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J. Biol. Chem., Vol. 279, Issue 18, 18535-18543, April 30, 2004

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Contribution of the 3'- to 5'-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis^*

Liping Song, Murari Chaudhuri, Charles W. Knopf, and Deborah S. Parris¶

From the Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210 and Institute for Applied Tumor Virology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120, Heidelberg, Germany

Received for publication, September 4, 2003 , and in revised form, February 6, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3'- to 5'-exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of K_m for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wild-type pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol·UL42 complex was much less efficient than by the mutant pol·UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol·UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Herpes simplex virus type 1 (HSV-1)¹ is the best characterized member of the large family of Herpesviridae pathogenic to humans, which also includes Epstein-Barr virus, varicella-zoster virus, human cytomegalovirus, and Kaposi sarcoma-associated herpes virus (reviewed in Ref. 1). Viruses in this family encode most of the proteins essential for and directly involved in DNA replication (2–4), including a well conserved DNA polymerase catalytic subunit (pol), which is a member of the polymerase B family (5, 6). HSV-1 pol possesses 5'- to 3'-polymerizing and 3'-to5'-exonuclease (exo) activities (7, 8), the latter of which is involved in the removal of incorrectly incorporated deoxyribonucleoside triphosphates (9–12). The importance of this proofreading activity for maintaining fidelity of DNA replication was suggested by studies from our laboratory that demonstrated the relatively poor ability of HSV-1 pol to discriminate between the correct and incorrect nucleotide for incorporation in single turnover experiments (13). That study (13) reported that selectivity of correct over incorrect dNTP was 300, whereas bacteriophage T4 and T7 DNA polymerases discriminate among dNTPs by a factor of >1000 and >100,000, respectively (14–16). Previous studies suggested that the exo activity of HSV-1 pol contributes substantially to the fidelity of HSV replication in vivo, because mutation of the conserved exo site III of the HSV-1 pol catalytic subunit was associated with a strong mutator phenotype (10, 17, 18). Moreover, such pol mutants are genetically unstable and revert or recombine with high frequency (10), suggesting strong in vivo selection against an HSV-1 pol lacking exo activity.

To gain a better understanding of the contribution of the associated exo activity of HSV-1 pol in maintaining fidelity of DNA replication, we compared misincorporation and mismatch extension by the wild-type pol with that of a mutant enzyme, which lacks exo function. We selected for study the D368A mutant pol, which converts an aspartate to alanine in the conserved exo site I domain. This site is thought to be involved in the coordination of two divalent metal ions responsible for the exo catalytic activity (19), and previous studies demonstrated that the D368A mutant pol lacked any detectable exo activity (9, 20). A previous study with this mutant pol used a limited set of primer/templates (P/Ts) and concluded that the enzyme possessed a defect in mismatch extension (11). Because polymerization, misincorporation, and mismatch extension frequency are dependent upon sequence context (21, 22), the contribution of the exo activity for maintaining fidelity of DNA synthesis was assessed by varying the P/T at only one position at a time to reduce or eliminate contextual differences. Our results demonstrate that nucleotide selectivity is the major contributor to fidelity and is controlled predominantly at the level of increased affinity for correct versus incorrect nucleotide. Exo activity enhanced nucleotide selectivity by roughly an order of magnitude in multiple turnover reactions. However, the primary effect of exo activity on fidelity was manifest during processive DNA synthesis because mismatches impeded the extension ability of pol, particularly in the presence of UL42 processivity factor, and increased the probability for excision and repair of misincorporated nucleotides.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cells and Viruses—Recombinant baculoviruses were used to express the HSV-1 wild-type (gift of Dr. Robert Lehman, Stanford University) or exo-deficient (D368A) pol genes with or without the UL42 processivity factor gene and have been described elsewhere (9, 23). Recombinant viruses were propagated in Sf9 insect cells at 27 °C as described previously (24).

Protein Purification—Cells infected with recombinant baculovirus (10 plaque-forming units/cell) were harvested at 40-h postinfection and lysed, and soluble extracts were prepared as described previously (24). Protein fractionation was conducted with the aid of a fast protein liquid chromatography system (Amersham Biosciences) at room temperature as described below, and fractions were collected on ice. The presence of HSV-1 wild-type or exo-deficient pol in fractions was monitored by enzyme assay using activated calf thymus DNA as template (25) with the exception that salt concentration in reactions was reduced to 50 mM KCl. The identity of the pol activity was confirmed by the presence of a polypeptide of 136 kDa following denaturing gel electrophoresis. Cellular extracts were dialyzed against B-2 buffer (20 mM Tris-HCl, pH 8.2, 50 mM NaCl, 5 mM 2-mercaptoethanol, 1 mM EDTA, and 10% glycerol), clarified by centrifugation at 70,000 x g, and loaded onto a 15-ml DEAE-Sepharose column. Following extensive washing of the column in B-2 buffer, bound proteins were eluted with a 60-ml linear salt gradient of 0–0.5 M KCl in B-2 buffer. The peak fractions containing the relevant HSV-1 pol eluted between 0.12 and 0.33 M KCl. Pol-containing fractions were pooled and applied to a 5-ml hydroxyapatite, type II column equilibrated with buffer D (10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 5 mM 2-mercaptoethanol). The pol proteins were found in the unbound fractions, which were pooled, dialyzed against buffer C (20 mM Hepes, pH 7.6, 50 mM NaCl, 5 mM 2-mercaptoethanol, 1 mM EDTA, and 10% glycerol), and loaded onto a 10-ml heparin-Sepharose column equilibrated with buffer C. Bound proteins were eluted with a 60-ml linear gradient from 0–1 M KCl in buffer C. The wild-type and mutant pol eluted between 0.42 and 0.54 M KCl. Peak fractions were collected, diluted with buffer C to achieve a salt concentration of <0.1 M KCl, and loaded onto a 10-ml cellulose phosphate column equilibrated with buffer C. Bound proteins were eluted with a 60-ml linear salt gradient from 0 to 1 M KCl. The pol proteins eluted between 0.25 and 0.34 M KCl and were pooled, concentrated by ultrafiltration using Centricon-100 units (Amicon, Bedford, MA), diluted with an equal volume of buffer E (200 mM KCl, 20 mM Tris, pH 8.0, 1 mM EDTA, and 60% glycerol), flash-frozen in liquid nitrogen, and stored at -80 °C. The wild-type and mutant pol proteins were also purified as a complex with UL42 as described previously (24). Protein concentrations were determined by the Bradford protein assay (Bio-Rad) using bovine serum albumin as a standard.

DNA Substrates—Gel-purified synthetic oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). The DNA sequences of the oligonucleotides used in the studies described herein are shown in Table I. The primer strands used in each experiment were 45-mers of identical sequence except at the 3'-nucleotide, whereas the template strands with the exception of template 4 were 67-mers, which varied only at positions complementary to the 45^th or 46^th (extended) nucleotide on the primer strand. The primer strands were radioactively labeled at the 5' end with [-³²P]ATP and T4 polynucleotide kinase by standard procedures. Duplex P/T was obtained by mixing primer with a 20% molar excess of template in 20 mM Tris-HCl, pH 8.0, and 100 mM KCl, heating the mixture to 55 °C and slowly cooling it to room temperature over several hours. Annealed P/T was stored at 4 °C.

(Eq. 1)

where a is the amplitude of the first exponential, b is the rate constant of the rapid phase, c is the amplitude of the second exponential, and d is the rate constant of the slower phase.

Steady-state reactions were conducted manually using a ratio of enzyme:P/T of 1:100 in buffer as indicated above but lacking EDTA. Reactions were initiated by the addition of enzyme, portions were terminated at intervals from 10 s to 2 h with 0.3 M EDTA, and products were analyzed as indicated above. The apparent steady-state rate constant for excision (k) was determined by fitting the data to a single exponential function as shown in Equation 2,

(Eq. 2)

where A is the initial concentration of 45-mer.

Steady-state Incorporation Assays—Steady-state kinetics of correct or incorrect incorporation of dNTP to produce all 16 possible base pairs were determined by incubating exo-deficient or wild-type HSV-1 pol with the 44A/TN P/Ts, which differed only in the templating position for extension of the primer by one (Table I). Increasing concentrations of a single dNTP were incubated with enzyme and P/T (1:100) at 37 °C under multiple turnover conditions (no DNA trap) as described previously (26–28). A time course for extension of P/T with maximum concentrations of correct dNTP indicated that reactions with wild-type or mutant pol were steady-state for at least 2 min. Therefore, reactions were quenched with an equal volume of 0.3 M EDTA 2 min after initiation with enzyme and the observed rate (V_obs) was calculated by dividing the concentration of product formed by the reaction time. V_obs was plotted as a function of each dNTP concentration, and the apparent K_m and V_max values for incorporation of each dNTP into each P/T were estimated for those which approached saturation by fitting the data to the Michaelis-Menten function as shown in Equation 3.

(Eq. 3)

Steady-state Extension Assays—Steady-state kinetics of extension of all 16 base pairs were measured by incorporation of increasing concentrations of the next correct nucleotide (dTTP) into alternative P/Ts (44N/NA in Table I) at 37 °C for 2 min under multiple turnover conditions as described previously (26, 27, 29). The P/Ts differed only in the base pairs (matched and mismatched) at the 45^th position with respect to primer and were extended by incubation with increasing concentrations of dTTP. In some cases, the high dTTP concentrations used for extension of mismatches by the exo-deficient pol promoted utilization of >50% of the substrate during a 2-min incubation. In those cases, reactions were performed for shorter periods (30 s) to ensure steady-state kinetics. The observed rates of extension (V_obs) were obtained by dividing the concentration of 46-mer by the reaction time and plotting these rates as a function of dTTP concentration to estimate the apparent K_m and V_max as described above. The V_max/K_m ratio indicated the efficiency of extension of each base pair.

Processive Elongation in the Presence of Three dNTPs—The 45-mer primer strand (44A) was labeled at the 5' end with [³²P] as indicated above and annealed to template 4 (Table I). The P/T (100 nM) was incubated with 50 nM wild-type or exo-deficient pol in the presence of 2 mM EDTA for 10 min, and reactions were initiated by the addition of (final concentrations) MgCl₂ (6 mM), dTTP, dCTP, and dGTP (250 µM each) and activated calf thymus DNA trap (500 µg/ml). Reactions were terminated with excess 0.3 M EDTA at various intervals from 0 to 2 s with the aid of a rapid quench instrument, and reaction products were analyzed by electrophoresis through denaturing gels. The efficacy of the DNA trap was demonstrated by the lack of primer extension in control reactions in which the enzyme and DNA trap were added together prior to initiation of reactions and product formation was monitored over the 2-s time course (results not shown). The concentration of products of various sizes that were formed was calculated from the proportion of all of the primers extended to greater than or equal to that size and normalized to total radioactivity in the lanes. The plots of the concentration of each product ([product]) versus time (t) were fit to the exponential burst equation as shown in Equation 4,

(Eq. 4)

to estimate the amplitude (A) and observed burst rate constant (k) except as indicated under "Results." For each experiment, the concentration of enzyme productively engaged with the P/T was the amplitude of the curve for the plot of products extended by at least one nucleotide (to 46 nt in length) as a function of time. The concentration of primer extended to an indicated length at 2 s was calculated from the curve fits and divided by the concentration of productively engaged enzyme to estimate the proportion of enzyme capable of correct extension (48 nt), misincorporation (49 nt), or mismatch extension with correct nucleotide (50 nt).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Nucleotide Excision Activity—A mutation in the conserved exo site I domain of the HSV-1 pol (D368A) had been reported previously to lack significant exo activity (9, 20). To confirm the complete absence of exo activity in D368A pol and to ensure that the mutation did not compromise the polymerizing activity of the enzyme, we purified the mutant and wild-type enzymes from Sf9 insect cells infected with recombinant baculoviruses, which expressed these genes as described under "Experimental Procedures." Comparable purities were achieved for the D368A and wild-type enzymes, which were estimated to be >95% by comparison of Coomassie Blue-stained bands to bovine serum albumin controls (13) and by the absence of other polypeptides on silver-stained gels (data not shown). Active site titration of D368A and wild-type pol preparations indicated that each contained a comparable proportion of active enzyme (40 and 50% active, respectively).

The exo activities of the purified pols were analyzed under sensitive pre-steady-state conditions using 5' end-labeled 45-mer ssDNA (10 nM) and 50 nM enzyme. Reactions were initiated by the addition of MgCl₂, and the products were separated by denaturing gel electrophoresis. Concentration of 45-mer remaining as a function of time was fit to a double exponential function (Equation 1), and the results demonstrated rapid removal of nucleotide from the 3' end of 20% ssDNA (rate constant of 21.5 ± 6.6 s^-1) by the wild-type pol (Fig. 1A). This was followed by slower excision (rate constant of 0.065 ± 0.006 s^-1) of the remainder of the DNA, most probably reflecting multiple turnovers of enzyme with DNA substrate. Under the same conditions, no excision of the 45-mer by D368A mutant pol was observed over a period of 50 s (Fig. 1A). To better assess whether the D368A mutant was capable of digesting the ssDNA after extensive periods of incubation, wild-type or mutant enzymes were incubated with the ssDNA at a 1:100 ratio to allow for sustained multiple turnovers. The steady-state rate constant for the removal of the 3'-nucleotide from the ssDNA by the wild-type enzyme was estimated at 0.047 ± 0.003 s^-1 by fitting the data to Equation 2 (Fig. 1B) in close agreement with that observed for slow excision with high enzyme:DNA ratios (Fig. 1A). However, no loss of 45-mer was detected even when incubated with D368A pol for up to 2 h (Fig. 1B). The D368A pol also failed to excise nucleotide from mismatched P/T under pre-steady-state or steady-state conditions (results not shown but see Fig. 4A, below). Thus, D368A pol is completely deficient in 3'- to 5'-exo activity.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Exonuclease activity of purified polymerases. The ability of the mutant () and wild-type (•) pols to degrade a 5' end-labeled 45-mer ssDNA substrate (44A primer strand in Table I) was monitored over time using enzyme:DNA concentrations of 5:1 (A) and 1:100 (B). Products were separated by electrophoresis through denaturing sequencing style gels, and the amount of 45-mer remaining was quantified and plotted as a function of time. A, enzyme and DNA were preincubated in the presence of EDTA, and reactions were initiated by the addition of MgCl2. The data for the wild-type pol were fit to a double exponential function to estimate a fast rate of 21.5 s-1 for 20% of the substrate and a slower rate of 0.065 s-1 for 76% of the substrate. B, reactions were initiated by the addition of enzyme. The data for the wild-type pol was fit to a single exponential function to estimate a steady-state rate constant of 0.047 s-1.

View larger version (29K): [in this window] [in a new window]   FIG. 4. Mismatch extension assays. The D368A exo-deficient mutant (A, C, and D) or wild-type pol (B, E, and F) (1.25 nM) was incubated with primer/templates (125 nM) as shown (44N/TA in Table I) where N is the 3'-nucleotide on the primer strand. Reactions contained increasing concentrations (long open triangle) of next correct nucleotide (dTTP), and the amount of primer extended following a 30-s or 2-min incubation period (for D368A or wild-type pol, respectively) was quantified. A and B, representative autoradiograms of the products of extension are shown. The positions the primer (P) and other products are as indicated in the legend to Fig. 2. For the 44G/TA P/T, extension by D368A approached saturation with the lower concentration range (0–250 µM) of dTTP shown on the autoradiogram compared with that shown (0–1000 µM) with the other mismatched P/Ts. The rate of extension at each concentration was plotted as a function of time, and if saturation was approached, the data were fit to Equation 3 to estimate the apparent Vmax and Km. For those plots in which dTTP concentrations did not approach saturation, the slope of a linear fit of the data was used to estimate extension efficiency (Vmax/Km). The plots for extension of matched (C and E) and mismatched (D and F) primer termini are shown.

Steady-state Correct and Incorrect dNTP Incorporation—Because the D368A mutation did not alter the polymerization function of HSV-1 pol, we reasoned that the impact of the pol-associated exo activity on nucleotide selectivity could be determined by comparing the efficiency of incorporation of correct and incorrect dNTPs by the wild-type and D368A pols. The D368A or wild-type pol was incubated at 37 °C with a 100-fold excess of 5' end-labeled P/T (44A/TA) and increasing concentrations of the next correct nucleotide (dTTP). The products formed during a 2-min incubation time, previously determined to reflect steady-state rates of accumulation, were assessed qualitatively by autoradiography (Fig. 2, A and B), and extension was quantified by phosphorimaging analysis (Fig. 2, C–F). As found for pre-steady-state rates of extension, the steady-state rate for correct extension by the wild-type pol was slower than that of D368A pol (see Table II). Incubation of the wild-type pol under these conditions resulted not only in extension but also in degradation of the primer strand (Fig. 2B), confirming that the slower steady-state rate of extension was due to competition between the polymerization and exo activities. This competition also resulted in a 6-fold higher apparent K_m for dTTP and an 8-fold reduced efficiency (V_max/K_m) for extension by the wild-type compared with the exo-deficient pol (Table II and Fig. 2).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Steady-state incorporation of correct and incorrect nucleotides into a model primer/template. The D368A exo-deficient mutant (A, C, and D) or wild-type pol (B, E, and F) was incubated with a 100-fold excess of P/T 44A/TA and increasing concentrations (long open triangle) of correct (dTTP) or incorrect (dCTP, dGTP, and dATP) dNTP, and the amount of extended primer following a 2-min incubation period was quantified. A and B, representative autoradiograms of gels of products formed with the indicated dNTP. P denotes the migration of the 45-mer primer strand. Positions of extension products are marked as +1, +2, or +3, whereas positions of degradation products are marked as -1 and -2. The observed rates for correct (C and E) and incorrect (D and F) incorporations were determined and plotted as a function of nucleotide concentration, and if nucleotide concentrations approached saturation, the data were fit to the Michaelis-Menten function (Equation 3) to estimate the Vmax and apparent Km.

The efficiencies (V_max/K_m) with which all of the 12 possible mispairs were formed with the D368A and wild-type pols are shown in Fig. 3. The absence of exo activity resulted in an increased efficiency, ranging from 1.6- to 170-fold, for all of the quantifiable misincorporation events. On average, the exo activity of the wild-type pol reduced the efficiency of misincorporation <30-fold, although differences were not significant for the formation of four mispairs. There was no detectable misincorporation of dCTP opposite a C residue on the template (C:C mispair) for either the wild-type or mutant pol, a result that was observed on other P/Ts with a different sequence context.² Incorporation of dGTP opposite an A residue was also among the least favored misincorporation events for both enzymes, although formation of G:G mispairs was even less efficient for the wild-type pol (Fig. 3). For D368A pol, the most efficient misincorporation events included dTTP opposite G- or C-templating residues. Whereas efficiency of misincorporation of dTTP opposite G or C was also among the highest for the wild-type pol, similar efficiencies were observed for the formation of four other mispairs.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Efficiencies for incorporation of incorrect dNTPs into various P/Ts. Steady-state assays were performed as indicated in the legend to Fig. 2 using four different P/Ts and monitoring incorporation of each incorrect dNTP to form the various mispairs shown (incorporated dNTP:template base). The efficiencies of incorporation (min-1) of incorrect dNTPs from Table II were calculated (Vmax/Km) and are displayed as mean ± S.D. for the exo-deficient pol (open bars) and for the wild-type pol (hatched bars). In the cases in which concentrations of nucleotide used did not approach saturation, the efficiency was reported as the slope of the data fit to a linear function by regression analysis.

Steady-state Mismatch Extension Efficiency—The ability of the 44A/TA P/T to be extended by three nucleotides in the presence of dCTP indicated that both the wild-type and exodeficient pols were capable of extending mismatched primer termini, at least at high concentrations of dNTP (Fig. 2, A and B). To determine the efficiency by which each enzyme could extend different mismatched P/Ts, we used P/Ts with termini composed of each possible base pair and measured their respective abilities to be extended with increasing concentrations of the next correct dNTP (dTTP). An example of the results of this assay for the four different 44N/TA primer/templates is shown in Fig. 4, and the results for all of the P/Ts are summarized in Fig. 5. As observed for misincorporation, mismatch extension by the exo-deficient pol varied most prominently at the level of K_m of the nucleotide, although more variation in the V_max for extension of mismatches was observed than that for misincorporation (data not shown). The exo-deficient pol displayed a wide range of efficiencies for extending different mismatched base pairs, whereas the wild-type pol exhibited uniformly low efficiencies with the exception of the 44N/AA P/T. For this P/T, mismatches were removed by the exo activity of the wild-type pol and then repaired and extended with the single nucleotide added (dTTP) (Fig. 5). This resulted in extension efficiencies by the wild-type enzyme similar to those that were matched. By contrast, the D368A pol was inefficient at extending all of the mismatched termini, confirming the absence of editing function.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Efficiencies for extension of mismatched primer termini with next correct nucleotide. Steady-state reactions were performed as described in the legend to Fig. 4 using increasing concentrations of the next correct nucleotide, dTTP, to extend each of the P/Ts indicated. The apparent Vmax and Km for dTTP incorporation into each P/T were determined as described in the legend to Fig. 4. The efficiency of extension (min-1) with dTTP (Vmax/Km) by the exo-deficient pol (open bars) and by the wild-type pol (hatched bars) is displayed as mean ± S.D. for each set of mismatched P/Ts.

Mismatch Extension during Processive DNA Synthesis—Because removal of a mismatch by the wild-type pol would be favored in multiple turnover reactions if initial binding of the P/T was to the pol exo site, we examined the ability of the wild-type and mutant pol to extend mismatches under conditions that did not permit enzyme cycling to new P/Ts. Running start reactions were performed in the presence of only three dNTPs (250 µM of each) using a P/T designed to allow correct incorporation of several nucleotides before a templating residue for which no matched nucleotide would be encountered by the pol (Fig. 6A). The pol was allowed to bind stably to the P/T prior to initiation of reactions with MgCl₂, and excess activated calf thymus DNA as a trap. In the absence of dATP, processive extension with correct dNTP was possible only up to 48 nt in length. Extension of the primer to 49 nt required a misincorporation, whereas extension to 50 nt required extension of a mismatch (Fig. 6, A–C). Fig. 6, D and E, shows plots for formation of these products as a function of time (from 10 ms to 2 s) for the wild-type and D368A pol, respectively. The data for extension of the primer by both enzymes to form products of 48 or 49 nt in length fit well to the exponential burst function (Equation 4). Nearly all of the P/T productively engaged with either pol was extended from 3 to 48 nt in length without dissociation (Fig. 6, D and E). However, only 76 and 49% P/T productively engaged by the wild-type and exo-deficient pol, respectively, was extended at least 1 nt further by 2 s, presumably with incorrect dNTP. Table III demonstrates that the apparent burst rate constants for extension with correct dNTP to form 46- or 48-nt products did not vary significantly with respect to the pol, although the apparent burst rate constants for the formation of the 49-mer mispaired product was 4 times slower for D368A compared with wild-type pol. Surprisingly, the plot for extension of mismatched P/T by the exo-deficient pol to form a 50-nt product did not fit an exponential function but did fit well to a linear function, suggesting a different and/or substantially slower rate-limiting step than observed with the wild-type pol (Fig. 6, D and E, and Table III). Similar results were observed with lower dNTP concentrations (100 µM), although the proportion of P/T extended to 49 and 50 nt was somewhat reduced (results not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 6. Misincorporation and mismatch extension during processive DNA synthesis with catalytic subunits. A, a P/T was used that consisted of a 5' end-labeled 45-mer primer (44A in Table I) and a 67-mer template strand with sequence modified as shown. The wild-type (B and D) or D368A mutant (C and E) pol catalytic subunit (50 nM) was incubated with 100 nM P/T, and reactions were initiated with 6 mM MgCl2, a mixture of dCTP, dGTP, and dTTP (250 µM each) and activated calf thymus DNA trap (500 µg/ml) to ensure single turnovers of enzyme with P/T. Reactions were terminated with EDTA from 0.005 to 2 s later, and the products were separated by denaturing gel electrophoresis and quantified as described under "Experimental Procedures." B and C, autoradiograms of the separated products are shown. D and E, plots of the concentration of products formed greater than or equal to 48 (•), 49 (), or 50 () nt in length as a function of time are shown. The data were fit to Equation 4 with the exception of the plot for formation of 50-nt products by D368A, which was fit to a linear function. The percentages shown to the right of each plot represent the amount of primer extended to that length or greater in 2 s as a proportion of the P/T productively engaged by the respective polymerases.

View larger version (33K): [in this window] [in a new window]   FIG. 7. Misincorporation and mismatch extension during processive DNA synthesis in the presence of UL42 processivity factor. Reactions were performed and extension products were quantified as described in the legend to Fig. 6 with the exception that the enzymes used were purified complexes of wild-type pol·UL42 (A and C) or D368A pol·UL42 (B and D). A and B, autoradiograms of products formed at the indicated times. C and D, plots of the concentration of products formed greater than or equal to 48 (•), 49 (), or 50 () nt in length as a function of time. The data were fit to Equation 4 or a linear function as detailed in the legend to Fig. 6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The relatively high misincorporation frequency of the wild-type HSV-1 pol (1 in 300 incorporation events, Ref. 13) predicts that >500 mismatches could be introduced per genome replicated, surely to be lethal if fixed in the genome. However, failure of the pol to extend mismatches would reduce misincorporation frequency in vivo. Steady-state kinetic analysis of the D368A exo-deficient pol demonstrates that mismatches do impose a kinetic barrier to the polymerizing function of the pol (Figs. 4 and 5), independent of the presence or absence of exo activity. For an exo-deficient pol, this barrier would be expected to promote stalling and/or dissociation of the enzyme prior to extension. For the wild-type pol with an associated exo activity, the kinetic barrier to mismatch extension increases the probability that the mismatch will be switched to the exo site and then removed. That this switching occurs is demonstrated by the fact that the low efficiency for mismatch extension by the wild-type pol in steady-state reactions is due in large part to excision of the mismatch (Fig. 4). Because the wild-type HSV-1 pol preferentially excises mismatched compared with matched primer termini (13), these steady-state reactions do not accurately measure the relative probability for extending a mismatched compared with a matched primer terminus for that enzyme.

To more directly determine the ability of each pol to extend mismatches during processive synthesis, we forced misincorporation and prevented repair of excised primer termini by the wild-type pol by limiting the nucleotide pool to three dNTPs. Under these conditions, both the wild-type and exo-deficient pol catalytic subunits not only readily incorporated an incorrect dNTP, they also extended a substantial proportion of mismatched primer termini (Fig. 6, D and E). Baker and Hall (11) have suggested that the D368A mutant pol possesses a defect in mismatch extension. In that study, the authors were actually comparing steady-state extension of a mismatch by the exo-deficient mutant pol with extension of a matched primer terminus by the wild-type pol due to the presence of repair nucleotide. Our results demonstrate conclusively that D368A pol can extend mismatches, although mismatch extension by the mutant is slower, compared with wild-type enzyme.

The linear kinetics for mismatch extension by the exo-deficient pol suggested that a different parameter was rate-limiting for extension of mismatched compared with matched primer termini. That this rate does not reflect multiple turnovers of enzyme with P/T was confirmed in control experiments, which demonstrated that no P/T was extended if the DNA trap was added prior to the addition of labeled P/T (results not shown). We speculate that this rate-limiting step is the rate by which the D368A pol switches the mismatched P/T between the polymerizing and exo sites on the enzyme. Because the exo site I mutation is thought to prevent the coordination of metal cations to effect excision (19), the D368A pol could hold the primer terminus at the exo site for an extended period of time prior to moving it back to the pol site. Transfer of the primer terminus from the exo to pol site is likely to be faster (and therefore not rate-limiting) for the wild-type pol because excision would produce a matched end, which has a higher affinity for the polymerizing domain than does a mismatched end.

In the absence of correct dNTP, the wild-type pol can engage in idling reactions to successively misincorporate and then remove another misincorporated nucleotide with its associated exo activity (9, 33). The results in Fig. 6 demonstrate that there is a high probability for mismatch extension by the wild-type pol in the absence of correct dNTP despite the presence of an active exo function. However, in the presence of the UL42 processivity factor, mismatch extension is rare, occurring in <6% P/Ts productively engaged by the pol·UL42 holoenzyme (Fig. 7). Although the initial rate for mismatch extension by the mutant compared with the wild-type pol·UL42 complex was slower (Table III), three time more P/T was extended to a size of 50 nt in length or greater by the mutant holoenzyme in 2 s. These results further demonstrate the importance of the exo activity in maintaining fidelity during processive DNA synthesis.

UL42 may also promote a more rapid transfer of the primer terminus from the pol to the exo site. If so, a reduced proportion of mismatched P/T would be predicted to accumulate following extension by the wild-type pol in the presence versus the absence of UL42, thereby reducing the substrate for mismatch extension. The kinetic barrier imposed by the mismatch that promotes switching from the pol to exo site, enhancement of the rate of switching by UL42 perhaps in conjunction with reduced dissociation, and the presence of an active exo function would favor excision and repair when all four dNTPs are present. Together, these factors suggest the means by which overall mutation frequency observed in vivo is significantly lower than that predicted by the poor nucleotide selectivity of the HSV-1 pol.

	FOOTNOTES

 
^* This work was supported in part by Grant GM34930 from the National Institutes of Health (to D. S. P.) and core services provided by the Ohio State University Comprehensive Cancer Center Core Grant P30 CA16058. 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 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 614-292-0735; Fax: 614-292-9805; E-mail: parris.1{at}osu.edu.

¹ The abbreviations used are: HSV-1, herpes simplex virus type 1; pol, HSV-1 DNA polymerase catalytic subunit; exo, exonuclease; dNTP, 2'-deoxyribonucleoside 5'-triphosphate; P/T, primer/template; ss, single-stranded; nt, nucleotide.

² R. Strick and C. W. Knopf, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Zetang Wu, Houleye Diallo, and Maria Chang for expert assistance in conducting some of these experiments.

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