Herpes Simplex Virus Type I DNA Polymerase

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 16, lssue of August 25, pp Printed m U S.A. Herpes Simplex Virus Type I DNA Polymerase KINETIC PROPERTIES OF THE ASSOCIATED 3-5 EXONUCLEASE ACTIVITY AND ITS ROLE IN araamp INCORPORATION* (Received for publication, March 26, 1981, and in revised form, May 4, 1981) David DerseS and Yung-Chi Chengg From the Department of Pharmacology and Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina and the Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York An exonuclease activity copurified with herpes sim- copurify with HSV DNA polymerase (4,6, 7) which liberates plex virus type I (HSV-1) DNA polymerase through 5 -deoxynucleoside monophosphates from labeled DNA (6). A DNA-cellulose column chromatography and comi- physical association of HSV DNA polymerase with a unique grated with DNA polymerase activity on nondenatur- exonuclease activity was indicated by their cosedimentation ing gel electrophoresis at varied polyacrylamide con- on glycerol gradients at high ionic strength (6, 7) and by their centrations. A gapped duplex DNA was the preferred identical heat denaturation kinetics (7). A functional associasubstrate for this exonuclease activity since the hydro- tion was inferred from the ability of HSV-1 DNA polymerase lytic activity on this type of DNA was much greater to catalyze the template-dependent conversion of deoxynuthan the hydrolysis of either native or heat-denatured cleoside triphosphates to monophosphates (6). DNA. Using 3 -terminally labeled activated calf thymus HSV DNA polymerase has previously been shown to be DNA as substrate, the exonuclease activity was found activated by salt (4, 5). In addition, certain polyamines were to be activated by salt and spermidine in a manner identical with HSV-1 DNA polymerase. This activation shown to stimulate DNA polymerase activity in a manner was accompanied by increases in apparent K,,, and V,, dependent on ionic strength (7). Phosphonoformic acid invalues of the activated DNA substrate. hibited HSV DNA polymerase activity (7) also in an ionic Phosphonoformic acid inhibited both DNA polymer- strength-dependent fashion (7). The kinetic behavior of the ase and exonuclease activities uncompetitively with 3 4 exonuclease activity in the presence of these compounds respect to activated DNA and had a Ki of 2.4 PM at an has not previously been reported. ionic strength of 0.25 p. Of the nucleoside 5 -monophos- A proofreading role has been proposed for the 3 3 exonuphates tested only the purine ribonucleotides inhibited clease activity associated with mammalian DNA polymerase the exonuclease activity. The inhibition was noncom- S and several procaryotic DNA polymerases (10, 17). It could petitive with respect to DNA, and GMP was about twice increase the fidelity of DNA replication by excising misas potent as AMP or IMP. matched nucleotides and might, in a similar way, influence 9-8-D-arabinosyladenine 5 -monophosphate (araamp) the inhibition of DNA synthesis caused by the incorporation could be incorporated into DNA by HSV-1 DNA polym- of fraudulent nucleotides. erase; however, 9-8-D-arabinosyladenine B -triphos- The nucleoside analogue 9-j?-D-arabinofuranosyladenine is phate would not replace datp in supporting in vitro a potent and selective inhibitor of HSV replication in vivo HSV-1 DNA synthesis. AraAMP incorporated into (14-16). The active metabolite, 9-P-D-arabinosyladenine 5 - primer termini caused a significant decrease in the rate triphosphate, inhibits HSV DNA polymerase in vitro and is of subsequent primer elongation. These 3 -terminal competitive with respect to datp (7). The mechanism of araamp residues could be removed by the HSV-1 DNA araa inhibition of DNA replication in vivo and especially its polymerase-associated exonuclease activity in a manantiviral selectivity relative to host is still unclear. It has been ner dependent on GMP concentration. proposed that in addition to a competitive inhibition of DNA polymerase by araatp, 3 -terminal incorporation of 9-/3-~arabinosyladenine 5 -monophosphate prohibits subsequent Herpes simplex virus induces a unique DNA polymerase primer elongation (8). Arguments posed for and against a activity in virus-infected cells (1-3). This DNA polymerase chain termination mechanism in vivo have been based on the has been purified to varying degrees and characterized with presence of araamp residues either at 3 termini or in interrespect to its substrate specificity, reaction optima, and kinetic nucleotide linkage, respectively, in DNA extracted from araabehavior (4-7). An exonuclease activity has been reported to treated cells (8, 9). Interpretations of such experiments were made on the assumption that 3 terminal araamp residues were not actively excised. Recent experiments performed with DNA polymerase 6 indicated that the incorporation of araamp into primer-tem- * This workwas supported in part by Research Project Grant CH29C from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ This work was submitted in partial fulfillment for the degree of Doctor of Philosophy to the faculty of the graduate school, State University of New York at Buffalo, Program of Pharmacology, Hoswell Park Memorial Institute Division. 9 Scholar of the American Leukemia Society and to whom reprint requests should be addressed plate was the basis for inhibition of DNA synthesis in vitro and that the 3 4 exonuclease activity could suppress this The abbreviations used are: HSV, herpes simplex virus; HSV-I, herpes simplex virus type I; araa, 9-0-D-arabinofuranosyladenine; araamp, 9-P-~-arabinosyladenine 5 -monophosphate; araatp, 9-p- D-arabinofuranosyl adenine 5 triphosphate; PFA, phosphonoformic acid.

2 8526 Herpes Simplex Virus Type I DNA Polymerase inhibition by removing 3'-terminal araamp residues (11). These data corroborate a current hypothesis that arabinosylnucleotides are not absolute chain terminators, but rather terminated primers are very slowly elongated. An examination of the primer-template effects of araamp incorporation on in vitro HSV DNA synthesis, with attention to the ofthe role 3'- 5' exonuclease, has not been reported. In this paper we present further evidence for the physical association of a 3'4' exonuclease activity with HSV-1 DNA polymerase and describe its substrate preference and kinetic properties. Furthermore, the in vitro inhibition of HSV-1 DNA synthesis by araatp was studied with respect to the elongation of araamp-terminated primers and the excision of 3"terminal araamp residues. MATERIALS AND METHODS Chemicals-All chemicals were of reagent grade or better. Unlabeled deoxynucleoside triphosphates, deoxyribo- and ribonucleoside monophosphates, dithiothreitol, spermidine, calf thymus DNA, and bovine serum albumin were purchased from Sigma. 9-B-D-Arabinofuranosyl adenine triphosphate was supplied by P-L Biochemicals. ICN was the source of ["HIdCTP, ['HIdGTP, [3H]dTTP, and [3H]- datp. Moravek Co. was the source of [3H]araATP. Pancreatic DNase I was purchased from Worthington Biochemicals. Phosphonoformic acid was a gift from Astra Pharmaceuticals. Agarose was from Bethesda Research Laboratories. Supercoiled PBR322 DNA was a gift of Dr. Koji Nakayama of this laboratory. DNA Substrates-Calf thymus DNA was activated by DNase I digestion by the method described by Baril et at. (12). The reaction was incubated at 25 "C for 15 min and then stopped by the addition of EDTA to 1 mm and heated at 70 "C for 20 min. The activated DNA was dialyzed extensively versus 10 mm Tris-C1, ph 8.0. Native and heat-denatured Escherichia coli [I4C]DNA were prepared as previously described (13). Gapped E. coti [''CIDNA was prepared in exactly the same way as activated calf thymus DNA, described above. In the experiments where 3'-5' exonuclease activity was compared on native, heat-denatured, or gapped E. coli [I4C]DNA, the DNA substrates were carried through the same preparative steps except that DNase I was omitted for the first two substrates. 3'-TerminaJly labeled activated calf thymus DNA was prepared by HSV-1 DNA polymerase-catalyzed incorporation of ['HIdNTPs in a 5-ml reaction which contained 50 m~ Tris-C1, ph 8.0, 4 mmmgc12, 0.5 mm dithiothreitol, 0.5 mg/ml of albumin, 200 m~ KC1, 1.7 mg of activated calf thymus DNA, 8 p~ each r3h]datp, ['HIdGTP, ['HIdCTP, and ['HIdTTP, all at 4 pci/ml, and 120 units of HSV-1 DNA polymerase. After a 30-min incubation at 37 "C the reaction was heated to 65 "C for 15 min, protein was removed by two phenolchloroform extractions, and DNA was separated from unreacted nucleotides by Sephadex G-25 column chromatography. The DNA was then precipitated in 3 volumes of absolute ethanol at -70 "C, lyophilized, and resuspended in 1 ml of IO mm Tris-C1, ph 8.0. The specific activity of the DNA was 1.2 X 10' cpm/pg. Activated calf thymus DNA containing 3"terminal ["HIaraAMP was prepared in a I-ml reaction containing the same buffers and salts as above plus 0.12 mg of activated calf thymus DNA, 1.25 p~ [3H]- araatp (1.25 pci/ml), 0.5 mm GMP, and 12.5 units of HSV-1 DNA polymerase. After a 20-min incubation at 37 "C the reaction was stopped by the addition of EDTA to 10 mm followed by 1 ml of phenol-chloroform (1:l). After phenol-chloroform extractions DNA was dialyzed extensively versus 10 m~ Tris-C1, ph 8.0, 100 mm KCl. ['HIaraAMP-terminated DNA had a specific activity of 672 cpm/pg. Enzymes-HSV-1 (KOS strain) DNA polymerase was purified from infected HeLa Bu cells by the procedure described by Ostrander and Cheng (7). The DNA-cellulose step enzyme preparation used here had a specific activity of at least 1.5 X lo4 units/mg. Enzyme Assays-All enzyme assays were performed at 37 "C. HSV DNA polymerase was assayed as previously described (7). The standard assay mix contained in a volume of 0.1 ml, 50 mm Tris-C1, ph 8.0, 4 m~ MgC12, 0.5 mm dithiothreitol, 0.5 mg/ml of albumin, 200 mm KCl, 120 pg/ml of activated calf thymus DNA, 0.1 mm each datp, dgtp, dctp, and 2 p~ ['HIdTTP. One unit of DNA polymerase activity is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of dtmp into activated DNA per h. HSV alkaline DNase was assayed as described previously (13). DNA polymerase-associated 3'-5' exonuclease was assayed by the same method used for alkaline exonuclease. The reaction mixture of 0.2 ml contained the same components as for HSV DNA polymerase except that dntps were absent and activated DNA was replaced by 10 pg/ml of 3"termindy labeled activated calf thymus DNA (1.2 X 104 cpm/pg). Endonuclease activity was determined by measuring the conversion of supercoiled PBR322 DNA to relaxed and linear forms as demonstrated on agarose gel electrophoresis. Reaction mixes of 40 pl contained 50 IIIM Tris-C1, ph 8.0, 0.2 M KCl, 4 mm MgC12,0.5 mm dithiothreitol, 0.5 mg/ml of serum albumin, 1 pgof supercoiled PBR322 DNA, and 2 units of HSV-1 DNA polymerase. Incubations were done at 37 "C for 30 min; reactions were then stopped by the addition of 10 pl of5% (w/v) sodium dodecyl sulfate, 25% (v/v) glycerol, and 0.25 mg/ml of bromphenol blue. Samples were applied to 1% (w/v) agarose slab gels. Electrophoresis and DNA visualization were performed as described (19). Polyacrylamide Gel Etectrophoresis-Nondenaturing polyacrylamide gel electrophoresis was performed by the method of Fisher and Korn (18). The ratio of acrylamide to methylene bisacrylamide was kept constant at 501. Samples were dialyzed against 100 mm KPO,, ph 7.5,2 mm dithiothreitol, 1 m~ thioglycolic acid, 1 mm EDTA, 20% (v/v) ethylene glycol, and 30% (w/v) sucrose. Aliquots of 0.15 ml were applied to gels (5 X 70 mm). For recovery of enyzme activity gels were sectioned at 4 "C into 2-mm slices, and each slice was eluted in 0.2 ml of 100 m~ KPO4, ph 7.5, 2 mm dithiothreitol, 1 mm EDTA, 1 mg/ml of serum albumin, and 20% (v/v) glycerol at 4 "C. Electrophoretic mobility is expressed relative to bromphenol blue tracking dye. RESULTS Polyacrylamide Gel Electrophoresis-HSV-1 DNA polymerase, DNA-cellulose fraction, was analyzed electrophoretically, as described under "Materials and Methods" on 5%, 6%, and 8% polyacrylamide gels (Fig. 1, A, B, and C, respectively). The eluted 2-mm gel sections were assayed for HSV DNA polymerase and 3'-5' exonuclease activities. As seen in Fig. 1, DNA polymerase activity was coincident with 3'-5' exonuclease activity at each polyacrylamide concentration. In addition, fractions from the 5% polyacrylamide gel were assayed under HSV alkaline DNase reaction conditions. A small but significant DNA hydrolytic activity was observed only in fractions which showed 3'-5' exonuclease activity.2 The HSV-1 DNA polymerase preparation used here was also assayed for the presence of endonuclease activity using PBR322 DNA as described under "Materials and Methods." Using reaction conditions optimal for HSV DNA polymerase activity, but in the absence of deoxynucleoside triphosphate, no endonuclease activity was detected. Substrate Specificity and Kinetic Properties of the 3'4' Exonuclease-Native, heat-denatured, and gapped E. coli [I4C]DNA were compared for their ability to serve as substrates for the DNA polymerase-associated exonuclease activity. Activity was measured at ionic strengths of 0.05 or 0.25 p. As shown in Table I, the hydrolysis of all substrates at the lower ionic strength was poor, and little substrate preference was observed. Assays at the ionic strength of 0.25 p revealed that gapped duplex DNA was digested about seven times faster than either native or heat-denatured DNA. As noted under "Materials and Methods" the gapped E. coli [ 14C]DNA was prepared in exactly the same way as the activated DNA used for DNA polymerase assays. Activated calf thymus DNA, radiolabeled by HSV-1 DNA polymerase-catalyzed incorporation of ["HjdNTPs, was used as the substrate for all subsequent studies of the HSV DNA polymerase 3'-5' exonuclease activity. The effects of ionic strength and spermidine on the associated exonuclease activity were examined. Fig. 2A shows that both HSV-1 DNA polymerase and its associated exonuclease activity were acti- vated by KC1. Maximal activity was observed at 200 mm KCl, or an ionic strength of 0.25 p, for both activities. Fig. 2 B shows ' D. Derse and Y-C. Cheng, data not shown.

3 255 p. B C Herpes Simplex Virus Type I DNA Polymerase 8527 and V,,, values were calculated from least squares analysis of these plots. For HSV-1 DNA polymerase K, (pg/ml) and V,,, (pmol of dtmp incorporated/h) values in the presence of KC1 were: 100 mm, K, = 0.8, V,,, = 38; 125 mm, K, = 0.95, V,,, = 81; 150 mm, K,,, = 2.0, V,,, = 153; 200 mm, K, = 8.6, V,,, = 296. In the presence of100 m~ KC1 and the indicated concentrations of spermidine these values were: 2.5 mm, K,,, = 1.4, V,, = 121; 10 mm, K,,, = 9.9, V,, = 322. For 3-5 Rf FIG. 1. Nondenaturing polyacrylamide gel electrophoresis exonuclease K,,, (pg/ml) and Vmax (ng of DNA hydrolyzed/h) values in the presence of the following concentrations of KC1 of HSV-1 DNA polymerase ml of HSV-1 DNA polymerase, were: 100 mm, K, = 1.4, V,,, = 73; 150 mm, K,,, = 1.7, V,,, DNA-cellulose step, was applied to 5% (A), 6% (B), and 8% (C) = 357; 200 mm, K, = 7.8, VmX = 909. In the presence of 100 polyacrylamide gels prepared and run as described under Materials mm KC1 and the following concentrations of spermidine these and Methods. Current was maintained at 2 ma/gel until the tracking dye band reached the bottom of the gel. After 1 week of elution at values were: 2.5 mm, K, = 1.0, V,, = 208; 10 mm, K, = 4.6, 4 C, aliquots from 2-mm gel slices were assayed for HSV-1 DNA VmaX = 709. polymerase (u) and 3-5 exonuclease activities (M) as Inhibition of the 3-5 Exonuclease-Phosphonoformic described. Activities are expressed in counts per rnin of [JH]dTMP acid, a pyrophosphate analogue, inhibits HSV DNA polymincorporated (M) or counts per min released from 3 labeled erase activity in a manner noncompetitive with respect to activated DNA (M) deoxynucleoside triphosphates and uncompetitive with respect to DNA (7). The HSV exonuclease activity is TABLE I Substrate specificity of HSV-1 DNA polymerase 3-5 exonuclease inhibited by both PFA (7) and its congener, phosphonoacetic acid (6). The kinetics of PFA inhibition of HSV exoactivity nuclease activity is shown in the Lineweaver-Burk plot of Fig. E. coli [I4C]DNA substrates were prepared and exonuclease assays 4. PFA was uncompetitive with respect to activated DNA and were performed as described under Materials and Methods. E. coli had a Ki of 2.4 p~ at the ionic strength of 0.25 p. Like DNA [14C]DNA(specific activity 7200 cpm/pg) was present in all assays at 10 pg/ml. Five units of HSV-1 DNA polymerase were used, and ionic polymerase activity (7), the degree of inhibition of exonuclease strength was adjusted with KCl. activity by PFA was dependent on ionic strength. The effects of nucleotides on the polymerase-associated Ionic strength Native DNA Degf$?d Gapped DNA pmol nucleotide rendered acid soluble 0.05 p u 29 KC1 and spermidine is based on the same kinetic mechanism, that is, increasing concentrations of either compound increase apparent V,,, and K, values of activated DNA. Apparent K,,, exonuclease activity are listed in Table 11. Deoxynucleoside triphosphates caused a reduction in the amount of labeled nucleotides released due to primer elongation and thus protection of labeled 3 terminal deoxynucleotides. Of the various mm KC1 I p mm Spermidine FIG. 2. Effects of KC1 and spermidine on 3-5 exonuclease activity. A, HSV-1 DNA polymerase (A-A) activity and 3-5 exonuclease (t.) activity were assayed as described under Materials and Methods. 0.8 unit of HSV-1 DNA polymerase was used at the indicated concentrations of KCl. B, 3-5 exonuclease activity was assayed as described in A using 1.25 units of HSV-1 DNA polymerase. Spermidine was varied in the presence of 0 (U), 50 mm (A-4,100 mm (o o), and 200 mm (t.) KC1. the interaction of spermidine and ionic strength in modulating exonuclease activity. Identical with the published effects of spermidine on HSV DNA polymerase activity (7), at low ionic strengths spermidine stimulates exonuclease activity but is inhibitory at high ionic strength. Apparently spermidine mimics salt in its effects on DNA polymerase and exonuclease but is effective at about 10-fold lower concentrations. The effects of fixed concentrations of KC1 and spermidine on HSV-1 DNA polymerase and its 3-5 exonuclease activity in the presence of variable concentrations of activated DNA are presented in the form of Hanes-Woolf plots in Fig. 3. The Hanes-Woolf plots revealed that the activation of both enzyme activities by m (SI IO I (SI IO FIG. 3. Effects of KC1 and spermidine on apparent K, and V,, values of activated DNA for HSV-1 DNA polymerase and 3-5 exonuclease activity. The data are presented in the form of Hanes-Woolf plots for clarity. HSV-I DNA polymerase activity is shown in A and B. (S) is micrograms per ml of activated DNA, and u is expressed in nanomoles of dtmp incorporated into DNA per h. C and D show 3-5 exonuclease activity; (S) is micrograms per ml of 3 -terminally labeled activated DNA, and u is equal to micrograms of DNA hydrolyzed per h. All assays were performed as described under Materials and Methods and included 1.5 units of HSV-1 DNA polymerase and variable amounts of 3 terminally labeled or unlabeled activated DNA as indicated. In A and C activated DNA was varied at KC1 concentrations of 100 mm (M), 125 mm (M), 150 mm (A-A), and 200 mm (H). In B and D reactions contained 100 m~ KC1 plus spermidine at 0 (M), 2.5 mm (A-A), and 10 mm (H).

4 8528 Herpes Simplex Virus Type I DNA Polymerase HSV-1 DNA Polymerase-catalyzed Incorporation and Excision of araamp in DNA-The effects of araamp incorporation, catalyzed by HSV-1 DNA polymerase, on subsequent primer-template utilization were examined. The time course of [3H]araAMP incorporation into activated DNA is shown in Fig. 6A. When the 3-5 exonuclease activity was inhibited by GMP the amount of [ HIaraAMP stably incorporated was increased about 1.5-fold. Activated DNA was prepared which contained [ HIaraAMP residues at 3 -termini. The excision of these residues by the HSV exonuclease activity, as a function of time, is shown in Fig. 6B. 3 -Terminal [ HIaraAMP was readily removed, and this excision was inhibited by GMP. Because araamp was incorporated into DNA by HSV-1 DNA polymerase, the ability of araatp to replace datp in in vitro DNA polymerase reactions was examined. HSV-1 GMP] 0.3 AMP I/DNA Ag/ml FIG. 4. Inhibition of 3-5 exonuclease activity on 3 terminally labeled DNA by phosphonoformic acid. 3-5 Exonuclease activity was measured as described under Materials and Methods using 1.25 units of HSV-1 DNA polymerase and the indicated concentrations of 3 labeled activated DNA. Phosphonoformic acid was present at 0 (O -O), 1.0 p~ (A-A), and 3.0 p~ (H). The data are presented in the form of Lineweaver-Burk plots. Straight lines were determined by the method of least squares. The inset shows a replot of apparent V,& obtained from the double reciprocal plot uersus PFA concentration. TABLE I1 Effects of nucleotides on HSV-1 DNA polymerase 3l-5 exonuclease activity 3-5 exonuclease activity was assayed as described under Materials and Methods. 2.5 units of HSV-1 DNA polymerase were used. 100% activity corresponds to the liberation of 6 X lo3 cpm from 3 labeled activated calf thymus DNA which was present at 10 pg/ml. datp and araatp were present at 10 p ~ all ; other nucleotides were present at I mm. Addition % activity of control None 100 datp 48 araatp 48 dtmp 100 damp 87 CMP 92 UMP 85 AMP 54 GMP 39 IMP 55 aracmp 100 araamp 81 nucleoside 5 -monophosphates tested, only purine ribonucleotides inhibited exonuclease activity but did not affect DNA polymerase. The mode of exonuclease inhibition by GMP and AMP is shown in the double reciprocal plot of Fig. 5. Both nucleotides were noncompetitive inhibitors with respect to activated DNA with K,, values of 0.3 and 0.7 m and K,, values of 0.7 and 1.3 m for GMP and AMP, respectively. The values for IMP were identical with AMP. The 5 -monophosphates of deoxynucleosides, pyrimidine ribonucleosides, and arabinosylnucleosides showed no significant inhibition I I/DNA,ug/ml FIG. 5. Inhibition of 3-5 exonuclease activity on 3 terminally labeled activated DNA by AMP and GMP. 3-5 Exonuclease activity was determined as described under Materials and Methods using 1.25 units of HSV-1 DNA polymerase at the indicated concentrations of 3 -terminally labeled activated DNA in the absence of inhibitors (t.), or in the presence of 0.5 mm AMP (A-A), or 0.5 mm GMP (M). The Lineweaver-Burk plot of the data is shown; strazght lines were fitted by least squares analysis. The kinetic constants were determined from slope and intercept replots of the double reciprocal plot. A - 2 p y,, irn: 201,,, 0 E, IO 15 t (min) B t (mid [3H]araAMP in acti- FIG. 6. Incorporation and excision of vated DNA by HSV-1 DNA polymerase. A, reaction mixtures of 0.4 ml contained 50 lll~ Tris-C1, ph 8.0, 4 mm MgC12, 0.5 mm dithiothreitol, 0.5 mg/ml of albumin, 0.2 M KCl, 124 pg/ml of activated calf thymus DNA, 0.1 mm each dgtp, dctp, dttp, 2.5 p~ [ HI- araatp (1240 cpm/pmol), 5 units of HSV-1 DNA polymerase, and 0 (M) or 0.5 mm (A-A) GMP. At the indicated times 50-pl aliquots were removed, and incorporation of radiolabel into DNA was determined as described under Materials and Methods. B, a 0.2-ml 3-5 exonuclease reaction mixture identical with that described under Materials and Methods except for the presence of 4 pg of [ HIaraAMP-terminated activated calf thymus DNA (672 cpm/pg) was used. 2.5 units of HSV-1 DNA polymerase and either 0 (t.) or 0.5 mm GMP (A-A) were present.

5 I eincubation Min 70r A r 60 - v L g 50- P P I r IO Min FIG. 7. Effects of araamp incorporation on primer-template utilization by HSV-1 DNA polymerase. A, substitution of datp by araatp in HSV-1 DNA polymerase reactions. Reaction mixtures of 0.4 ml contained 50 mm Tris-C1, ph 8.0, 4 mm MgC12, 0.5 mm dithiothreitol, 0.5 mg/ml of albumin, 0.2 M KCI, 125 pg/d of activated calf thymus DNA, 0.1 rn~ each of dgtp and dctp, 5 p~ [ HIdTTP (312 cpm/pmol), 2.5 units of HSV-1 DNA polymerase, and either no addition (&-A), 5.0 p~ datp (M), or 5.0 PM araatp (H). At the indicated times 50-p1 aliquots were removed, and dtmp incorporation into DNA was determined. B, effect of araamp incorporation on subsequent primer-template utilization by HSV-1 DNA polymerase. Reaction mixtures of 0.1 ml contained 50 mm Tris- CI, ph 8.0, 4 mm MgC12, 0.5 mm dithiothreitol, 0.5 pg/ml of albumin, 0.2 M KCl, 21 pg/ml of activated calf thymus DNA, 0.1 mm each dgtp and dctp, 2.5 units of HSV-1 DNA polymerase, 0 or 1.0 mm GMP (M), 2.5 mm araatp (H), or 1.0 mm GMP plus 2.5 m araatp (A-A). At the indicated times after addition of enzyme the reactions were terminated by heating to 65 C for 10 min and then allowed to cool slowly to 25 C. Primer-template utilization was assayed after the addition of 0.1 rn~ datp, 5.0 p~ [JHIdTTP (320 cpm/pmol), and 0.8 unit of HSV-1 DNA polymerase, described under Materials and Methods. DNA polymerase reactions limited by the omission of datp proceeded at a slower rate in the presence of araatp (Fig. 7A). AraATP would not substitute for datp in supporting HSV-1 DNA synthesis, although both were incorporated, and it inhibited synthesis even in the absence of datp. This experiment suggested that araamp-terminated primers were negligibly elongated by HSV-1 DNA polymerase. To further test this and to determine the possible role of the 3-5 exonuclease activity, a preincubation experiment was performed. Activated DNA was preincubated for varied times with datp or araatp and HSV-1 DNA polymerase to generate damp- or araamp-terminated DNA. These primer-templates were then assayed for their ability to serve as substrates for HSV- 1 DNA polymerase in the presence or absence of the exonuclease inhibitor GMP (Fig. 7B). The rate of DNA synthesis after varied times of prior incubation with araatp alone decreased to about 90% of the rate observed on DNA preincubated with datp. When GMP was present with araatp, subsequent DNA synthesis decreased with preincubation time to about 75% of the control after 40 min. DISCUSSION A DNase activity which copurified with HSV-1 DNA polymerase through phosphocellulose chromatography was Fist observed by Weissbach et al. (4). Two other laboratories have since reported that a 3-5 exonuclease activity is associated with HSV DNA polymerase after DNA-cellulose chromatography and glycerol gradient centrifugation at high ionic strength (6, 7). The peak of HSV DNA polymerase activity recovered after Centrifugation catalyzed the template-depend- Herpes Simplex Type Virus I DNA Polymerase 8529 ent conversion of dgtp to dgmp and the hydrolysis of oligo(c H]dG) poly(dc) to [ HIY-dGMP. The DNA polymerase used in the present studies was free of alkaline exonuclease contamination, and no endonuclease activity was detected when assayed under DNA polymerase reaction conditions. The previously reported cosedimentation of DNA polymerase and exonuclease activities (6, 7), their identical heat denaturation kinetics (7), and the present observation that both activities comigrated on nondenaturing gel electrophoresis at varied polyacrylamide concentrations, indicated a physical association of the two activities. It was reported that 3 -terminally labeled primer-templates were more sensitive substrates for the DNA polymerase-associated exonuclease than uniformly labeled double or single stranded DNA (4, 6). To determine more precisely the substrate specificity of the 3-5 exonuclease, its activity on several forms of E. coli DNA, uniformly labeled with [I4C]dThd, was assayed. At an ionic strength optimal for HSV-1 DNA polymerase activity a gapped duplex DNA was preferred over native or heat-denatured DNA. Using 3 -terminally labeled activated calf thymus DNA as substrate, it was found that KC1 and spermidine activated the 3-5 exonuclease activity in a manner identical with DNA polymerase. Both compounds caused increases in apparent V,,, and K, values of activated DNA for both HSV-1 DNA polymerase and 3-5 exonuclease activities. Apparently spermidine mimics the effects of salt but is effective at much lower concentrations. The apparent K,, values of DNA for both activities were very similar. It has been reported that the HSV-specific alkaline DNase activity was inhibited by concentrations of salt or polyamine optimal for the 3 3 exonuclease activity (13). Phosphonoformic acid, a pyrophosphate analogue, has been shown to inhibit HSV DNA polymerase in vitro (7), probably by forming a dead-end enzyme-dna-pfa complex (20). PFA was an uncompetitive inhibitor of the associated 3-5 exonuclease activity with a Kt value of 2.4 PM, at an ionic strength of 0.25 p, identical with the value published for HSV-1 DNA polymerase (7). This laboratory previously reported that the degree of inhibition of HSV-1 DNA polymerase activity by PFA decreased with increasing ionic strength; this was also observed here for the 3-5 exonuclease activity. Knopf observed that the template-dependent conversion of dgtp to dgmp, catalyzed by HSV-1 DNA polymerase, was inhibited by GMP but not by other nucleoside 5 -monophosphates (6). Of the nucleoside monophosphates tested here only the purine ribonucleotides inhibited the associated exonuclease activity noncompetitively with respect to 3 -labeled activated DNA. GMP had a K,., of 0.3 mm and a Kt, of 0.7 mm and was about twice as potent as AMP and IMP. The inhibition of the HSV exonuclease activity, exclusively by purine ribonucleoside 5 -monophosphates, is identical with the inhibition of the 3-5 exonuclease activities associated with other DNA polymerases (10). Because these nucleotides have no effect on DNA polymerase activity they are potentially useful in probing the influence of the 3-5 exonuclease on in vitro DNA synthesis under various conditions. The role of the DNA polymerase-associated exonuclease activity in affecting the inhibition of in vitro HSV DNA synthesis by araatp was investigated. Of particular interest was the relationship of the exonuclease activity to th effects of incorporated araamp residues on primer-template utilization by HSV-1 DNA polymerase. AraATP is a competitive inhibitor of HSV DNA polymerase with respect to datp (7). Whether this is the sole mechanism by which araa inhibits virus replication in vivo is still uncertain. An in uiuo mode of action based on the truncation of primer elongation by terminally incorporated araamp has been debated (8, 9). The

6 8530 Herpes Simplex Type Virus I DNA Polymerase interpretations of experiments testing this hypothesis have been based primarily on the location of araamp residues in expect that increases in the pools of purine ribonucleoside 5 - monophosphates might increase the amounts of mismatched host and viral DNA extracted from araa-treated cells. The or fraudulent nucleotides found in viral DNA by inhibiting assumption was made that araamp was stably incorporated the DNA polymerase-associated exonuclease activity. Some into DNA, that is, araamp was not actively removed. The in antiherpes virus agents may exert their action by inhibiting vitro data presented here show that araamp was incorporated the 3-5 exonuclease activity. For example, ribivirin, which into DNA by HSV-1 DNA polymerase. The amount of inhibits IMP dehydrogenase and thus increases IMP pools araamp incorporated was increased when the 3-5 exonucle- (21), may work in this way. ase activity was partially inhibited by GMP. Moreover, REFERENCES [3H]araAMP present at 3 4ermini of activated DNA was 1. Keir, H. M., and Gold, E. (1963) Biochim. Biophys. Acta 72,263- readily removed by the associated exonuclease activity. These 276 data are consistent with the recent observation that more 2. Keir, H. M., Hay, J., Morrison, J. M., and Subak-Sharpe, J. H. araamp was found in host compared to viral DNA extracted (1966) Nature 210, from araa-treated HSV-1-infected cells (9). 3. Keir, H. M., Subak-Sharpe, J. H., Shedden, W. I. H., Watson, D. Although araamp was incorporated into activated DNA H., and Wildy, P. (1966) Virology 30, by HSV-1 DNA polymerase, araatp would not replace datp 4. Weissbach, A,, Hong, S. C. L., Aucker, J., and Muller, R. (1973) J. Biol. Chem. 248, in supporting in vitro DNA synthesis. In fact, the rate of DNA 5. Powell. K. L., and Purifoy, D. J. M. (1977) J. Virol. 24, synthesis in reactions limited by the omission of datp was 6. Knopf, K. W. (1979) Eur. J. Biochern. 98, depressed in the presence of araatp, indicating that araamp- 7. Ostrander, M., and Cheng, Y.-C. (1980) Biochim. Biophys. Acta terminated primers were poorly extended. Activated DNA 609, preincubated with HSV-1 DNA polymerase served as a poorer substrate in subsequent DNA polymerase reactions when araatp was present in the preincubation. The primer-template utilization was depressed further when the 3-5 exonuclease activity was inhibited by GMP. These data indicated that araamp-terminated primers were elongated at a very slow rate and that most araamp residues were probably hydrolyzed by the DNA polymeraseassociated exonuclease before elongation could occur. This is consistent with a current hypothesis that while arabinosylnucleotides are not absolute chain terminators, their incorporation into DNA significantly decreases the rate of primer elongation. Previous data (9) showing that araamp was found exclusively in internucleotide linkages in HSV DNA extracted from araa-treated cells most likely reflects the very slow elongation but active removal of 3 -terminal araamp residues by HSV-1 DNA polymerase. In conclusion the data presented here verify that HSV-1 DNA polymerase has an intrinsic 3-5 exonuclease activity. The substrate preference and kinetic behavior of this exonuclease parallels that of the DNA polymerase with respect to DNA substrate. We propose that araamp severely retards primer elongation and that most araamp residues are removed before elongation can occur, Moreover, one would 8. Muller, W. E. G., Zahn, R. K., Beyer, R., and Falke, D. (1977) Virology 76, Pelling, J. C., Drach, J. C., and Shipman, C. (1981) Virology 109, Byrnes, J. J., Downey, K. M., Que, B. G., Lee, M. Y. W., Black, V. L., and So, A. G. (1977) Biochemistry 16, TsangLee, M. Y. W., Byrnes, J. J., Downey, K. M., and So, A. G. (1980) Biochemistry 19, Baril, E., Mitchener, J., Lee, L., and Baril, B. (1977) Nucleic Acids Res. 4, Hoffmann, P. J., and Cheng, Y. (1978) J. Biol. Chem. 253, Shannon, W. M. (1975) in Adenine Arabinoside: An Antiuiral Agent (Pavan-Langston, E., Buchanan, R. A,, andalford,c. A,, Jr., eds) pp. 1-43, Raven Press, New York 15. Shipman, C., Jr., Smith, S. H., Carlson, R. H., and Drach, J. C. (1976) Antimicrob. Agents Chemother. 9, Schwartz, P. M., Shipman, C., Jr., and Drach, J. C. (1976) Antimicrob. Agents Chemother Brutlag, D., andkornberg, A. (1972) J. Biol. Chern. 247, Fisher, P. A., and Korn, D. (1977) J. Biol. Chem. 252, Gellert, M., Mizuuchi, K., ODea, M. H., and Nash, H. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, Boezi, J. A. (1979) Pharrnacol. Ther. 4, Miller, J. P., Kigwana, L. J., Streeter, D. G., Robins, R. K., Simon, L. N., and Roboz, J. (1977) Ann. N. Y. Acad. Sci. 284,