The Herpes Simplex Virus Type 1 DNA Polymerase Processivity Factor Increases Fidelity without Altering Pre-steady-state Rate Constants for Polymerization or Excision*

Murari Chaudhuri, Liping Song, and Deborah S. ParrisDagger

From the Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210

Received for publication, October 1, 2002, and in revised form, January 7, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pre-steady-state and steady-state kinetics of nucleotide incorporation and excision were used to assess potential mechanisms by which the fidelity of the herpes simplex virus type 1 DNA polymerase catalytic subunit (Pol) is enhanced by its processivity factor, UL42. UL42 had no effect on the pre-steady-state rate constant for correct nucleotide incorporation (150 s-1) nor on the primary rate-limiting conformational step. However, the equilibrium dissociation constant for the enzyme in a stable complex with primer-template was 44 nM for Pol and 7.0 nM for Pol/UL42. The catalytic subunit and holoenzyme both selected against incorrect nucleotide incorporation predominantly at the level of nucleotide affinity, although UL42 slowed by 4-fold the maximum rate of incorporation of incorrect, compared with correct, nucleotide. Pol, with or without UL42, cleaved matched termini at a slower rate than mismatched ones, but UL42 did not significantly alter the pre-steady-state rate constant for mismatch excision (~16 s-1). The steady-state rate constant for nucleotide addition was 0.09 s-1 and 0.03 s-1 for Pol and Pol/UL42, respectively, and enzyme dissociation was the rate-limiting step. The longer half-life for DNA complexes with Pol/UL42 (23 s) compared with that with Pol (8 s) affords a greater probability for excision when a misincorporation event does occur, accounting predominantly for the failure of Pol/UL42 to accumulate mismatched product at moderate nucleotide concentrations.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The HSV-11 DNA polymerase holoenzyme is a stable heterodimer composed of a large, 134-kDa catalytic subunit (Pol) and a smaller, 51-kDa subunit, UL42 (1-4). Although Pol possesses inherent 5' to 3' polymerizing activity (5-7), its processivity is greatly enhanced by UL42 (3, 8). Both subunits are absolutely essential for origin-dependent DNA synthesis and for productive viral replication (9-13), indicating the importance of processive DNA synthesis to viral replication. However, the precise mechanism by which UL42 increases Pol processivity is not known and may differ from that utilized by ring-shaped processivity factors such as proliferating cell nuclear antigen and the beta  subunit of Escherichia coli pol III (14).

HSV-1 Pol, similar to pol delta , possesses an inherent 3' to 5' exo activity that imparts proofreading ability to Pol (7, 15). Although the proofreading function of the HSV-1 DNA polymerase can reduce misincorporation frequency in vitro and in vivo (16-18), the impact of UL42 on the exo activity of Pol and its effect on the fidelity of DNA synthesis have not been well studied. In fact, there has been much disagreement regarding the general role of processivity factors on the fidelity of their cognate polymerases. For example, proliferating cell nuclear antigen has been shown to increase base substitution errors and translesion synthesis by pol delta  (19, 20). However, exo-deficient T7 bacteriophage DNA polymerase has a decreased frequency of base substitutions but an increased frequency of frameshift mutations at reiterated sequences in the absence, compared with presence, of thioredoxin (21). To understand better the effect of HSV-1 UL42 on the fidelity of Pol, we have analyzed the pre-steady-state and steady-state kinetics of nucleotide incorporation and excision. Surprisingly, UL42 does not alter the pre-steady-state rate constant for correct nucleotide incorporation by Pol nor that for excision of mismatched primer termini, although UL42 dramatically decreases the accumulation of misincorporated products under pre-steady-state conditions. Because UL42 decreases the dissociation of Pol from the primer-template, these results suggest that the editing of misincorporated nucleotides is facilitated by the increased time of residence of Pol/UL42 on DNA, compared with that of Pol.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymes-- HSV-1 Pol and Pol/UL42 were purified from Sf9 insect cells infected with recombinant baculoviruses that express the genes encoding these proteins as detailed previously (22). Recombinant viruses that contained the HSV-1 Pol or UL42 genes were the kind gifts of Robert Lehman (Stanford University) and Mark Challberg (National Institutes of Health), respectively. Proteins used for the studies described herein were determined to be >95% pure, and all preparations contained 50-75% active enzyme, based on active site titrations (see below).

Preparation of Synthetic Primer-Template-- Synthetic oligonucleotides (Integrated DNA Technologies, Inc., Coralville, IA) were purified by gel electrophoresis. The sequences of the 45-mer primers and 67-mer templates are shown in Fig. 1. For most studies, the primer strand was radioactively labeled at the 5'-end using [gamma -32P]ATP (ICN, Emoryville, CA) and T4 kinase (Invitrogen) by standard procedures (23). Duplex primer-templates were obtained by heating equimolar amounts of primer and template to 55 °C followed by slow cooling to at least 37 °C. Labeled and unlabeled annealed primer-templates were mixed to yield a final specific activity of 150-300 µCi/nmol.


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Fig. 1.   Synthetic model primer-templates. The sequences of the annealed 45-mer primer and 67-mer template strands are shown. The primer-templates shown in A and B are matched at the 3'-end of the primer and were used with dTTP or dATP, respectively, as the next correct nucleotide for incorporation analysis. The primer-template in C contains a single C:T mismatch at the 3'-end of the primer.

Pre-steady-state Single Turnover Reactions-- All pre-steady-state experiments were performed at 37 °C using the Kintec RQ-3 (Austin, TX) rapid quench apparatus described by Johnson (24). Except as indicated, all concentrations of enzyme refer to active concentrations. Enzyme and primer-template were incubated for at least 10 min on ice at the respective concentrations indicated for each experiment in buffer that contained 50 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA,400 µg/ml bovine serum albumin, and 50 mM KCl for Pol or 125 mM KCl for Pol/UL42. Pol and Pol/UL42 have been shown to be optimally active under these respective low and high ionic strength concentrations (22, 25, 26). Prebound 16-µl enzyme-DNA mixtures were equilibrated to 37 °C, and reactions were initiated by the addition of an equal volume of the same buffer to yield the following final concentrations: 6 mM MgCl2, 500 µg/ml activated calf thymus DNA as trapping agent, and dNTP as indicated for each experiment. Except as specifically indicated under "Results," the primer-template used was that shown in Fig. 1A, and the dNTP added was dTTP. Reactions were stopped at various times by the addition of a large volume of 0.3 M EDTA, pH 8.0. Products were separated by electrophoresis through gels containing 12% polyacrylamide and 7 M urea, and the gels were exposed to x-ray film or phosphor screens. The amounts of 45-, 46-, and 47-mer in each sample were quantified using a Molecular Dynamics PhosphorImager and ImageQuant software (Sunnyvale, CA) and normalized to total radioactivity in the sample to minimize errors caused by loading (27).

Active Site Titration-- Initial concentrations of purified preparations of Pol and Pol/UL42 were determined by absorbance measurements and confirmed by comparison of separated protein on Coomassie-stained SDS-polyacrylamide gels using bovine serum albumin as a standard. Pol, corresponding to a protein concentration of 46 nM, or Pol/UL42, corresponding to a protein concentration of 43 nM, was incubated with increasing concentrations of the primer-template shown in Fig. 1A. Reactions were initiated with MgCl2, 40 µM dTTP, and DNA trap and stopped with EDTA 10-500 ms later. The amount of primer extended was plotted as a function of time (t) for each primer-template concentration, and the amplitude of the burst (A) and observed rate constant (k) were determined by Equation 1 (28).


46-<UP>mer</UP>=A(1−e<SUP><UP>−</UP>kt</SUP>) (Eq. 1)
The burst amplitudes were then plotted versus primer-template concentration, and the data were fit to the quadratic equation to give the active enzyme concentration and the equilibrium dissociation constant (Kd) for formation of productive enzyme-DNA complex (27, 29),
[E · D]=0.5(K<SUB>d</SUB>+E<SUB>0</SUB>+D<SUB>0</SUB>)−(0.25(K<SUB>d</SUB>+E<SUB>0</SUB>+D<SUB>0</SUB>)<SUP>2</SUP>−(E<SUB>0</SUB>D<SUB>0</SUB>))<SUP>0.5</SUP> (Eq. 2)
where [E·D] is the concentration of enzyme and DNA in productive complex, and E0 and D0 are the initial enzyme and primer-template concentrations, respectively.

Determination of Kd of dTTP-- Reactions containing 50 nM active Pol or Pol/UL42 and 100 nM primer-template (Fig. 1A) were preincubated in buffer containing EDTA as described above, and reactions were initiated with increasing concentrations of the next correct nucleotide, dTTP (0.1-30 µM), MgCl2, and activated calf thymus DNA trap. The burst rate constant and amplitude for each concentration of dTTP were determined by fitting the data according to Equation 1. The resulting observed rate constants (kobs) were then plotted as a function of dTTP concentration, and the data were fit to the Michaelis-Menten function (27, 29),


k<SUB><UP>obs</UP></SUB>=(k<SUB>p</SUB>[<UP>dNTP</UP>])/([<UP>dNTP</UP>]+K<SUP><UP>app</UP></SUP><SUB>d</SUB>) (Eq. 3)
where kp corresponds to the pre-steady-state catalytic rate constant for formation of productive complex, [dNTP] is the concentration of nucleotide, and K<UP><SUB><IT>d</IT></SUB><SUP>app</SUP></UP> is the apparent equilibrium dissociation constant of dTTP from the enzyme-DNA complex. The amplitudes at each dTTP concentration were also plotted as a function of dTTP concentration and fit to a hyperbolic function to estimate the maximum product formed at saturating dTTP concentration (ampmax) and the true Kd for productive complex of dTTP with enzyme bound to primer-template.
<UP>amplitude</UP>=<UP>amp<SUB>max</SUB></UP>[<UP>dTTP</UP>]/([<UP>dTTP</UP>]+K<SUB>d</SUB>) (Eq. 4)

Pre-steady-state Kinetics of Incorporation of Incorrect dNTP-- Reactions were conducted as indicated above for correct incorporation, except that concentrations of an incorrect nucleotide ranged from 0.125 to 2 mM as indicated for each experiment. The amplitude and burst rate constant were determined by fitting the kinetic data to Equation 1, and the K<UP><SUB><IT>d</IT></SUB><SUP>app</SUP></UP> for an incorrect nucleotide (dATP) was determined by fitting the data to the Michaelis-Menten function (Equation 3).

Rate of Extension during Processive DNA Synthesis-- Reactions were conducted under pre-steady-state conditions in which 25 nM enzyme was preincubated with 50 nM end-labeled primer-template (Fig. 1A). Reactions were initiated with buffer to achieve final concentrations of 6 mM MgCl2; 50 µM each dATP, dCTP, dGTP, and dTTP; and 500 µg/ml activated calf thymus DNA. Reactions were incubated at 37 °C and quenched with 0.3 M EDTA 5-500 ms later. Extension products were resolved through denaturing polyacrylamide gels, and the largest extension product and products greater than 45 in length were quantified by phosphorimaging analysis and normalized as a function of total radioactivity in the lane. In control reactions, EDTA was added at the time of initiation.

Elemental Effect-- Pre-steady-state reactions were conducted by preincubating 50 nM active enzyme concentrations with excess primer-template indicated in Fig. 1B. Reactions with Pol contained 250 nM primer-template, whereas those with Pol/UL42 contained 100 nM primer-template. Reactions were initiated by the addition of 50 µM dATP or 50 µM dATPalpha S (Amersham Biosciences), 6 mM MgCl2, and 500 µg/ml activated calf thymus DNA trap, and the reactions were stopped with EDTA 5-300 ms later. Concentrations of materials used for initiating reactions refer to final concentrations after mixing. The production of extended primer was plotted as a function of time, and the data were fit to the burst equation (Equation 1) to determine the amplitude and rate constant of the burst phase.

Steady-state Kinetics of Correct Nucleotide Incorporation-- Reactions were performed at 37 °C by incubating bulk reactions (400 µl) containing 5 nM active Pol or Pol/UL42 with a 100-fold excess (500 nM) of primer-template. Reactions were initiated by the addition of MgCl2 to 6 mM and dTTP to 40 µM. No DNA trap was included to allow multiple association/dissociation events of enzyme with primer-template. Portions of the reaction were removed at intervals up to 350 s, quenched with EDTA, and the separated products were quantified by phosphorimaging analysis. Product formation as a function of time was plotted, and the data were fit to a straight line. The steady-state catalytic rate constant (kcat) was obtained by dividing the slope of the line by the concentration of enzyme present in the reaction mix.

Determination of koff-- An excess amount of labeled primer-template (Fig. 1A) was incubated with Pol in the presence of 1 mM EDTA, and reactions were initiated with buffer to achieve final concentrations of 5 nM enzyme, 50 nM primer-template, 50 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol, 400 µg/ml bovine serum albumin, 50 mM KCl, 6 mM MgCl2, and 250 µM dTTP. Activated calf thymus DNA (500 µg/ml) or buffer alone was added at intervals from 7 to 80 s later, and reactions were stopped with 0.3 M EDTA 90 s after initiation. The amount of extended product formed was plotted as a function of the time of addition of trapping agent, and the data were fit to a single exponential function (Equation 1), except that k is the dissociation rate constant and t is the time of the addition of DNA trap.

Pre-steady-state Kinetics of Excision-- Reactions were conducted with the rapid quench apparatus as described for incorporation experiments except that dNTP was not added. Excess Pol or Pol/UL42 (100 nM) was preincubated with 20 nM 5'-end-labeled matched or mismatched primer-template (Fig. 1, A and C, respectively), reactions were initiated by the addition of MgCl2 and activated calf thymus DNA trap, and they were incubated at 37 °C. Reactions were terminated at intervals from 10 ms to 8 s later by the addition of 0.3 M EDTA. Products were separated by denaturing gel electrophoresis, and the amount of 45-mer remaining compared with total radioactivity was quantified and plotted as a function of time (t). The data were fit to a double exponential function as follows (30, 31),


45-<UP>mer</UP>=ae<SUP><UP>−</UP>bt</SUP>+ce<SUP><UP>−</UP>dt</SUP> (Eq. 5)
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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Effect of UL42 on Extension Products in Single Turnover Reactions-- Insect cells infected with HSV-1 UL42- and/or Pol-expressing recombinant baculoviruses were the source material for purification of the catalytic subunit and holoenzyme to near homogeneity as detailed previously (22). To begin to understand the possible effects of the processivity factor on the catalytic properties of Pol, we measured the incorporation of correct dNTP in single turnover experiments using a defined model primer-template. This primer-template consisted of a 45-mer primer strand annealed to a 67-mer template strand (Fig. 1A). The size of the primer-template was designed to accommodate fully the amount of DNA bound by the holoenzyme based on DNA footprint analysis (32). We have demonstrated by electrophoretic mobility shift analysis that Pol/UL42 binds to the primer-template at a 1:1 stoichiometry (22). Pol also binds to the primer-template at a 1:1 stoichiometry except at extremely high ratios of protein to DNA, where it can also bind to the lower affinity double-stranded end (22, 33).

Pre-steady-state reactions were performed using a fixed enzyme concentration and varying amounts of the primer-template indicated in Fig. 1A, in which the primer was labeled at the 5'-end. The enzyme and primer-template were preincubated in the presence of EDTA to prevent premature initiation and degradation of the primer by the potent 3' to 5' exo activity. Reactions were initiated with MgCl2 and the next correct nucleotide, dTTP, and stopped with excess EDTA at various periods of time using a rapid quench apparatus. Control experiments (not shown) demonstrated that the addition of activated calf thymus DNA to a final concentration of 500 µg/ml at the time of initiation effectively prevented cycling of the enzyme to additional labeled primer-template. Moreover, the trap had no effect on the rate of incorporation at saturating dNTP concentrations in the time interval associated with the rapid burst. The accumulation of extended primers was monitored using a denaturing gel assay to separate products on the basis of size. Fig. 2 illustrates the products formed by Pol and Pol/UL42 (A and B, respectively) with 2-fold excess primer-template after increasing times of incubation at 37 °C. The respective enzymes were incubated in the low (50 mM KCl) and high (125 mM KCl) ionic strength buffer conditions determined to be optimum for the activity of each enzyme (22). Both Pol and Pol/UL42 extended the primer by one nucleotide in the presence of dTTP to form a 46-mer within 10-20 ms. However Pol, but not Pol/UL42, also accumulated a 47-mer product with somewhat delayed kinetics. Based on the sequence of the primer-template (Fig. 1A), the 47-mer product would correspond to a misincorporation of a T residue opposite a G. The formation of the 47-mer product is not caused by a low level of contamination of the dTTP with dCTP because incorporation of dCTP was shown to lead instead to the rapid appearance of a 48-mer (results not shown). The absence of 47-mer in reactions containing Pol/UL42 suggested that the processivity factor contributed to the overall fidelity with which the HSV-1 Pol incorporated dNTP. To assess better the means by which UL42 increased the fidelity of nucleotide incorporation, we examined the effect of UL42 on the individual kinetic parameters governing Pol incorporation and excision.


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Fig. 2.   Pre-steady-state active site titration. A constant amount of purified Pol or Pol/UL42 (46 and 43 nM, respectively) was incubated with different amounts of the primer-template shown in Fig. 1A, and reactions were initiated by the addition of MgCl2, dTTP, and activated calf thymus DNA trap as described under "Experimental Procedures." A and B, autoradiograms of separated radioactive products of reactions containing Pol or Pol/UL42, respectively, and 100 nM 5'-end-labeled primer-template. In control reactions (lanes 1 and 9), EDTA was added at the time of initiation or was used to terminate reactions after 10, 20, 30, 40, 50, 100, or 200 ms (A, lanes 2-8, respectively, and B, lanes 10-16, respectively). The lengths of resolved products are indicated. C and D, Pol or Pol/UL42 was incubated with the indicated concentrations (in nM) of primer-template, and the data for each plot were fit to Equation 1 to yield the burst amplitudes. E and F, burst amplitudes were plotted as a function of primer-template concentration and fit to Equation 2 to determine the active enzyme concentration with saturating primer-template and the Kd for formation of productive enzyme-DNA complex.

Active Site Titration-- To determine the pre-steady-state parameters for correct nucleotide incorporation, it was first necessary to determine the concentration of molecules in the purified enzyme preparations capable of extending the primer strand. A fixed concentration of Pol or Pol/UL42 (46 and 43 nM, respectively, based on protein determination) was incubated in single turnover reactions at primer-template concentrations ranging from 12.5 to 100 nM, and the formation of extended products was monitored over time. Products were separated from unextended primer by denaturing gel electrophoresis, and both were quantified by phosphorimaging analysis. Because Pol extended a portion of the primer by two nucleotides, we summed the amount of 46-mer and 47-mer formed by Pol to obtain the total amount of extended product. Fig. 2, C and D, shows the kinetics of primer extension by Pol and Pol/UL42, respectively, in the presence of 40 µM dTTP at different primer-template concentrations. The amplitude of the rapid burst at each primer-template concentration, indicative of the amount of active enzyme bound, was determined by fitting the data to Equation 1. In Fig. 2, E and F, the resulting amplitudes were plotted as a function of primer-template concentration, and the data were fit to Equation 2. The results revealed that the active enzyme concentration at saturating primer-template was 32.5 nM for Pol and 25 nM for Pol/UL42, demonstrating that the purified enzyme preparations were 70 and 58% active, respectively. The true Kd for formation of the productive complex between each enzyme and the primer-template was also calculated from these data and was found to be 44 ± 3 nM for Pol and 7.0 ± 2 nM for Pol/UL42. These Kd values were consistent for several independent preparations of Pol and Pol/UL42 with active protein concentrations ranging from 50 to 75% (results not shown).

Pre-steady-state Kinetics of Correct dNTP Incorporation-- We determined the dependence of polymerization on the concentration of the next correct dNTP using a 2-fold excess of model primer-template (100 nM) to active enzyme (50 nM). To ensure single turnover kinetics, enzyme and primer-template were preincubated in the presence of EDTA, and reactions were initiated by the addition of increasing concentrations of dTTP (0.1-30 µM), MgCl2, and activated calf thymus DNA to trap unbound enzyme or that dissociated from the primer-template. Reactions were terminated with 0.3 M EDTA at intervals ranging from 5 to 500 ms using a rapid quench apparatus, and the extension products were separated and quantified. The results are shown in Fig. 3 for Pol (A, C, and E) and Pol/UL42 (B, D, and F) and demonstrate that both the amount and rate of formation of extended primer are dependent upon dTTP concentration in the presence or absence of UL42. The observed rates of incorporation at each dTTP concentration by Pol and Pol/UL42 (Fig. 3, A and B, respectively) were calculated by fitting the data to Equation 1. The observed rate constants were then plotted versus dTTP concentration using the Michaelis-Menten function (Equation 3) to predict the pre-steady-state rate constant for polymerization (kp) at saturating dTTP and the apparent equilibrium dissociation constant for dTTP. The results (Fig. 3, C and D) demonstrate that the kp of Pol (157 ± 31 s-1) and Pol/UL42 (137 ± 21 s-1) did not differ significantly (p > 0.1), indicating that UL42 does not alter the inherent rate at which the next correct nucleotide is incorporated under single turnover conditions. The apparent dissociation constant of dTTP based on this analysis was 12.2 ± 5.7 µM for Pol and 6.4 ± 2.8 µM for Pol/UL42 (Fig. 3, C and D, respectively). However, the fact that the amount of product varied as a function of dTTP concentration (Fig. 3, A and B) suggested that some of the preformed enzyme-DNA complex dissociated and was trapped by the calf thymus DNA prior to the formation of a stable ternary complex with dTTP. Therefore, we plotted the amplitude of the reaction as a function of dTTP concentration and fit the data to a hyperbola (Equation 4) to estimate the maximum amount of enzyme-DNA complex that could be driven into productive complex with saturating dTTP (Fig. 3, E and F). The results demonstrate that the 2:1 ratio of primer-template to active enzyme was insufficient to drive all of the enzyme into productive complex because the maximum amplitude was 29.8 ± 1.2 nM for Pol (Fig. 3E) and 36.4 ± 4.7 nM for Pol/UL42 (Fig. 3F). The concentration of dTTP at half-maximum amplitude, therefore, provides the most accurate measure of the true Kd for dTTP in productive ternary complex with enzyme and primer-template. The results indicate that the true Kd for dTTP with Pol (1.57 ± 0.30 µM) was not significantly different from that with Pol/UL42 (2.84 ± 1.45 µM). The fact that amplitude changes less at high dTTP concentrations, compared with concentrations below the apparent Kd, supports a two-step model for the binding of nucleotide to the enzyme-DNA complex as suggested for the mechanism of HIV reverse transcriptase (34). This model predicts that only a proportion of initial binding events leads to a conformational change in the ternary complex which, in turn, stabilizes the complex prior to chemical catalysis. If multiple exchanges of nucleotide occur with a given enzyme-DNA complex, then high nucleotide concentrations would increase the probability for a binding event with a subsequent conformational change prior to the dissociation of the enzyme from primer-template, whereas subsaturating dTTP concentrations would favor more dissociation prior to the stabilizing conformational change.


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Fig. 3.   dTTP concentration dependence of pre-steady-state primer extension. A fixed active concentration (50 nM) of Pol (A and C) or Pol/UL42 (B and D) was incubated with 100 nM end-labeled primer-template as indicated in the legend to Fig. 2 except that reactions contained varying amounts of dTTP. A and B, the curves were determined by quantifying the extension products formed at each time and fitting the data to Equation 1 to determine the burst rate constants. The curves in ascending order were derived using 0.1, 0.5, 2.5, 5, 10, 20, or 30 µM dTTP. C and D, the rate constants (kobs) for the burst reactions were plotted as a function of dTTP concentration, and the data were fit to Equation 3 to determine the pre-steady-state catalytic rate constant for single incorporation (kp) and apparent Kd of dTTP as described under "Results." E and F, the amplitudes of the burst reactions were plotted as a function of dTTP concentration, and the data were fit to Equation 4 to determine the maximum amount of enzyme, primer-template, and dTTP in a productive ternary complex at saturating dTTP and the true Kd for dTTP in a productive ternary complex.

Pre-steady-state Kinetics of Incorrect dNTP Incorporation-- As indicated in Fig. 2, A and B, Pol, but not Pol/UL42, permitted accumulation of a 47-mer, apparently because of misincorporation. To quantify more accurately the ability of each enzyme to incorporate incorrectly base-paired nucleotides, we performed pre-steady-state kinetic analysis in single turnover reactions essentially as described above. The primer-template (100 nM) indicated in Fig. 1A, in which A is the templating residue, was preincubated with 50 nM Pol or Pol/UL42, and reactions were initiated with 1 mM (final concentration) incorrect nucleotide (dATP, dCTP, or dGTP), MgCl2, and activated calf-thymus DNA trap. Fig. 4, A and B, shows the reaction products formed by Pol and Pol/UL42, respectively, at increasing times after initiation with 1 mM dATP. The amount of primer extended by Pol and Pol/UL42 with the incorrect nucleotide (dATP) was quantified as a function of time, and the results demonstrate a burst in mismatched product formation (Fig. 4, C and D, respectively). Although both Pol and Pol/UL42 extended the primer with this high concentration of incorrect nucleotide (dATP), approximately half as much was extended by Pol/UL42 (3.57 ± 0.13 nM) compared with that extended by Pol (5.94 ± 0.31 nM). Moreover, the observed rate constant for misincorporation of 1 mM dATP by Pol/UL42 (34 ± 4 s-1) was approximately half that for Pol (76 ± 16 s-1). In fact, Pol extended the primer with each incorrect dNTP, with rate constants for dATP > dCTP > dGTP. Pol/UL42 also extended the primer with 1 mM dCTP, but no significant misincorporation was observed with 1 mM dGTP (results not shown).


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Fig. 4.   Pre-steady-state misincorporation of dATP. Reactions were performed by preincubating 50 nM Pol (A, C, and E) or Pol/UL42 (B, D, and F) with 100 nM end-labeled primer-template indicated in Fig. 1A. Reactions were initiated as described in the legend to Fig. 3 except that the nucleotide added was dATP, which forms a mismatch with the templating A residue. A and B, autoradiograms of gel showing the products of reactions containing 1 mM dATP and Pol or Pol/UL42, respectively, after incubation for the times indicated. The positions of the 45-mer primer and the primer extended by 1 nucleotide (46) are shown. C and D, product formation in the reactions shown in A and B above was quantified by phosphorimaging analysis. The curves were determined by fitting the data to Equation 1 to determine the burst rate constants. E and F, the burst rate constants were determined with various concentrations of dATP (0.125-2 mM) and were plotted versus the dATP concentration. The curves show the fit of the data to Equation 3 to determine the pre-steady-state catalytic rate constant for misincorporation (kmis) and the apparent Kd of dATP as described under "Results."

We performed detailed kinetic analysis with increasing concentrations of the most readily misincorporated nucleotide, dATP, as indicated above. Fig. 4, E and F, shows a plot of the resulting observed rate constants as a function of dATP concentration for Pol and Pol/UL42, respectively. The data were fit to the Michaelis-Menten function (Equation 3) to determine the rate constant for extension with saturating incorrect nucleotide (dATP) and the apparent Kd for dATP. The apparent Kd for formation of productive complex with dATP was 279 ± 83 µM for Pol and 229 ± 89 µM for Pol/UL42, approximately 2 orders of magnitude greater than that for correct nucleotide, dTTP (Fig. 3). Interestingly, the maximum rate for misincorporation of dATP (kmis) by Pol was 106 ± 9 s-1, only slightly less than the maximum rate for correct incorporation; however, the maximum rate for misincorporation of dATP by Pol/UL42 (37 ± 4 s-1) was approximately one-third that observed for Pol. Nevertheless the maximum rate of misincorporation of dATP by Pol/UL42 was only four times slower than the maximum rate of incorporation of correct dNTP. Thus, nucleotide discrimination occurs predominantly at the level of nucleotide affinity for both enzymes. If the selectivity index for incorporation of correct over incorrect dNTP is calculated as follows (34),
<UP>selectivity</UP>=((k<SUB>p</SUB>/K<SUB>d</SUB>)<SUB><UP>dTTP</UP></SUB>+(k<SUB><UP>mis</UP></SUB>/K<SUB>d</SUB>)<SUB><UP>dATP</UP></SUB>)/((k<SUB><UP>mis</UP></SUB>/K<SUB>d</SUB>)<SUB><UP>dATP</UP></SUB>) (Eq. 6)
then the selectivity of Pol/UL42 is 296, whereas that of Pol is 265. Thus, both Pol and Pol/UL42 clearly distinguish correct from incorrect dNTP under pre-steady-state conditions, but UL42 does not significantly enhance the overall ability of the catalytic subunit to discriminate correct (dTTP) from incorrect (dATP) nucleotide during a single cycle of polymerization.

Kinetics of Incorporation during Processive Synthesis-- For multiple incorporations to occur during processive DNA synthesis, the polymerase must translocate on the primer-template to the adjacent residue on the template without dissociating. If this rate were slower than the rate of extension of the primer by one nucleotide (~150/s), then the rate by which all subsequent nucleotides are incorporated would be substantially slower than the kp. We therefore tracked the extension of primer in the presence of near saturating amounts (50 µM) of all four dNTPs as a function of time. Multiple turnovers of enzyme with primer-template were prevented by the addition of excess activated calf thymus DNA trap at the time of initiation with MgCl2. Although the maximum number of nucleotides incorporated at each time was the same for Pol and Pol/UL42, the distribution of extension products was somewhat altered by UL42 in favor of larger products (compare Fig. 5, B and A). By 100 ms, 0.6 and 4.3% of the extension products were full-length with Pol and Pol/UL42, respectively. By 200 ms, 18% of the extension products were full-length for both enzymes. These results demonstrate a maximum extension rate, ranging from 110 to 220 nucleotides/s during processive synthesis, which is not limited by the rate of translocation of the polymerase. However, the efficiency of translocation among those primer-template molecules engaged by Pol may be greater in the presence of UL42, consistent with the role of UL42 as a processivity factor.


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Fig. 5.   Primer extension during processive DNA synthesis. Preincubation reactions contained 25 nM Pol (A) or Pol/UL42 (B) and 50 nM 5'-end-labeled primer-template. Reactions were initiated in the presence of DNA trap and incubated at 37 °C as described in the legend to Fig. 2 except that all four dNTPs were added to final concentrations of 50 µM. The figure shows an autoradiogram of products resolved on denaturing gels. The sizes of the unextended primer (45) and fully extended product (67) are indicated. In lanes 1 and 2, EDTA was added to the initiation buffer to prevent extension. Lanes 3 and 4 show products of reactions terminated with EDTA after 5 ms, and in lanes 5-14 reactions were terminated at 10, 20, 30, 40, 50, 60, 80, 100, 200, and 500 ms, respectively.

Rate-limiting Step for Pre-steady-state Incorporation of Correct dNTP-- The pre-steady-state rate constant we determined reflects the slowest step, which occurs after the binding of enzyme to primer-template and dNTP until formation of the extended product. To ascertain whether the rate-limiting step reflected the rate of chemical bond formation or occurred prior to catalysis, we measured the elemental effect caused by substituting a phosphothioate analog of dNTP. The rate of formation of the chemical bond with phosphothioate in intermolecular enzymatic and nonenzymatic interactions (the thio effect) has been estimated to be 4-100 times slower than with the alpha -phosphate of dNTP (27, 35-37). It has been argued that a smaller effect of thio substitution on rate indicates that a step other than, or in addition to, chemistry is rate-limiting (27, 36-40). We compared the pre-steady-state kinetics of incorporation of dATPalpha S with that of dATP (correct nucleotide) for Pol and Pol/UL42 into a primer-template containing a T residue on the template opposite the 46th position on the primer (Fig. 1B). The experiments shown in Fig. 6 contained a fixed concentration (50 nM) of Pol or Pol/UL42 preincubated with excess primer-template (100 nM for Pol/UL42 and 250 nM for Pol). A higher ratio of primer-template to enzyme was used for Pol reactions because of the lower affinity of Pol, compared with Pol/UL42, for primer-template (Fig. 2, E and F). Reactions were initiated with 6 mM MgCl2, 50 µM dATP or dATPalpha S (correctly matched dNTP), and 500 µg/ml activated calf thymus DNA trap (final concentrations), and the pre-steady-state burst rate of polymerization was determined by fitting the data to Equation 1. For Pol, the burst rate constant for dATP incorporation was 201 ± 28 s-1, whereas that for dATPalpha S was 62 ± 6 s-1, a 3.2-fold effect (Fig. 6A). Because there was little or no effect of the thio-substituted analog on the amplitude of the Pol reaction under these high nucleotide concentrations, there was a similar probability for the formation of a stable ternary complex of either nucleotide with enzyme and primer-template prior to enzyme dissociation (see discussion of Fig. 3 above). For Pol/UL42 complex, the burst rate constant for dATP was 261 ± 26 s-1 and for dATPalpha S, it was 185 ± 2 s-1, a 1.4-fold effect (Fig. 6B). The somewhat reduced amplitude of the Pol/UL42 reaction with the thio derivative is caused by enzyme dissociation prior to catalysis, which is consistent with a lower affinity of thio-substituted dATP, compared with oxy-dATP, for Pol/UL42 bound to primer-template. Thus, for both Pol and Pol/UL42, the thio effect is small and of a magnitude similar to that reported for incorporation of a correct nucleotide by other polymerases, where a rate-limiting conformational step has been proposed (27, 36, 38-41). Nevertheless, the thio effect for nonenzymatic intermolecular reactions of phosphate diesters, where there is no evidence for a conformational step, was determined to range from 4 to 11, arguing that even small thio effects occur when chemistry is rate-limiting (37). Although there is no consensus for the absolute amount of elemental effect which can completely rule out a rate-limiting chemistry step, an elemental effect of less than 4 is indeed small and suggests that the primary limitation to rate occurs prior to chemical catalysis; however, some contribution of chemistry to the rate-limiting step cannot be ruled out (27, 37). It is likely that the slow step prior to catalysis reflects the rate at which a conformational change occurs in the ternary complex of enzyme bound to both primer-template and dNTP (27, 38, 39).


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Fig. 6.   Elemental effect. Pre-steady-state reactions were conducted with 50 nM Pol and 250 nM 5'-end-labeled primer-template shown in Fig. 1B (A) or 50 nM Pol/UL42 and 100 nM primer-template (B). Reactions were initiated with MgCl2, calf thymus DNA trap, and 50 µM dATPalpha S () or dATP (black-square) and terminated at various times by the addition of EDTA. Extension products were separated, quantified, and plotted as a function of time, and the data were fit to the burst equation to produce the indicated curves.

Steady-state Kinetics of Incorporation-- To determine whether UL42 alters the steady-state kinetics for primer extension during multiple turnovers of enzyme with primer-template, we employed excess primer-template shown in Fig. 1A (500 nM) to enzyme (5 nM) and initiated reactions by the addition of dTTP, and MgCl2, but without trap DNA. Reactions were terminated at intervals from 10 to 350 s by the addition of EDTA. Fig. 7A shows the accumulation of extended primers as a function of time for Pol and Pol/UL42. The steady-state rate constant (kcat) was calculated as the ratio of the slope of the line derived by linear regression analysis to enzyme concentration and was found to be 0.090 ± 0.002 s-1 for Pol and 0.032 ± 0.002 s-1 for Pol/UL42. Because the steady-state rate reflects the rate of the slowest step in the reaction, and it is substantially slower than the kp, the kcat most likely reflects the dissociation of enzyme from the primer-template.


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Fig. 7.   Steady-state kinetics of incorporation. A, bulk reactions (400 µl) contained 5 nM Pol () or Pol/UL42 (black-square) and 500 nM labeled primer-template shown in Fig. 1A. No DNA trap was included, and reactions were initiated with 6 mM MgCl2 and 40 µM dTTP (final concentrations) and incubated at 37 °C. At the indicated times, a portion was removed and terminated with EDTA. Products were separated, quantified, and plotted as a function of time of reaction. The data were fit to a straight line by the least squares method. B, reactions with 5 nM Pol were assembled and initiated as described in A except that 50 nM primer-template was used. Activated calf thymus DNA was added to a final concentration of 500 µg/ml at the times indicated (), or an equivalent volume of buffer was added to parallel control reactions (black-square). All reactions were terminated 90 s after initiation, and the products were separated and quantified. The plot shows the concentration of extended primer as a function of the time of addition of DNA trap or buffer. The data for reactions trapped with calf thymus DNA were fit to Equation 1 to determine the koff. The dotted line indicates the average amount of primer extended after a 90-s reaction time in buffer controls.

To test this hypothesis directly, reactions containing 5 nM Pol and 50 nM labeled primer-template were initiated by the addition of MgCl2 and a saturating concentration of dTTP (250 µM). Activated calf thymus DNA trap (500 µg/ml) or buffer alone was added at various times after initiation to trap Pol that dissociated from the primer-template. All reactions were terminated 90 s after initiation. The addition of buffer alone had little or no effect on the amount of product formed at 37 °C (~30 nM). The addition of DNA trap at the same time as reactions were initiated led to formation of only 5 nM product, reflecting extension of primer-template to which Pol was bound at the time of initiation. Thus, the trap was effective in preventing additional turnovers of enzyme with primer-template. We plotted formation of extended primer as a function of the time (t) of addition of DNA trap (Fig. 7B) and fit the data to a single exponential function (Equation 1). The results demonstrate a koff of 0.073 ± 0.01 s-1, in excellent agreement with the steady-state kcat of 0.090 ± 0.002 s-1. The longer half-life for Pol/UL42 association with primer-template, coupled with strong exo activity, led to extensive degradation of the primer and prevented us from directly assessing its koff by this approach.

Pre-steady-state Exo Activity-- The above results demonstrate that UL42 does not influence fidelity by altering the kinetics of incorporation of correct dNTP by Pol under pre-steady-state conditions, and UL42 has little or no effect on the inherent ability of Pol to discriminate correct from incorrect dNTP for polymerization. However, it was possible that UL42 influenced the proof-reading 3' to 5' exo activity. Crystallographic studies of other DNA polymerases in association with primer-template have revealed that the polymerization and exo sites form physically distinct domains and that the exo site interacts with the primer as a single stranded entity (for review, see Ref. 42). We first examined whether UL42 altered the rate by which Pol degraded fully matched primer-template (Fig. 1A) or primer-template containing a single C:T mismatch at the 3'-terminus of the primer strand (Fig. 1C). We preincubated the respective primer-templates (20 nM) with 100 nM Pol or Pol/UL42 as described above for nucleotide incorporation. However, reactions were initiated by the addition of MgCl2 and calf thymus DNA trap, but no dNTPs were included to prevent polymerization. Reactions were performed at 37 °C and were stopped at intervals by the addition of EDTA. The amount of 45-mer remaining was plotted as a function of time, and the data were fit to a double exponential function (Equation 5) to determine the rate constant for the initial rapid phase and the proportion of primer-template which was cleaved at that rate (30, 31). Pol cleaved 88% of the mismatched primer-template with a rate constant of 16.7 ± 3.4 s-1, whereas Pol/UL42 cleaved 73% of the mismatched primer-template with a rate constant of 15.7 ± 3.7 s-1 (Fig. 8B). Thus, when incubated to equilibrium with mismatched primer-template, both Pol and Pol/UL42 efficiently recognized and cleaved the 3'-terminal mismatched nucleotide at rate constants that were indistinguishable. By contrast, matched primer-template was less efficiently recognized and cleaved by both enzymes (Fig. 8A) compared with mismatched primer-template (Fig. 8B). Pol cleaved only 29% of the matched primer-template at a rate constant of 5.9 ± 2.2 s-1, whereas the Pol/UL42 complex cleaved 19% of the matched primer-template with a rate constant of 2.2 ± 1.6 s-1.


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Fig. 8.   Pre-steady-state excision of matched and mismatched primer-template. Excess (100 nM) Pol () or Pol/UL42 (black-square) was incubated with 20 nM end-labeled matched (A) or mismatched (B) primer-template in the presence of EDTA. Reactions were initiated by the addition of MgCl2 and DNA trap as indicated in the legend to Fig. 2, but without dTTP. Reactions were terminated with EDTA at various times using a rapid quench apparatus, and the products were separated and quantified. The amount of 45-mer remaining was plotted as a function of time and fit to a double exponential function (Equation 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of UL42 on Pre-steady-state Kinetic Parameters Governing Nucleotide Incorporation by Pol-- Factors that increase the processivity of their cognate DNA polymerases could do so by decreasing the dissociation rate from DNA, increasing the catalytic rate, and/or increasing the affinity or selectivity for correct nucleotide. Although UL42 increases the affinity of Pol for model primer-template 6-fold as demonstrated by pre-steady-state single turnover reactions (Fig. 2), it does not alter the pre-steady-state catalytic rate constant (150 s-1) at saturating correct nucleotide (dTTP) concentrations (Fig. 3). E. coli thioredoxin, which forms a stable complex with T7 DNA polymerase catalytic subunit and increases its affinity for DNA, also fails to change the rate constant for polymerization (27, 43). We also found that the UL42 processivity factor does not significantly alter the ground-state binding of correct nucleotide (Fig. 3, E and F). However, our results suggest that binding of correct nucleotide to a preformed enzyme-DNA complex is most likely a two-step process, regardless of the presence of UL42, with a conformational change occurring after initial binding which would serve to increase the stability of enzyme-DNA complexes prior to catalysis. A similar nucleotide stabilizing effect has been observed with HIV reverse transcriptase and pol delta  (34, 40).

UL42 does not significantly alter the affinity of correct dNTP for enzyme-DNA complex (Fig. 3). UL42 also does not alter the overall ability of Pol to discriminate between correct and incorrect nucleotide during polymerization, although the maximum rate of misincorporation of dATP by Pol is three times slower in the presence, compared with the absence, of UL42 (Fig. 4, E and F). Surprisingly, the rate constant for incorporation of incorrect nucleotide (dATP) by Pol at a saturating nucleotide concentration was only slightly (33%) slower than the rate constant for correct incorporation. Because misincorporation was associated with a burst under pre-steady-state conditions and the rate exceeded that for steady-state incorporation, dissociation of the enzyme from primer-template is not rate-limiting for single turnover misincorporation reactions. Inasmuch as we observed faster rates of dissociation at lower, compared with higher, concentrations of correct (Fig. 3, E and F) and incorrect (results not shown) dNTP, it is likely that there is a rate-limiting conformational step for both correct and incorrect incorporation. A rate-limiting conformational step prior to catalysis with correct nucleotide is also supported by the moderate elemental effects of a thio derivative of dNTP on the polymerization rate constant (kp) for Pol and Pol/UL42 (Fig. 6), although a partial effect of chemistry on the rate-limiting step cannot be excluded.

The kp for dNTP incorporation observed in single turnover conditions approximates that for multiple additions of nucleotides in a single binding of enzyme to primer-template. The latter confirmed an elongation rate of 110-220 nucleotides/s for the most rapidly extended molecules when all 4 dNTPs were added, and DNA trap was present to prevent rebinding of dissociated enzyme to labeled primer-template. The results further demonstrate that the rate of translocation of the polymerase along DNA is more rapid than the rate of nucleotide incorporation. In experiments similar to those we report in Fig. 5, Weisshart and co-workers (33) estimated an elongation rate of 26-33 nucleotides/s. The lower estimate most likely reflects the wide distribution of product sizes (Fig. 5) which would lead to an underestimation of elongation rate if calculated from the average length of extension in a given time period. Indeed, in our experiments only 29 and 25% of the extension products were full-length after 500 ms when incubated with Pol or Pol/UL42, respectively. This would correspond to an average elongation rate of 44 nucleotides/s for those molecules, similar to the extension rates that were estimated by Weisshart and co-workers over a similar time interval (33).

UL42 Reduces the Steady-state Rate Constant for Nucleotide Incorporation-- Although UL42 does not substantially alter the pre-steady-state parameters for correct nucleotide incorporation, it does alter steady-state rates. Direct measurement of the koff of Pol from primer-template (0.073 s-1) predicted a half-life of ~10 s for enzyme-DNA complexes. This is in excellent agreement with the 8-s half-life predicted by the steady-state rate constant (0.09 s-1). Interestingly, the dissociation of Pol and Pol/UL42 from primer-template is much slower under static conditions employed for nitrocellulose filter binding assays (no MgCl2 or dTTP), although the latter demonstrated a koff twice that for Pol compared with Pol/UL42 (22). The differences observed between the static and kinetic koff values could indicate that MgCl2, dTTP, or both alter the conformation of the enzyme bound to primer-template, as observed for complexes of DNA with reverse transcriptase or with pol delta  and proliferating cell nuclear antigen (34, 40, 44). Alternatively, kinetically active molecules may have a greater propensity for dissociating from primer-template than static ones.

Because processivity is defined as kp/koff, our results demonstrate a processivity, under their respective optimum salt concentrations, of 1,670/turnover for Pol and 5,000/turnover for Pol/UL42, the latter of which is consistent with the processivities reported for other replicative DNA polymerases (43, 45, 46). The 3-fold increase in processivity of Pol when complexed to UL42 may not be significant enough to explain fully why UL42 is absolutely essential for HSV-1 DNA synthesis in infected cells (12, 13). UL42 has been shown to interact with the origin-binding protein, another essential DNA replication protein (10, 11, 47), and may be required for the formation of a functional replisome in vivo. Indeed, immunofluorescence analysis has revealed defects in the formation of replication compartments at the nonpermissive temperature in cells infected with an HSV-1 UL42 temperature-sensitive mutant (48).

UL42 Does Not Alter the Pre-steady-state 3' to 5' Exo Activity of Pol-- When Pol, with or without UL42, was bound to mismatched primer-template prior to initiation of reactions, the 3'-mismatched nucleotide was rapidly excised from the bulk of bound primer-template. Although we observed no significant difference in the rate constants for cleavage of the mismatched nucleotide in the rapid phase, Pol/UL42 complexes cleaved a slightly smaller proportion of the bound primer-template than did Pol. Likewise, Pol/UL42 cleaved a smaller proportion of matched primer-template compared with Pol; however, the rates of cleavage of a matched 3'-primer terminus by Pol, with or without UL42, in this rapid phase were ~3 times slower than rates of cleavage of mismatched primer termini (Fig. 8). The biphasic excision curves also revealed a subsequent slow rate of cleavage of matched primer-template by Pol and Pol/UL42, despite the presence of competitor DNA in the reactions. This rate was slower for Pol/UL42 than for Pol and may result from slower switching of the primer-template from the polymerase to the exo site (30, 49). Thus, the cost of the exo activity is low, with polymerization favored over excision of matched termini ~25:1 and 70:1 for Pol and Pol/UL42, respectively.

Relationship between Enzyme Dissociation and Accumulation of Mismatched Products-- The accumulation of mismatched termini in pre-steady-state reactions with Pol was surprising given its potent exo activity. The results suggest that the inherent nucleotide selectivity of Pol is poor. Nevertheless, comparison of the discrimination index for incorporation of correct (dTTP) versus incorrect (dATP) nucleotide revealed a relative selectivity of ~265 for Pol, not substantially different from the selectivity of 296 observed for Pol/UL42 under pre-steady-state conditions. This level of nucleotide selectivity by Pol and Pol/UL42 was lower than reported for several other replicative polymerases, including the HIV reverse transcriptase, which lacks 3' to 5' proofreading exo activity (29, 34, 50). Nevertheless, the role of the exo function is to moderate the effects of misincorporation events. Analysis of idling turnover reactions reveals that multiple rounds of excision and polymerization at the 3'-terminus of the primer occur with each binding event of Pol and Pol/UL42.2 Thus, the longer half-life of the Pol/UL42 on primer-template coupled with a kinetic barrier to extend mismatches would be expected to increase further the probability that excision of a misincorporated nucleotide would occur prior to dissociation of Pol/UL42 (30). Based on these results, we think it likely that the failure of Pol/UL42 to accumulate mismatched products at moderate nucleotide concentrations is due largely to its decreased dissociation rate, compared with Pol, from primer-template.

    ACKNOWLEDGEMENTS

We thank Zetang Wu and Houleye Diallo for excellent technical assistance and Smita Patel for valuable advice during the initial phase of these studies.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM34940. Facilities, equipment, and core services were supported in part by the Ohio State University Department of Molecular Virology, Immunology, and Medical Genetics and by Comprehensive Cancer Center Core Grant P30 CA16058.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, 2198 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210. Tel.: 614-292-0735; Fax: 614-292-9805; E-mail: parris.1@osu.edu.

Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M210023200

2 D. S. Parris, unpublished results.

    ABBREVIATIONS

The abbreviations used are: HSV-1, herpes simplex virus type 1; dATPalpha S, 2'-deoxyadenosine 5'-O-(1-thiotriphosphate); exo, exonuclease; HIV, human immunodeficiency virus; Pol, HSV-1 DNA polymerase catalytic subunit; Pol/UL42, Pol complexed with its processivity factor UL42; pol III, E. coli DNA polymerase III; pol delta , mammalian DNA polymerase delta .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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