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INTRODUCTION |
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
subunit
of Escherichia coli pol III (14).
HSV-1 Pol, similar to pol
, 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
(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.
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EXPERIMENTAL PROCEDURES |
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
[
-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.
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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).
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(Eq. 1)
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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),
|
(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),
|
(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
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.
|
(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
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 dATP
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),
|
(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.
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RESULTS |
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.
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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.
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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."
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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),
|
(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.
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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
-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 dATP
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 dATP
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
dATP
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 dATP
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
dATP S ( ) or dATP ( ) 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.
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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 ( ) 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 ( ). 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.
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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 ( ) 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).
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DISCUSSION |
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
(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
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.