Vaccinia Virus DNA Polymerase IN VITRO ANALYSIS OF PARAMETERS AFFECTING PROCESSMTY*

The polymerization and proofreading activities of the vaccinia virus DNA polymerase reside within a 116-kDa catalytic polypeptide. We report here an investigation of the intrinsic processivity of this enzyme on both natural and homopolymeric DNA templates. Inclusion of the Escherichia coli helix destabilizing protein allowed the viral enzyme, which lacks strand displacement activity, to utilize a singly primed M13 DNA template. In the presence of either 10 m~ MgCl, or 1 m~ MgCl, + 40 m~ NaCl, synthesis was achieved in a highly distributive manner. RFII formation required a significant excess of enzyme, and 510 nucleotides (nt) were added per primer-tem- plate binding event. The apparent rate of primer elongation varied with the enzymehemplate ratio and reached a maximum of 8 nus. A similar lack of proces- sivity was observed on a poly(+,)-oligo(dT,,,,) template. In contrast, highly processive synthesis was achieved on both templates in the presence of 1 m~ MgCl, and the absence of NaCl. A primer extension rate of 30 nus was observed, and ~ 2 0 0 0 nt were added per binding event. These studies suggest that the catalytic polypeptide of the vaccinia virus DNA polymerase will require accessory protein(s) to form a stable enzyme-template interaction and direct processive DNA synthe- sis under isotonic conditions in vivo. DNA polymerases represent the core of the complex enzy-matic machinery responsible for the faithful duplication of genomic DNA. These enzymes bind to a

DNA polymerases represent the core of the complex enzymatic machinery responsible for the faithful duplication of genomic DNA. These enzymes bind to a primer-template junction and catalyze the addition of the correct nucleotide to the nascent strand. Analysis of viral, procaryotic and, more recently, eucaryotic polymerases has provided a growing insight into the regulation of polymerase-template interactions. The emerging generalization is that there is an inherently weak interaction between polymerases and their templates, perhaps to facilitate the rapid translocation of polymerases during nascent strand synthesis. Processivity, the ability of polymerases to synthesize long stretches of DNA without dissociating from the template, is usually conferred upon the catalytic subunit by association with accessory proteins (see Refs. 1-5). In Herpes simplex virus DNA replication, the catalytic polymerase (UL30) is rendered processive by a single accessory protein, UL42; similarly, processivity is conferred on the bacteriophage * This work was supported by National Institutes of Health Grant AI 21758 (to l? T.) and by contributions from D. Cofrin and a special group of donors from the Dorothy Rodbell Cohen Foundation. 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. T7 polymerase by association with thioredoxin. In yeast and mammalian cells, processivity is conferred by the homotrimeric protein PCNA. Analogous to the p subunit of the Escherichia coli polymerase I11 holoenzyme and the gene 45 protein of bacteriophage T4, PCNA acts as a sliding clamp to tether replicative DNA polymerases to the primed template. The processivity factor and polymerase are recruited to the primer-template junction by additional accessory proteins (the RF-C complex in yeast and mammalian cells, the y complex in E. coli, the g44l62 complex in bacteriophage T4). By interacting with both template and polymerase, these processivity factors serve as a bridge that sustains a stable association and permits e acient synthesis of long nascent chains.
The studies presented here concern the DNA polymerase encoded by vaccinia virus, the prototypic member of the poxvirus family. The cytoplasmic localization of vaccinia's infectious cycle, and its genetic autonomy from the host, are singular among DNAviruses. The 192 kb' vaccinia genome is thought to encode all of the functions required for DNA replication (6)(7)(8).
In addition to the DNA polymerase, the current repertoire of gene products with demonstrated or potential roles in DNA replication includes the D5 ATPase, B1 protein kinase, uracil DNA glycosidase (D4), thymidine kinase (521, thymidylate kinase (A48), ribonucleotide reductase (F4, 14), DNA ligase (A501 and topoisomerase I (H6). The polymerase, encoded by the E9 gene, is a 116-kDa protein with both polymerization and 3'-5' exonuclease activity (6,(9)(10)(11)(12). It has strong homology t o the OJS family of DNA polymerases (10, 13,14). We have recently reported the overexpression of the enzyme using the hybrid vaccinia-T7 expression system and its purification to apparent homogeneity (12). The availability of pure enzyme will facilitate the development of an in vitro replication system, which may provide the basis for the identification of polymerase accessory proteins. Preliminary to the latter goal, we present here our characterization of the mode of synthesis catalyzed by the purified polymerase on homopolymeric and natural templates.  pressed in BSC40 cells using the hybrid vaccinia virus-T7 overexpression system (15). Clarified cytoplasmic lysates were fractionated sequentially on DEAE-cellulose, phosphocellulose, hydroxyapatite, and heparin-agarose, and DNA polymerase was assayed on an activated salmon sperm DNA template. Fraction V contained a single 116-kDa protein species as visualized on silver-stained SDS-polyacrylamide gels. The purified enzyme had a specific activity of 3023 units/mg (~2 8 5 pmol/unit), where 1 unit is that amount of enzyme that can incorporate 10 nmol of dNTP into acid precipitable material in 30 min at 37 "C.

, .
DNA Polymerase Assays Singly Primed M I 3 DNA-% construct a primed template, a 24-mer oligonucleotide primer (5'-CGCCAGGG"TCCCAGTCACGAC-3') was annealed to M13mplO ssDNAat a molar ratio of 20:l. To construct a 5'-"2PP-primed template, the primer was radiolabeled with T4 polynucleotide kinase and [y3'P]ATP prior to annealing to M13mplO ssDNA. Radiolabeled primer/template was purified on a Bio-Gel A1.5 m sizing column to remove free label and unannealed primer. Unless indicated otherwise, DNA polymerase was assayed in reactions (25 pl) that contained 10 mM Ms-HCI, pH 7.5, 40 mg/ml bovine serum albumin, 4% glycerol, 0.1 mM EDTA, 5 mM dithiothreitol, 8 mM MgCI,, 25 fmol of singly primed M13mplO ssDNA, 750 ng of E. coli SSB (10 pmol of tetramer), 60 PM each of dCTP, dGTP, and dATP, and 20 PM ["'PlTTP (2400 counts/midpmol). Reactions were preincubated with enzyme and two of the four dNTPs (dCTP and dGTP) at 30 "C for 3 min. Primer extension was initiated by the addition of dATP and [32PldTTP, and incubation was continued a t 30 "C. To measure the incorporation of radiolabeled nucleotide into acid precipitable material, DNA synthesis was stopped by the addition of an equal volume of 20% trichloroacetic acid, 0.2 M NaPP,. Trichloroactetic acid precipitates were collected and washed on glass fiber filters, and radioactivity was quantitated by Cerenkov counting. To visualize primer extension products, reactions were quenched with an equal volume of 1% SDS, 40 mM EDTA and fractionated on 0.8% agarose gels containing 0.125 pg/ml EtBr. Gels were cast and run in 1 x TBE (50 mM Tris, 50 mM boric acid, and 1 mM EDTA), dried, and subjected to autoradiography.

RESULTS
Single-stranded M13 phage DNA annealed to an oligonucleotide primer has served as a useful template for the biochemical characterization of diverse DNA polymerases. An advantage to using this molecule over activated (nicked and gapped) DNA is that enzyme processivity, or the ability of polymerases to syn- thesize long stretches of DNA, can be assessed directly. However, most DNA polymerases are sensitive to the barriers of secondary structure present within this DNA template. "he inability of the vaccinia DNA polymerase to overcome these barriers, and hence to direct significant primer elongation, was described some years ago in the classic biochemical study of this enzyme (16). In the intervening years, it has become clear that utilization of such a template can be facilitated by inclusion of a helix destabilizing SSB in experimental assays. Because no vaccinia-encoded SSB has yet been identified, and because the E. coli SSB has been shown to support DNA synthesis by a variety of heterologous polymerases (17-201, we explored the use of the E. coli protein in our analysis of the processivity of the purified vaccinia DNA polymerase (12).

Primer Extension by the Vaccinia Enzyme on M13 ssDNA Is
Stimulated by E. coli SSB-A fixed amount of primed M13 DNA (25 fmol), uncoated or coated with E. coli SSB, was incubated with various amounts of the purified vaccinia DNA polymerase (Fraction V) (12). After a 3-min preincubation designed to permit the polymerase to bind to the primer terminus, DNA synthesis was initiated and allowed to proceed for 30 min at 30 "C. Reactions were terminated and products were analyzed by quantitation of the incorporation of radiolabeled dNTP into acid-precipitable material and by fractionation of the prod- ucts on neutral agarose gels under conditions that maximize separation of template from product. As seen in Fig. lA, dNTP incorporation by the vaccinia enzyme was greatly increased in the presence of E. coli SSB. When the vaccinia polymerase was present in a 7-fold excess over template, a maximum stimulation of 40-fold was observed. Electrophoretic analysis of the radiolabeled products (Fig. lB) elucidated the nature of this stimulation. Only in the presence of SSB could the vaccinia enzyme synthesize the full complement of the template strand to form products whose migration was indistinguishable from RFII (double-stranded nicked circle). Although a significant amount of dNTP incorporation was detected in the absence of SSB, the radiolabeled material co-migrated with (or migrated slightly above) the single-stranded DNA template. These products, in which the primer was only extended a short distance, reflect the inability of the enzyme to displace barriers of secondary structure in the naked template.
To determine whether the presumed RFII product obtained in the presence of enzyme excess and SSB was completely du- plex in nature, a modified experiment was performed. Reactions contained unlabeled dNTPs and a population of primertemplates in which some of the primers contained a "Pradiolabeled 5' terminus. A molar excess of the vaccinia DNA polymerase was incubated with 25 fmol of this template, and DNA synthesis was initiated and terminated as described previously. The DNA products were fractionated electrophoretically on neutral agarose gels containing ethidium bromide. Examination of the total DNA population by ultraviolet illumination revealed that all of the single-stranded template was converted to RFII DNA in 15' at 30 "C ( Fig. 2 A ) . Under these conditions, primer elongation by the vaccinia enzyme occurred at an average rate of 8 nus. To confirm that the completed DNA products contained nicks and not gaps, bacteriophage T4 DNA ligase and ATP were added subsequent to the production of RFII, and ligation was allowed to proceed for 1 h at 30 "C. Visualization of the products by autoradiography revealed that the radiolabeled RFII molecules (i.e. those products extended from a primer containing a phosphorylated 5' terminus) were completely converted to the rapidly migrating RFI form (Fig. 2 B , lane 8). The covalently closed RFI molecules become positively supercoiled during migration through EtBrcontaining gels and hence migrate more rapidly than nicked RFII molecules. Because the majority of the annealed primers contained unphosphorylated 5' termini, and the vaccinia enzyme has no detectable 5'-3' exonuclease activity (121, most of the RFII products (those which are not radiolabeled) were not converted to RFI (compare the products seen by ultraviolet illumination (Fig. 2 A , lane 8) with those seen by autoradiography (Fig. 2B, lane 8)). These results demonstrate that vaccinia polymerase-catalyzed DNA synthesis on SSB-coated M13 templates is indeed complete and results in the formation of true RFII molecules.
Characterization of Polymerization Efficiency on Singly Primed M13 DNA and Poly(dA)-Oligo(dT)-In the analysis described above, we failed to observe completely extended M13 products when enzyme levels below 185 fmol were incubated with 25 fmol of an SSB-coated M13 template for 30 min at 30 "C ( Fig. 1). At lower enzyme concentrations (<7-fold excess), all of the available primed template, as visualized by EtBr staining of fractionated replication products (data not shown), was acted upon by the enzyme, yet no complete RFII product was detected. These findings suggested that DNA synthesis was catalyzed by the vaccinia polymerase in a distributive, or nonprocessive, fashion. Further support for this conclusion came from our observation that the time required to complete RFII formation depended upon the amount of enzyme added; whereas RFII formation was evident by 15 min when 674 fmol were assayed, more than 20 min were required when 185 fmol were assayed (data not shown).
To address the issue of distributivity versus processivity more directly, we performed a template challenge experiment. Enzyme was preincubated with an equimolar amount of SSBcoated, 32P-primed-M13 template in the presence of 2 dNTPs. Upon initiation of DNA synthesis by the addition of the other two dNTPs, a 5-fold molar excess of an unlabeled challenge template was added to half of the reaction. Aliquots were removed at various times, DNA synthesis was terminated, and reaction products were analyzed on denaturing polyacrylamide gels. As shown in Fig. 3, an examination of primer elongation on M13 DNA revealed distinct termination sites. Such polymerase pause sites are typically observed with natural DNA templates and may reflect residual M13 secondary structure. Under the conditions used in this experiment we observed an average elongation rate of one nt every 2 s. However, in the presence of challenge DNA, a 15-fold decrease in the rate of primer elongation was seen: approximately one nt was incorporated every 30 s. The majority of the extended products were one nt longer than the original primer. As G is the first nucleotide to be added with the primer used in these studies, it is probably incorporated during the preincubation (idling) step of the reaction when both dCTP and dGTP are available. These results indicated that the enzyme readily dissociated from the first template shortly after the addition of the second. The enzyme displayed a low processivity number, with only one to two nt incorporatedlpolymerase binding event.
To eliminate any effect E. coli SSB might have on the stabil- ity of the enzyme-template interaction, the processivity of the vaccinia enzyme was also examined using a poly(dA)-oligo(dT) template. This homopolymeric template lacks the secondary structure found in M13 DNA and therefore should serve as an effective template for DNA synthesis in the absence of a helix destabilizing protein. The primer template was constructed by annealing 5'-S2P-oligo(dT),,-,, to poly(dA),,, at a 1:l molar ratio in order to maximize the regions of single-stranded DNA available. Reactions containing 20 VM TTP were initiated by the addition of various amounts of enzyme and terminated after 5 min a t 30 "C. Extension products were separated on denaturing urea-acrylamide gels. As seen in Fig. 4, the vaccinia DNA polymerase could efficiently utilize poly(dA)/oligo(dT) in the absence of E. coli SSB. At equimolar concentrations of enzyme and template, extension products of >lo0 nucleotides were evident (Fig. 4, lane l 1. The addition of E. coli SSB to these reactions had no effect on polymerase activity (data not shown). As expected for a nonprocessive (distributive) enzyme, a decrease in the average length of the extended products was observed with decreasing amounts of polymerase. At limiting enzyme concentrations, where unreacted "P-oligo(dT) primers remained following a 5-min incubation, the number of nucleotides incorporatedpolymerase binding event could be determined. Assaying 9 fmol of enzyme (35-fold molar excess of template over polymerase) resulted in extension products that were maximally 24 nt in length (Fig. 4, lane 6). Given an initial population of primers that were 12-18 nt in length, the number of T residues incorporatedminding eventlpolymerase molecule (processivity number) could therefore be approximated a t less than six and, on average, two to four.
The Effect of MgCl, on Polymerase Processivity-The distributive mode of synthesis observed with the vaccinia enzyme suggested that a weak interaction between the enzyme and primer-template was responsible for frequent dissociation of the synthetic complex. In other systems, the stability of this enzyme.DNAcomplex has been shown to be acutely sensitive to ionic conditions (18, [21][22][23][24]. We therefore investigated the impact of varying the concentrations of either MgCl, or NaCl on polymerase processivity. Primer elongation on a poly(dA)-oligo(dT) template was monitored in the presence of varying concentrations of MgC1,.
In 5-min reactions performed with limiting amounts of pure polymerase, a decrease in divalent cation concentration from 10 to 1 mM increased the processivity number of the polymerase (nt incorporatedpolymerase binding event) from <lo to >lo0 (Fig. 5). That significant levels of unreacted primers remained a t lower concentrations of MgCI, was evidence of fewer enzyme reinitiation events, confirming that a reduction in divalent cation concentration increased the processivity rather than the catalytic rate of the enzyme. That enzyme activity per se does not vary significantly between 1 and 10 mM MgCI, was confirmed in a parallel experiment by quantitating the incorporation of radiolabeled TTP into acid-precipitable material (Fig.  5B); varying the concentration of MgCl, had only a 3-fold effect on overall TTP incorporation, with maximum synthesis occurring a t 4 mM MgCI,. As expected, no productive primer extension was detected in the absence of MgCI, (Fig. 5A).
The effect of MgCI, on enzyme processivity was not specific to the homopolymeric template. A dramatic increase in the rate and processivity of primer elongation on a singly primed M13 template was detected when the MgCI, concentration was reduced to 1 mM (Fig. 6). In agreement with our earlier findings, only short extension products consistent with a distributive mode of synthesis were observed in reactions containing <7-fold excess enzyme and 8 mM MgCl, (Fig. 6B). In the presence of 1 mM MgCI,, we observed the formation of complete RFII products within 4 min (Fig. 6B). Comparison of the data shown in panels A and B of Fig. 6 indicates that the appearance of RFII products after 4 min of synthesis occurred before all of the templates had been extended: a t 4 min, only 70 pmol of nucleotide had been incorporated, whereas full replication of the available 25 fmol of template requires the incorporation of 180 pmol of nucleotides. These data suggest that enzyme-template interactions on singly primed DNA templates were prolonged at low concentrations of MgCI,, permitting processive synthesis. Because reassociation with a rare primer-template junction (1/7200 base pairs) would be presumed to be rate-limiting, a faster apparent rate of primer elongation would be predicted. Indeed, reducing the MgCl, concentration from 8 to 1 mM increased the apparent elongation rate during RFII formation from 8 nus (RFII in 15 min, Fig. 2, time course) to 2 30 ntls (RFII in 4 min, Fig. 6B). The data in Fig. 6A show that total dNTP incorporation in a given time period increased 10-fold when the MgCI, concentration was decreased. As discussed above, such an impact of reduced MgCI, concentration on overall synthetic activity was not seen when a poly(dA)-oligo(dT) template was used. The relative abundance of primer-template junctions (1/390 base pairs of template) in the homopolymeric template must reduce the contribution of primer-template reassociation to the net rate of DNA synthesis.
To further demonstrate that the dramatic increase in primer elongation rate at lower MgCI, concentrations was due to an increase in the processivity of the enzyme, extension products from single polymerase binding events were analyzed (Fig. 7). Replication reactions containing M13 template, various amounts of enzyme, and 1 mM MgCI, were incubated for 10 min. Analysis of the extension products on alkaline agarose gels revealed the synthesis of DNA products ranging in size from 2 to 7 kb. With decreasing amounts of polymerase, a reduction in the amount, but not the length, of these products was observed. Because the template was in excess at most enzyme concentrations examined, the products seen reflected single enzyme binding events. Thus, at low concentrations of MgC1, the processivity of the polymerase appeared to be extremely high, with 2-7 kb of synthesis completed per polymerase binding event.
The Effect of NaCl on Enzyme-Template Interaction-The effect of NaCl on the processivity of the purified polymerase was determined by monitoring the conversion of "'P-primertemplate to RFII in the presence of 1 mM MgCI, and the pres- ence or absence of 40 mM NaCl. In the absence of NaCI, processive conversion of M13 ssDNA to RFII was apparent (Figs.  6B, 7, and 8). Under conditions of template excess, complete RFII products were observed when most of the "P-primer-template remained untouched. In the presence of 40 mM NaCI, a reversion of this processive activity to a distributive one was seen (Fig 8). All of the available "P-primer-templates were elongated slowly and to a similar extent, and no RFII products were formed during the reaction time allowed.

DISCUSSION
Based on sequence homology with other eucaryotic DNA polymerases, the vaccinia virus DNA polymerase has been classified as a member of the a16 family. Seven domains of the vaccinia protein are shared with DNA polymerase CY from human and yeast, and an additional five domains, which include three putative exonuclease motifs, are shared with 6 enzymes (10, 13,14,25). A structure-function analysis designed to explore the relationship between these conserved regions and enzyme function has begun to yield structural information regarding active sites of the protein. To date, seven drug-resistant and two temperature-sensitive alleles have been isolated and their lesions mapped within the gene (10, 14, 26-28). Several drug-resistant mutations that display altered mutation rates in vivo (14,26) are predicted to contain lesions that affect nucleotide binding and exonuclease activity.
The purification and biochemical characterization of the vaccinia DNA polymerase were originally reported by Challberg and Englund (9,16). In addition to defining the 5'-3' polymerization and intrinsic 3'-5' exonuclease activities of the enzyme, their studies documented a lack of strand displacement capability. This inability of the viral polymerase to pass through regions of DNA secondary structure precluded an in depth analysis of enzyme processivity on a natural DNA template. In the analysis presented here, removal of structural barriers by the inclusion of the E. coli SSB protein enabled processivity determinations to be performed on natural as well as homopolymeric DNA templates. The vaccinia polymerase was pu-rified from BSC40 cells infected with vaccinia virus recombinants permitting inducible overexpression of the DNA polymerase (12,15). Homogeneous preparations of the induced protein displayed both polymerization and exonuclease activities and consisted of a single protein species of 116 kDa (12).
In our analysis of enzyme processivity, we found that primer elongation on both natural and homopolymeric DNA templates was achieved in a distributive or nonprocessive manner under reaction conditions that were optimal for DNA synthesis on gapped DNA (9). Conversion of singly primed M13 DNA to the duplex RFII F-oduct required the presence of a helix destabilizing protein (E. coli SSB). RFII formation was completed at a synthetic rate that vaned with enzyme concentration; higher polymerase concentrations resulted in faster apparent rates of primer elongation. A maximal rate of dNTP incorporation of 8 nWs at 30 "C was detected when enzyme concentrations were in vast excess over the primed DNA template. A clue to the relative slowness of this elongation rate was provided by template challenge experiments, which indicated that perhaps only one or two nucleotides were incorporated during a single cycle of enzyme-template binding.
A distributive mode of DNA synthesis was also observed using a poly(dA)-oligo(dT) template, whose full extension did not require a helix destabilizing protein. To determine the number of consecutive nucleotides incorporated during each interaction of the polymerase with this template, assays were performed under conditions of template excess. At a template to enzyme ratio of 35:1, incorporation of fewer than 10 dTMPs/ primer was observed. As the homopolymeric template studies were performed in the absence of E. coli SSB, we conclude that the distributive behavior of the vaccinia DNApolymerase (processivity number 510) is an intrinsic property of the catalytic enzyme and not due to the presence of the E. coli protein.
The effect of individual reaction components on polymerase activity was examined to determine their influence, if any, on enzyme processivity. A significant increase in processivity was detected when MgC1, concentrations were reduced in assays where primed M13 DNA or poly(dA)-oligo d(T) served as replication templates. Analysis of extension products from single interactions of the enzyme with the M13 template revealed a >200-fold increase in enzyme processivity when the concentration of MgC1, was reduced from 8 to 1 m~. A corresponding increase in the apparent rate of primer elongation from 8 nus (with excess polymerase) to a n average rate of 30 nus at 30 "C was also observed. That the reduction in MgC1, was augmenting enzyme processivity, and not catalysisper se, was confirmed in studies using poly(dA)-oligo(dT) as a template. TTP incorporation was maximal at 4 mM MgC1,; approximately 2-fold lower levels of incorporation were obtained in the presence of either 1 or 8 mM MgCl,. Moreover, varying the MgC1, concentration for DNApolymerases from other systems (18, [21][22][23][24]. The processivity of calf thymus DNA polymerase a can be reduced 20fold by raising the Mg2' concentration from 1 to 10 m~ (from 50-100 nt incorporateainding event to 2-5, pH 8.0) (23, 24). In a recent study of the Drosophila mitochondrial DNA polymerase (221, an increase in processivity with decreasing MgC1, and NaCl concentrations, and an inverse relationship between polymerase processivity and enzyme activity, were demonstrated. However, no reaction conditions were described that allowed for maximal processivity and polymerase activity simultaneously. We did observe an increase in DNA synthesis with increasing processivity in assays where MgC1, and NaCl concentrations were reduced and singly primed M13 DNA served as a template. Because this substrate presents a low concentration of primer-template junctions (1/7200 nt), the contribution of enyzme recycling to the apparent rate of DNA synthesis is likely to be significant. It is therefore not surprising that ionic conditions which reduce enzyme dissociation (low ionic strength) stimulate the rate of dNTP incorporation.
It is clear from the analyses presented here that the vaccinia DNA polymerase must associate with additional factors in order to replicate a template such as the 192 kb viral genome efficiently. The need for a helix destabilizing activity and the lack of duplex invasion capabilities indicate that an SSB and perhaps a helicase are essential. In addition, the weak enzymetemplate interactions seen in the presence of moderate levels of salt suggest that other accessory factors are needed for ensuring processive DNA synthesis under isotonic conditions. Because the purified enzyme was overexpressed within vacciniainfected cells, viral factors that stimulate polymerase activity or processivity might be expected t o copurify with the catalytic subunit. In our purification protocol, however, assays containing an activated DNA template and moderate salt levels were used to follow polymerase activity. In retrospect, this approach would have selectively enriched for a distributive catalytic activity. To investigate further the potential role of accessory proteins in processive viral DNA synthesis, a careful fractionation of the vaccinia enzyme from wild-type infected cells is underway. Using a primed M13 ssDNA template to monitor polymerase activity over the course of purification, we have partially purified a processive form of the vaccinia enzyme., This activity catalyzes DNA synthesis rapidly and processively under reaction conditions that permit only a distributive mode of synthesis with the purified enzyme. Although significant progress has been made in the purification of this processive activity, subunit composition has not been determined and awaits further biochemical analysis.