate tubes containing pl of HzO, pl of 44 p~ phosphonoacetic acid, or pl of. mm phosphonoacetic acid. Samples ( pl) were removed at the times indicated and quenched immediately upon adsorption to a polyethyleneiminecellulose strip. Chromatography and quantitation of dtmp and products of synthesis (origin) were as described for the 3 4 exonuclease assay under Experimental Procedures. PAA, phosphonoacetic acid. A) DNA POLYMERASE 8) 3 5 EXONUCLEASE 0 / ADDTON / NO 5 0 MNUTES 5 0 MNUTES TABLE ComDarison of HSV- DNA Dolvmerase with DNA Dolvmerase 0 Mr. of subunits Primase 3 +5 exonuclease 5 +3 exonuclease Rate of polymerization (nucleotides/s/enzyme)b Processivity Effect of 0. M NaC nhibition by PAAd HSV- DNA polymerase 36.000 i Yes 4.5 >5000 Stimulates Yes DNA polymerase a 80,000 4 Yes 5 nhibits DNA polymerase from Drosophila embryos (33). * Singly primed 4X ssdna coated with E. coli SSB as template. e Activated calf thymus DNA template. PAA, phosphonoacetic acid. t is not surprising that the 3 45 exonuclease and polymerase activities share the same polypeptide chain. Other polymerases with associated exonuclease activity have both active sites on a single polypeptide (). However, the roughly equal sensitivity of the herpes polymerase and 3 +5 exonuclease activities to inhibition by phosphonoacetic acid during DNA synthesis was unexpected. n the case of the E. coli DNA polymerase large fragment (3), crystallographic anal- ysis has demonstrated that the two active sites are physically separated from each other by approximately 5 A (3). Possibly, phosphonoacetic acid binds to one active site and thereby prevents switching of the primer 3 terminus to the other site. The herpes polymerase differs in many important respects from DNA polymerase (Y (Table ). These differences may reflect differences in the complexity of replicating host chromosomes organized into a complex nucleosomal structure as compared with the relatively simple 50-kb HSV- genome. n both cases, however, the DNA polymerase is very likely associated with accessory replication proteins that could very significantly alter their catalytic properties. Acknowledgment-We are grateful to Ed Mocarski for expert advice and assistance in the handling of cells and viruses. REFERENCES. Kornberg, A. (980) DNA Replication, W. H. Freeman, San Francisco. Roizman, B., and Betterson, W. (985) in Virology (Fields, B. N., ed) pp. 497-56, Raven Press, New York 3. Powell, K. L., and Purifoy, D. J. (977) J. Virol. 4, 68-66 4. Gibbs, J. S., Chiou, H. C., Hall, J. D., Mount, D. W., Retondo, M. J., Weller, S. K., and Coen, D. M. (985) Proc. Natl. Acad. Sci. U. S. A. 8, 7969-7973 5. Quinn, J. P., and McGeoch, D. J. (985) Nucleic Acids Res. 3, 843-863 6. Conley, A. J., Knipe, D. M., Jones, P. C., and Roizman, B. (98) J. Virol. 37, 9-06 7. Weller, S. K., Lee, K. J., Sabourin, D. J., and Shaffer, P. A. (983) J. Virol. 45, 354-366 8. Shaffer, P. A., Bone, D. R., and Courtney, R. J. (976) J. Virol. 7,043-048 9. Ruyechan, W. T. (983) J. Virol. 46,66-666 0. O Donnell, M. E., Elias, P., Funnell, B. E., and Lehman,. R. (987) J. Bwl. Chem. 6,460-466. Eisenberg, S., Harbers, B.. Hours,. C... and Denhardt. D. T. (975).. J. Mol.-koi. 99, 07-3. Aposhian, H. V., and Kornberg, -. A. (96). J. Biol. Chem. 37. 59-55 3. Soltis, D. A., and Lehman,. R. (983) J. Bwl. Chem. 58,6073-6077 4. McHenry, C., and Kornberg, A. (977) J. Biol. Chem. 5,6478-6484 5. Panasenko, S. M., Alazard, R. J., and Lehman,. R. (978) J. Bwl. Chem. 53,4590-459 6. Post, L. E., Mackem, M., and Roizman, B. (98) Cell 4, 555-565 7. Elias, P., O Donnell, M. E., Mocarski, E. S., and Lehman,. R. (986) Proc. Natl. Acad. Sci. U. S. A. 83, 63-636 8. Alberts, B., and Herrick, G. (970) Methods Enzymol. 3, 908-97 9. Laemmli, U. K. (970) Nature 7,680-685 0. Read, M., and rthcote, D. H. (98) Anal. Biochem. 6,53-64. Sanger, F., Coulson, A. R., Friedmann, T., Air, G. M., Barrell, B. G., Brown, N. L., Fiddes, J. C., Hutchison, C. A.,, Slocombe, P. M., and Smith, M. (978) J. Mol. Biol. 5,5-46. O Donnell, M. E., and Kornberg, A. (985) J. Bwl. Chem. 60, 875-883 3. Lehman,. R. (974) Science 86, 790-797 4. Weissbach, A., Hong, S.-C. H., Aucker, J., and Muller, R. (973) J. Biol. Chem. 48,670-677

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