DNA Structure and Aspartate 276 Influence Nucleotide Binding to
Human DNA Polymerase
IMPLICATION FOR THE IDENTITY OF THE RATE-LIMITING CONFORMATIONAL
CHANGE*
Brian J.
Vande Berg,
William A.
Beard, and
Samuel H.
Wilson
From the Laboratory of Structural Biology, NIEHS, National
Institutes of Health,
Research Triangle Park, North Carolina 27709
Received for publication, April 5, 2000, and in revised form, October 4, 2000
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ABSTRACT |
Structures of DNA polymerase (pol)
bound to
single-nucleotide gapped DNA had revealed that the lyase and pol
domains form a "doughnut-shaped" structure altering the dNTP
binding pocket in a fashion that is not observed when bound to
non-gapped DNA. We have investigated dNTP binding to pol
-DNA
complexes employing steady-state and pre-steady-state kinetics.
Although pol
has a kinetic scheme similar to other DNA polymerases,
polymerization by pol
is limited by at least two partially
rate-limiting steps: a conformational change after dNTP ground-state
binding and product release. The equilibrium binding constant,
Kd(dNTP), decreased and the insertion
efficiency increased with a one-nucleotide gapped DNA substrate, as
compared with non-gapped DNA. Valine substitution for
Asp276, which interacts with the base of the incoming
nucleotide, increased the binding affinity for the incoming
nucleotide indicating that the negative charge contributed by
Asp276 weakens binding and that an interaction between
residue 276 with the incoming nucleotide occurs during ground-state
binding. Since the interaction between Asp276 and the
nascent base pair is observed only in the "closed" conformation of
pol
, the increased free energy in ground-state binding for the
mutant suggests that the subsequent rate-limiting conformational change
is not the "open" to "closed" structural transition, but instead is triggered in the closed pol conformation.
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INTRODUCTION |
Proficient DNA repair systems are critical to maintaining the
stability of the human genome. Many forms of endogenous base damage,
including alkylation, oxidation, and deamination, result in the
formation of abasic sites. In particular, normal and damaged DNA bases
are lost spontaneously or removed by lesion-specific DNA glycosylases.
The resulting abasic sites are repaired by the base excision repair
(BER)1 pathway to prevent the
accumulation of this miscoding lesion. Following base removal,
apurinic/apyrimidinic (AP) endonuclease incises the damaged strand 5'
to the abasic site, leaving a single-nucleotide gap with a
5'-deoxyribose phosphate (dRP) flap. The gap is then processed by DNA
polymerase (pol)
to create nicked DNA that will be ligated by DNA
ligase I or III. DNA polymerase
possesses both the
nucleotidyltransferase activity to fill the one-nucleotide gap and
lyase activity required to remove the dRP flap. The central role of pol
in BER has been established (1, 2), and, in the absence of pol
,
alternate BER pathways have been reported (3, 4).
DNA polymerase
is an attractive model to study polymerase
mechanisms employed to assure efficient and faithful DNA synthesis. Its
small size and lack of accessory proteins has facilitated its
biochemical, kinetic, and structural characterization. Although pol
appears to have evolved separately from other classes of DNA/RNA
polymerases of known structure (5), it shares many general structural
and mechanistic features. Each of the polymerases possesses a groove
along a broad face of the enzyme where nucleic acid binds. The
polymerase domain of these enzymes has been likened to a right hand and
is composed of finger, palm, and thumb subdomains (6). Polymerases have
at least two acidic residues in the palm subdomain that bind
catalytically essential metals. The crystal structures of the substrate
complexes of pol
(7, 8), T7 DNA polymerase (9), Klentaq DNA
polymerase (10), and HIV-1 reverse transcriptase (11) indicate that the
reactive groups (i.e. metals, dNTP, and template-primer)
have a similar three-dimensional arrangement. These structures are
consistent with a "two metal ion" mechanism for nucleotidyl
transfer (12).
In general, pol
also utilizes a similar kinetic mechanism as most
other DNA polymerases. Steady-state kinetic analyses indicate that pol
follows an ordered addition of substrates (13). Employing pre-steady-state kinetics, it has been shown that Escherichia coli Klenow fragment (14, 15), T4 DNA polymerase (15, 16), T7 DNA
polymerase (17), HIV-1 reverse transcriptase (18), and pol
(19)
utilize a two-step nucleotide binding mechanism. Initial dNTP binding
to a pol-DNA complex places the nucleotide within the active site. A
subsequent conformational change results in the alignment of the
catalytic atoms and rapid chemistry. Binding of the correct nucleotide
facilitates this conformational change, whereas binding of the
incorrect nucleotide does not. This "induced-fit" mechanism is
consistent with the numerous structural differences that are observed
upon binding of a correct nucleotide (8). In particular, the
carboxyl-terminal subdomain (residues 262-335) is observed to
reposition itself after binding a correct dNTP by rotation about an
axis,
-helix M, that positions
-helix N so that several side
chains can interact with the nascent base pair in this "closed" conformation.
Replicative DNA polymerases and reverse transcriptases often have an
intrinsically associated exonuclease or endonuclease activity that
complements their fundamental nucleotidyltransferase function. DNA
polymerase
also has an associated accessory activity that
complements its DNA synthesis step in BER. Mild proteolysis of pol
separates a 31-kDa fragment, which possesses the nucleotidyltransferase activity, from an amino-terminal 8-kDa fragment (20). This smaller domain binds strongly to 5'-phosphate groups in gapped DNA (21) and
possesses the dRP lyase activity that is needed for removal of the dRP
flap remaining after AP endonuclease cleavage of the abasic site
intermediate in BER (22, 23). In the absence of a downstream DNA strand
(i.e. no DNA gap), a crystal structure of a pol
-DNA-ddNTP complex revealed that the 8-kDa domain does not interact
with the DNA or the 31-kDa domain, but instead is positioned some
distance from the 31-kDa domain (Fig.
1A). The crystal structure of pol
bound to
one-nucleotide gapped DNA demonstrates that the 8-kDa domain binds to
the 5'-phosphate in the DNA gap and interacts with the carboxyl
terminus of the 31-kDa domain (Fig. 1A) (8). This results in
a more compact, doughnut-shaped structure forming a channel with
dimensions appropriate for dNTP diffusion (Fig. 1B) (24).
Thus, this conformation of the 8-kDa domain is potentially significant
due to its proximity to the nucleotide binding pocket of pol
. The
experiments described here were performed to examine nucleotide binding
to pol
and to ascertain the influence of a gapped DNA structure.
Additionally, the role of Asp276 and
-helix N in dNTP
binding was investigated by comparing the effect of valine substitution
for Asp276 on nucleotide binding with alternate DNA
substrates. Asp276 is observed to form van der Waals
contact with the base of the incoming nucleotide triphosphate in the
closed pol
ternary substrate complex. Employing steady-state and
pre-steady-state kinetics, we examine nucleotide binding to pol
.
The results indicate that residue 276 interactions with the incoming
nucleotide influence ground-state nucleotide binding during template
base recognition. The implication is that
-helix N and
Asp276 influence ground-state binding in the closed
conformation that occurs prior to the rate-limiting conformational
change and that subdomain closure is very rapid and not
rate-limiting.

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Fig. 1.
Structure of DNA polymerase
bound to DNA and ddCTP.
A, the palm subdomain of DNA polymerase (yellow ribbon) bound to non-gapped DNA (data not
shown) (7) is superimposed with the palm of pol (blue-green 31-kDa pol domain and white 8-kDa
lyase domain) bound to a one-nucleotide gapped DNA (8). The
template strand of the one-nucleotide gapped DNA is red,
while the primer and downstream strands are pink. The
pink incoming ddCTP is illustrated in a Corey-Pauling-Koltun
representation and identifies the 3'-primer terminus. The 8-kDa domain
of pol bound to non-gapped DNA does not interact with DNA, but
instead is positioned some distance from the 31-kDa domain. A
transparent molecular surface of pol that is bound to the
one-nucleotide gapped DNA substrate is shown and illustrates the
doughnut-shaped structure of pol in this conformation.
B, a view of the channel that is formed when the
amino-terminal 8-kDa lyase domain (white) interacts with the
carboxyl terminus of the 31-kDa pol domain (blue-green).
This channel has dimensions appropriate for dNTP diffusion (24). This
figure was made with GRASP (60) and Molscript (61) and rendered with
Raster3D (62).
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EXPERIMENTAL PROCEDURES |
Materials--
Ultrapure deoxynucleoside triphosphates,
[
-32P]ATP, [
-32P]dCTP,
[
-thio]dCTP, and MicroSpin G-25 columns were from Amersham Pharmacia Biotech. DE-81 filters were from Whatman.
A 34-mer oligonucleotide substrate containing a single nucleotide gap
was prepared by annealing three gel-purified oligonucleotides (Oligos
Etc., Wilsonville, OR) to create a gap at position 16. Each
oligonucleotide was resuspended in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and the concentration determined from their
UV absorbance at 260 nm. The annealing reactions were carried out by
incubating a solution of 10 µM primer with 11 µM each of downstream and template oligonucleotides at
90 °C for 2 min followed by slow cooling to room temperature. The
sequence of the gapped DNA substrate was as follows: primer,
5'-CTGCAGCTGATGCGC-3'; downstream oligonucleotide, 5'-GTACGGATCCCCGGGTAC-3'; template,
3'-GACGTCGACTACGCGGCATGCCTAGGGGCCCATG-5'.
The upstream primer in each case was 5'-labeled with
[
-32P]ATP (specific activity = 6.6 × 106 dpm/pmol) using T4 polynucleotide kinase (New England
Biolabs) and contaminating radioactive ATP was removed with a MicroSpin G-25 column. The downstream primer was synthesized with a 5'-phosphate. A non-gapped DNA substrate was prepared by omitting the downstream oligonucleotide. A nicked substrate was prepared by annealing a 16-mer
primer, where the 15-mer primer had a dCMP added to the 3'-end, and
downstream oligonucleotide with template as described above.
Human recombinant pol
(wild type and D276V) was overexpressed in
Escherichia coli cells and purified as described (25). The
enzyme concentration was determined from the absorbance at 280 nm using
an extinction coefficient of 21,200 cm
1
M
1 (26).
Kinetic Assays--
Rapid-quench assays were performed at
37 °C using a KinTek Model RQF-3 rapid-quench-flow apparatus (KinTek
Corp., Austin, TX). Unless noted otherwise, all concentrations refer to
the final concentration after mixing. The final reaction mixture
typically consisted of 50 mM Tris-HCl, pH 7.2, 100 mM KCl, 100 µg/ml bovine serum albumin, 10% glycerol,
and 5 mM MgCl2. The final concentration of
enzyme and substrates are given in the text or figure legends.
Enzyme and substrate DNA were preincubated for 1 min and rapidly mixed
with dCTP and MgCl2 to initiate each reaction. After various time periods, reactions were stopped by the addition of 0.25 M EDTA. The quenched samples were mixed with an equal
volume of formamide dye and the products separated on 12% denaturing polyacrylamide gels. The dried gels were analyzed using a
PhosphorImager (Molecular Dynamics) to quantify product formation.
Steady-state time courses were measured by manual mixing and quenching
at 37 °C to determine steady-state constants (i.e.
apparent kcat and Km).
Following the appropriate incubation period, individual reactions were
quenched with EDTA and the quenched reaction mixtures were applied to
DEAE cellulose filters (DE-81). The unincorporated [
-32P]dCTP was removed, and radioactive nucleotide
incorporation was quantified as described previously (27).
Pyrophosphorolysis--
A nicked DNA substrate (300 nM) was preincubated with 1 nM pol
in
reaction buffer. The reaction was initiated by the addition of 3 mM pyrophosphate and 5 mM MgCl2.
The reaction was quenched at various time periods with EDTA.
Data Analysis--
Time courses were fitted to appropriate
equations by nonlinear least squares methods. These equations are
described under "Results." Progress curves were also fitted to
kinetic models with KinTekSim, which is based on the programs KINSIM
(28) and FITSIM (29).
 |
RESULTS |
Influence of DNA Structure on Steady-state Efficiency of dCTP
Incorporation--
The efficiency of correct nucleotide
(i.e. dCTP) incorporation by pol
was dependent on the
nature of the template-primer (Table I).
DNA polymerase
catalyzed single-nucleotide gap filling at a
steady-state rate of 0.8 s
1. A similar rate
of polymerization was observed when pol
was bound to non-gapped DNA
(i.e. the downstream oligonucleotide was omitted from the
annealing reaction). In contrast, the dCTP concentration dependence of
DNA synthesis indicated that Km(dCTP)
with a single nucleotide gap DNA substrate was 12-fold lower than for a
substrate without a gap (Table I), suggesting that the enzyme might
possibly have a higher affinity for nucleotide when bound to gapped
DNA. As a result of the lower Km, catalytic efficiency
(kcat/Km(dCTP))
was over an order of magnitude greater for the gapped DNA substrate
assayed under steady-state conditions than for the non-gapped DNA
substrate.
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Table I
Steady-state kinetic constants for wild-type pol -DNA complexes
Gapped and non-gapped DNAs (300 nM) were preincubated with
1 nM pol prior to addition of dCTP. Initial velocity
data were fitted to the Michaelis equation by nonlinear least squares
methods.
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Equilibrium Nucleotide Binding--
Since steady-state kinetic
parameters are often the composite of several rate constants, we sought
to determine the equilibrium constant (Kd) for
nucleotide binding to pol
-DNA. Using rapid-mixing and quenching
techniques, we measured pre-steady-state product formation. When a high
concentration of pol
is preincubated with excess single-nucleotide
gapped DNA (DNA/enzyme = 3) and rapidly mixed with a high
concentration of the complementary nucleoside triphosphate
(i.e. dCTP) and Mg2+, product formation is
biphasic. A rapid burst of product formation precedes a slower apparent
linear rate (Fig. 2, dashed
line). The biphasic nature of the time course indicates that a
step following chemistry is rate-limiting during steady-state
catalysis. The slow step has been attributed to dissociation of the
extended pol-DNA complex (19). The rates of product formation observed in Fig. 2 indicate that in contrast to processive replicative polymerases, kpol is not considerably greater
than koff(DNA+1) for pol
. Scheme I describes
the basic kinetic mechanism for single-nucleotide incorporation
utilized by all DNA polymerases that have been examined. Following an
ordered binding of substrate DNA and dNTP, a conformational change
(kpol) precedes rapid chemistry (see below).
Pyrophosphate release is rapid, but dissociation of the extended
product DNA is usually slow and at least partially rate-limiting.
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Since dNTP binding is in rapid equilibrium,
kpol is dependent on dNTP concentration and is
given by Equation 1.
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(Eq. 1)
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Fitting the time course in Fig. 2 to an equation with rising
exponential and linear terms, as shown by Equation 2,
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(Eq. 2)
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results in a burst rate constant (kobs) of
12.3 s
1 and an apparent linear rate
(vss) of 120 nM/s (dashed
line). With this analysis, the burst amplitude (A)
represents the "apparent" active concentration of enzyme
(A = 40 nM) so that
kss
(vss/polactive) would be 3.0 s
1. Yet, since kobs is
similar to kss for pol
, this analysis is not
satisfactory and inappropriate. According to Scheme I, pol
will
cycle quickly (kpol is similar to
koff(DNA+1)), so that the burst amplitude is an
underestimate of the active enzyme concentration. The amplitude is
given by Equation 3 (30).
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(Eq. 3)
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Thus, even when kpol is 1 order of
magnitude greater than koff(DNA+1), the burst
amplitude is still only 83% of the true active fraction of enzyme. The
apparently linear portion of the time course also suffers from the high
concentration of free product DNA (DNA+1) that is
accumulating after the first turnover, therefore competing with
extendable substrate DNA (i.e. product inhibition). Correct
analysis of the time course in Fig. 2 requires a model that takes into
account product competition. Assuming rapid ligand binding and an
"active" enzyme concentration of 70%, the data in Fig. 2 were
fitted to the model outlined in Scheme I to estimate
kpol and koff(DNA+1)
(solid line). The fitted values for
kpol (7.7 ± 0.5 s
1) and koff(DNA+1)
(2.8 ± 0.2 s
1) are similar to the
values determined independently below.

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Fig. 2.
Pre-steady-state incorporation of dCTP by DNA
polymerase . DNA polymerase (100 nM) was pre-incubated with 300 nM gapped DNA
substrate prior to mixing with an equal volume of 50 µM
dCTP and 5 mM MgCl2 to initiate the reaction.
Reactions were stopped at time intervals and the products isolated and
quantified as described under "Experimental Procedures." The time
course of product formation appears to be biphasic with an initial
rapid exponential phase followed by a linear phase. The dashed
line represents the best fit to an equation (Equation 2 under
"Results") with rising exponential and linear terms. The observed
rate constant of the burst phase was 12.3 s 1
followed by an apparent linear rate (vss) of 120 nM/s. With this analysis, the burst amplitude represents
the apparent active concentration of enzyme (40 nM) so that
kss
(vss/polactive) would be 3.0 s 1. However, since
kpol/koff(DNA+1) is not
considerably greater than 1, this analysis is not appropriate (see
"Results" for details). Assuming rapid ligand binding and an
"active" enzyme fraction of 70%, the data can be fitted to the
model outlined in Scheme I to derive kpol and
koff(DNA+1) of 7.7 and 2.8 s 1, respectively (solid line). The
simulated time course also indicates that the steady-state phase is
short-lived (i.e. time course is non-linear).
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To eliminate interference of enzyme cycling in determining the
equilibrium nucleotide dissociation constant, an excess of enzyme was
preincubated with DNA (enzyme/DNA = 5). Under this condition,
nearly all of the substrate DNA is bound to enzyme so that, upon
addition of dCTP/Mg2+, dNTP binding and incorporation limit
catalysis. Under these single-turnover conditions, the first-order time
courses were dependent on the concentration of dCTP
(Fig. 3). A secondary plot indicated that
the apparent first-order rate constants can be fitted to Equation 1
with a Kd and kpol of 5.6 µM and 10.0 s
1, respectively
(Fig. 3B and Table II).
Interestingly, however, the amplitude of the fast exponential phase was
only 50% (i.e. 50 nM) of that expected.
Extended incubation of the pol with substrates demonstrated that
greater than 90% of the primer could be extended (data not shown).
This result suggested that pol
can bind to the template-primer in a
catalytically nonproductive mode. This nonproductive binding form
competes with the excess free enzyme for productive DNA binding
resulting in a biphasic time course. To determine whether enzyme that
bound DNA nonproductively can isomerize to form productive enzyme-bound
DNA or must dissociate from DNA to allow enzyme the opportunity to bind
productively, we added excess unlabeled DNA (5 µM) with
50 µM dCTP to pol
(300 nM) preincubated
with 100 nM 5'-labeled DNA. The unlabeled DNA binds free
enzyme and enzyme that dissociates from the radioactively labeled DNA
to eliminate enzyme cycling. The excess non-radioactively labeled DNA
trap had no effect on the amplitude or rate of the rapid or slow phases
(data not shown), indicating that for the wild-type enzyme that the
nonproductive DNA binding form can isomerize to the productive form
without dissociating from the DNA.

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Fig. 3.
dCTP concentration dependence of
single-turnover incorporation of dCTP by DNA polymerase
. A, gapped DNA (100 nM) was preincubated with 500 nM pol and
then mixed with increasing concentrations of dCTP with 5 mM
MgCl2 to initiate the reaction. Reactions were quenched at
the indicated times and the products isolated and quantified as
described under "Experimental Procedures." The dCTP concentrations
were 1 ( ), 5 ( ), 10 ( ), 30 ( ), 50 ( ), and 100 µM ( ). The solid lines represents the best
fit of the data to a first-order process. B, a secondary
plot of the dCTP concentration dependence of the observed first-order
rate constants measured in A. The data were fitted to a
hyperbola (Equation 1) to yield Kd of 5.6 µM and kpol of 10.0 s 1 (Table II).
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Table II
Equilibrium nucleotide binding affinities for wild-type and D276V
pol
The equilibrium binding affinity (Kd) of pol -DNA
complexes with dCTP was measured by single-turnover analysis as
described under "Results."
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To ascertain whether dNTP incorporation during these single-turnover
conditions is limited by chemistry, the rate constant for the
incorporation of the
-thio-substituted analogue of dCTP was
determined. Due to the lower electronegativity of sulfur relative to
oxygen, a significant decrease in rate upon sulfur substitution would
suggest that chemistry is rate-limiting. Model studies with phosphate
triesters indicate a large elemental effect upon substitution of sulfur
at a nonesterified position, whereas studies with phosphate diesters
indicate smaller decreases in rate upon sulfur substitution (see Ref.
31 for discussion). From the dCTP
S concentration dependence of the
rate of incorporation, the affinity (Kd) and rate
constant for incorporation of the analogue were 9.8 µM and 4.7 s
1, respectively (data not shown).
Thus, the thioelemental effect observed for pol
is only 2.1, suggesting that a step other than chemistry is rate-limiting.
Steady-state kinetic characterization of mouse pol
failed to
demonstrate a reversal of the polymerization reaction (i.e. pyrophosphorolysis) in the presence of PPi with activated
DNA. Nonetheless, PPi was inhibitory for DNA synthesis
(13). To ascertain the possible significance of pyrophosphorolysis on
single-nucleotide gap filling DNA synthesis, nicked DNA was used as a
substrate to measure the magnitude of the reverse reaction. The
substrate was identical to that used in the one-nucleotide gap filling
reaction except that the primer was one nucleotide longer
(i.e. +dCMP). Pyrophosphorolysis was initiated by the
addition of excess PPi (3 mM) and
MgCl2 to pol
preincubated with DNA. The steady-state removal of dCMP to create a one-nucleotide gap occurred with an apparent rate constant of 0.01 s
1 (data not shown).
Affinity of DNA Polymerase
for Gapped DNA--
The biphasic
time course observed when DNA/pol > 1 (Fig. 2) indicates
formation of a "stable" pol-DNA complex. When
kpol
koff(DNA+1),
the amplitude of the rapid (burst) phase is equal to the active-enzyme
concentration, but in the case of pol
is only proportional to the
concentration of pol-DNA complex, as discussed above (see Equation 3).
To determine the equilibrium binding affinity of pol
for the gapped
DNA substrate, a fixed concentration of pol
(polt = 100 nM) was preincubated with increasing concentrations
of substrate DNA (DNAt = 25-200 nM). The
concentration of pol-DNA complex was estimated by rapidly mixing an
excess of dCTP (50 µM) and measuring the time course of
single-nucleotide incorporation. The time courses were biphasic
and the amplitude of the burst phase increased as a function of
increasing DNA concentration. The DNA concentration dependence of the
burst amplitude is illustrated in Fig. 4
and fitted to the quadratic equation (Equation 4).

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Fig. 4.
Titration of DNA polymerase
with gapped DNA. DNA polymerase (100 nM) was preincubated with increasing concentrations of
one-nucleotide gapped DNA and mixed with 50 µM dCTP and 5 mM MgCl2. Reactions were quenched at varying
times up to 1 s and the products isolated and quantified as
outlined under "Experimental Procedures." Time courses were fitted
to Equation 2 to estimate the apparent amplitude. As discussed under
"Results," the amplitude is proportional to the concentration of
pol-DNA complex. The amplitudes are plotted against the concentration
of one-nucleotide gapped DNA. The solid line is a fit of the
data to the quadratic equation (Equation 4 under "Results"), which
gave a Kd of 22 ± 13 nM and an
apparent active-fraction of enzyme of 70 ± 10 nM.
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(Eq. 4)
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The Kd and maximum burst amplitude were 22 and
70 nM, respectively. From Equation 3 and
kpol (Fig. 3B and Table II), the
maximum burst amplitude can be used to calculate
koff(DNA+1). This results in a dissociation rate
constant of 1.9 s
1 and in turn can be used to
estimate an association rate constant of 8.6 × 107
M
1 s
1
(Kd = koff/kon).
Influence of DNA Gap on Nucleotide Affinity--
As shown in Table
I, pol
fills single-nucleotide gapped DNA substrates nearly 15 times more efficiently than non-gapped substrates under steady-state
conditions. To determine if the binding affinity of the incoming
nucleotide (i.e. Kd(dCTP)) is
influenced by the single-nucleotide gap, single-turnover analysis (pol/DNA = 5) was employed to quantify nucleotide binding to a non-gapped DNA substrate (the downstream primer was omitted). Using
this non-gapped DNA substrate, pol
was found to bind and incorporate dCTP with Kd of 22 µM and
kpol of 5 s
1 (Table
II). This represents a 4-fold decrease in binding affinity as compared
with a gapped DNA complex and an 8-fold decrease in insertion efficiency.
Role of Residue 276 in the Nucleotide Binding Pocket--
The
structure of the pol
-DNA-ddCTP complex reveals that there are van
der Waals interactions between the base moiety of the incoming
nucleotide and the C
of Asp276,
Fig. 5 (7, 8). Steady-state kinetic
analysis and cross-linking studies previously suggested that removal of
the electronegative side chain by valine or glycine substitution
resulted in an apparent increase in binding affinity (32). To determine
whether the binding affinity for the incoming nucleotide is altered by
removing the negative charge, but retaining van der Waals interactions, the Kd(dCTP) was determined for the
D276V mutant. Single-turnover analysis employing both non-gapped and
gapped DNA substrates indicated that the Kd for the
incoming nucleoside triphosphate decreased 3.6- and 9-fold,
respectively, as compared with wild-type enzyme (Table II). Thus, the
binding affinity for the incoming nucleotide is increased nearly
40-fold for the mutant enzyme when utilizing a gapped DNA substrate as
compared with wild-type enzyme on a non-gapped DNA substrate.

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Fig. 5.
Asp276 of DNA polymerase
interacts with the incoming dNTP. In the
ternary pol /one-nucleotide gapped DNA/ddCTP complex (8), the C
of Asp276 (D276, Corey-Pauling-Koltun representation) makes
van der Waals contact with the base of the incoming nucleotide (ball
and sticks). Asp276 also forms a hydrogen bond with
Arg40 (R40) of the amino-terminal 8-kDa lyase domain.
Arg40 and Asp276 are displaced from one another
in the absence of the incoming nucleotide or with non-gapped DNA. The
van der Waals surface of the incoming nucleotide is also illustrated.
This figure was made with Molscript (61) and rendered with Raster3D
(62).
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Incorrect Nucleotide Binding--
To investigate whether
Asp276 participates in the binding of an "incorrect"
nucleotide (e.g. dTTP), the binding affinity of thymidine triphosphate with a gapped DNA substrate was determined for wild-type pol
and the D276V mutant. For the kinetic mechanism described above
(Scheme I), when kpol becomes very slow
(i.e. rate-limiting), then
Km(dNTP) is equivalent to
Kd(dNTP) (33-35). DNA polymerases
generally discriminate against incorrect nucleotides by binding them
weakly and incorporating them slowly. Thus, incorporation of an
incorrect nucleotide is always rate-limiting for polymerases that
exhibit low processivity, where processivity is defined as a
competition between nucleotide incorporation and polymerase dissociation from the template-primer (i.e.
kpol/koff(DNA)). This analysis does
not require that the active fraction of enzyme be known. When the dTTP
concentration dependence of incorporation opposite the template
deoxyguanine in the single-nucleotide gapped DNA substrate was
examined, the Km(dTTP) was 330 ± 60 and 130 ± 20 µM (five independent
determinations) for the wild-type and the D276V enzymes, respectively.
Therefore, valine substitution at Asp276 modestly increased
(2.5 ± 0.6-fold) the binding affinity of dTTP opposite a
templating deoxyguanine.
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DISCUSSION |
Nucleic acid polymerases must select (bind and incorporate) the
correct nucleotide from a pool of structurally similar molecules to
ensure accurate and efficient nucleic acid synthesis. DNA polymerase binding of the incoming nucleoside triphosphate has been
mechanistically and kinetically described as occurring in at least two
steps. After initial complex formation, an isomerization of the
polymerase ternary complex leads to a productive catalytic complex
where chemistry occurs rapidly (Scheme II). Each of the steps
illustrated in Scheme II (ground-state binding, isomerization,
chemistry) offers an opportunity for selection of the correct
nucleotide.
The contribution of each of these steps to the accuracy of
nucleotide selection for DNA synthesis depends on the specific DNA
polymerase. Whereas polymerases generally bind the incorrect nucleotide
weakly and incorporate (kpol) them slowly
(e.g. T7 DNA polymerase and HIV-1 reverse transcriptase),
the Klenow fragment of E. coli pol I does not appear to
utilize ground-state binding to increase selectivity
(Kd(correct)
Kd(incorrect)) (36). The lack of a
significant elemental effect when the non-bridging oxygen on the
-phosphorus is substituted with sulfur is consistent with a
rate-limiting conformational change in a two-step dNTP binding
mechanism for human DNA polymerase
. This interpretation is
supported by fluorescence changes of the template base analogue 2-aminopurine that occur upon nucleotide binding (37). The environment of the templating base, as monitored by fluorescence, changes at a rate
that is similar to nucleotide incorporation even when incorporation is
prevented (i.e. no chemistry) by using a
dideoxynucleotide-terminated primer. A similar approach had been used
to separate chemical and non-chemical steps of nucleotide binding with
Klenow fragment and T4 DNA polymerase (15).
DNA polymerase
utilizes ground-state binding to selectively
incorporate dCTP opposite a templating deoxyguanine. The selectivity over thymidine triphosphate
(Kd(dTTP)/ Kd(dCTP)) is approximately 60. This is similar to the selectivity of 25 (38) and
120 (39) reported for HIV-1 reverse transcriptase, but less than the
selectivity of 410 for T7 DNA polymerase (40), when considering
formation of the same mispair. The selectivity of the D276V mutant was
moderately increased to 220 by binding the correct deoxynucleoside
triphosphate more tightly than the incorrect thymidine triphosphate
relative to wild-type enzyme.
Steady-state and pre-steady-state kinetics indicate that DNA
polymerases bind substrates in an ordered fashion; DNA binding precedes
nucleotide binding. However, this is clearly not obligatory. For
example, utilizing photoreactive nucleotide analogues, it is possible
to covalently cross-link these analogues to pol
and other DNA
polymerases in the absence of DNA. Upon the addition of DNA, only
analogues cross-linked at the polymerase active site are incorporated,
resulting in DNA that becomes cross-linked to the polymerase (32).
Additionally, crystal structures of binary pol-dNTP complexes reveal
that the dNTP is bound in the active site through its triphosphate
moiety (41-43). The triphosphate moiety, therefore, offers minimal
specificity toward accuracy for correct nucleotide binding. The sugar
also affords some specificity, since 2'-deoxyribose is preferred over
ribose in the presence of DNA. However, discrimination during dNTP
binding originates mainly from the identity of the base and its
hydrogen bonding capacity and steric complementarity with the
templating base. Ground-state binding of a nucleotide offers the
opportunity to check proper Watson-Crick hydrogen bonding and/or steric
complementarity. Formation of a base pair with good geometry induces a
conformational change that results in rapid incorporation of the
nucleotide. Thus, the rate of insertion is limited by a conformational
change that triggers chemistry. The identity in structural terms of
this rate-limiting conformational change will be discussed here.
Several significant structural changes occur upon substrate (DNA and
dNTP) binding to pol
and other DNA polymerases as inferred from
comparison of structures of apoenzyme with substrate bound complexes.
These conformational changes occur with both the polymerase and the
substrate. The rate-limiting conformational change observed kinetically
has been postulated to be the large subdomain movement that is observed
by comparing the crystal structures of open pol and pol-DNA complexes
with those of the closed ternary pol-DNA-dNTP complexes (see Ref. 44
for a review). For pol
, in addition to significant movement of the
8-kDa lyase domain upon binding gapped DNA, the carboxyl-terminal
subdomain is observed to "close" upon the exposed face of the
nascent base pair. Rotation around an axis,
-helix M, positions
-helix N to probe correct Watson-Crick geometry (7, 8, 24).
Superimposing the polymerase catalytic subdomains of the binary
one-nucleotide gapped DNA substrate with that including the incoming
ddCTP (ternary complex) illustrates this subdomain movement
(Fig. 6A) and reveals that the
8-kDa domain moves slightly to form an even more compact structure (not
shown) (24). Numerous active site side chain and substrate motions are
associated with these conformational differences. Assuming that valine
substitution for Asp276 does not produce additional active
site structural changes, the finding that interactions between
Asp276 and the incoming dNTP influence ground-state dNTP
binding implies that the conformational change limiting insertion
during the first turnover may not involve movement of
-helix N. This follows because C
of Asp276 must be in
close contact with the base of the incoming dNTP during template base
annealing. The van der Waals interaction between C
of
Asp276 and the correct dNTP is observed in the closed
conformation indicating that the closed conformation itself may trigger
the rate-limiting conformational change. A "structural signal" to
indicate that positioning of
-helix N in the closed position has
occurred could be transmitted to the catalytic metal-binding site
through the altered positioning observed with Arg283,
Glu295, Arg258, and Asp192 in the
open and closed forms of pol
(Fig. 6B). From a
comparison of open and closed forms of pol
ternary complexes, it
has also been suggested that thumb closure repositions the
mononucleotide binding motif (residues 179-189) closer to the primer
3'-hydroxyl (8).

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Fig. 6.
Comparison of open and closed DNA
polymerase -substrate complexes.
A, the catalytic subdomains of the binary pol-DNA
complex (1bpx, blue-green) and ternary complex (+ddCTP;
1bpy, purple) were superimposed and indicate that rotation
around helix M (about 30°) positions helix N next to the new base
pair. The DNA backbone, templating base, and incoming ddCTP (closed
ternary complex) are illustrated. Asp276 (D276) is
illustrated in a ball-and-stick representation and stacks with the base
of the incoming ddCTP in the closed complex. B, the position
of -helix N can be structurally transmitted to the catalytic metals
through the altered interactions between Arg283 (R283), in
helix N, and Asp192 (D192) that coordinates both active
site Mg2+ ions (A and B). This may occur through altered
interaction observed between Glu295 (E295) and
Arg258 (R258) in the open (inactive) and closed (active)
forms of pol . Phe272 (F272) is postulated to
transiently interfere with interactions between Asp192 and
Arg258 permitting an interaction with Glu295.
Any of these subtle structural movements inferred from comparison of
the open and closed complexes could kinetically limit chemistry and
represent the rate-limiting conformational change. This figure was made
with Molscript (61) and rendered with Raster3D (62).
|
|
DNA polymerase
is an integral component of the BER pathway. It is
responsible for removing the 5'-dRP flap and filling the one-nucleotide
gap generated during BER (for a review, see Refs. 45 and 46). DNA
polymerase
processively fills short DNA gaps (= 5 nucleotides)
where the downstream DNA is 5'-phosphorylated (23, 47). In contrast,
DNA synthesis occurs in a distributive fashion with non-gapped DNA.
This is attributed to the 5'-phosphate binding activity of the
amino-terminal 8-kDa domain (23). The 8-kDa domain interacts with the
carboxyl-terminal domain when bound to a one-nucleotide DNA gap (Fig.
1A). This interaction buries
approximately 130 Å2 of surface area. In the closed
ternary complex of the one-nucleotide gap DNA structure, this
solvent-inaccessible interface increases to 300 Å2.
We have examined the influence of the template-primer structure on
nucleotide binding by pol
. Previous studies that examined the
influence of template-primer structure on the catalytic efficiency of
pol
have reported dissimilar effects. In one study, the catalytic efficiency was increased over 2500-fold when using a single-nucleotide gap, as compared with a non-gapped DNA substrate (48). In contrast, only a modest influence (10-fold) of gap structure on catalytic efficiency has also been reported (49). In the current study, the
catalytic efficiency increased about 10-fold as determined from
steady-state (Table I) or pre-steady-state kinetics (Table II) with a
one-nucleotide DNA gap, as compared with non-gapped DNA. With a
template-primer system that is typically employed for kinetic study of
DNA polymerases (non-gapped DNA), pol
binds the correct nucleotide
with a lower affinity than when bound to gapped DNA (Table II). This
could be due to the role that the amino-terminal 8-kDa domain may play
in forming the dNTP binding pocket. In the absence of downstream DNA
(i.e. no gap), the dNTP binding site is "exposed"
relative to that observed in the ternary complex with a one-nucleotide
gapped DNA (Fig. 1). As illustrated in Fig. 1, this results in a
doughnut-like structure that apparently gates dNTP binding through a
cavity leading to the polymerase active site. This type of polymerase
architecture is similar to that observed with B-type replicative
polymerases (50, 51), as well as with the RNA-dependent RNA
polymerase from hepatitis C (52, 53).
The modestly higher dNTP binding affinity when pol
utilizes a
one-nucleotide gapped DNA substrate indicates a role for the 8-kDa
domain in dNTP binding with the one-nucleotide gapped DNA substrate.
Additionally, both wild-type enzyme and the D276V mutant exhibit a
2-fold increase in the rate of nucleotide insertion (kpol) with a one-nucleotide gapped DNA
substrate (Table II). A strong hydrogen bond between Asp276
of
-helix N and Arg40 (
-helix B) of the 8-kDa lyase
domain in the closed ternary complex is the only electrostatic
interaction between the 8-kDa domain and
-helix N. The increased
binding affinity with gapped DNA suggests that the negative charge
contributed by Asp276 restricts binding (reduced
association and/or increased dissociation) and that Arg40
may increase binding affinity in the gapped structure by charge compensation. Except for that contributed by Asp276, the
electrostatic surface potential around the sugar and base of the
incoming nucleotide is positive (Fig. 7).
It is noteworthy that most other X family polymerases have an arginine
or lysine at this position (54). Additionally, Arg72 of
HIV-1 reverse transcriptase is observed to stack with the incoming dNTP
and form hydrogen bonds with the
-phosphate. It is highly conserved
among retroviral reverse transcriptases and telomerase. In contrast to
the results reported here for Asp276, modification of the
Arg72 side chain results in mutant enzymes that have
dramatically lower catalytic efficiency (55, 56). In the case of pol
, it appears that the surrounding positive potential
(i.e. blue) can facilitate dNTP binding with a
one-nucleotide gapped DNA substrate. This interpretation is consistent
with the observation that removal of the acidic side chain by valine
substitution (D276V) results in ground-state binding affinity on
non-gapped DNA similar to that of wild-type enzyme on gapped DNA (Table
II). In this situation, the positive potential is removed by side chain
replacement, whereas in the latter situation, the highly basic 8-kDa
domain facilitates binding through charge neutralization.

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Fig. 7.
Electrostatic surface potential of the dNTP
binding pocket of pol . The view is
similar to that of Fig. 1. Blue and red represent
positive and negative potentials, respectively. The template and gapped
(primer and downstream DNA) strands are red and
yellow, respectively. The incoming ddCTP (yellow)
is situated in a cavity at the 3'-primer terminus. Asp276
(D276) contributes negative potential in the vicinity of the incoming
ddCTP. It is surrounded by several basic residues (e.g.
Lys27 (K27), Arg40 (R40), and
Lys280 (K280)). This figure was made with GRASP (60) and
Molscript (61) and rendered with Raster3D (62).
|
|
When a one-nucleotide gapped DNA substrate is utilized by the D276V
mutant polymerase, the binding affinity for the incoming nucleotide is
increased even further (Table II). This result indicates that
additional interactions and conformational changes give rise to an
increased free energy of binding. The additional methylene-group in the
valine side chain, as compared with aspartate, may increase the van der
Waals contacts with the base of the incoming nucleotide.
The low processivity of pol
makes it difficult to accurately
analyze the biphasic time courses of product formation when DNA is in
excess by pre-steady-state methods (enzyme/DNA < 1; Fig.
2). This is because
kpol and koff are
both partially rate-limiting, and product inhibition makes
it difficult to estimate the linear steady-state rate of product
formation. Processivity is kinetically defined as the probability of
inserting a nucleotide (i.e. kpol) to that of
dissociating from the DNA substrate (i.e.
koff). Thus, with a one-nucleotide gapped DNA
substrate the processivity of DNA polymerase
is modest
(kpol/koff = 10 s
1/1.9 s
1 = 5.3).
This is consistent with the qualitative gel assay that had indicated
pol
can processively fill short gaps of up to 6 nucleotides (47).
Single-turnover analysis (enzyme/DNA
1) of dNTP binding to DNA
polymerase
eliminates enzyme cycling. From the
nucleotide-dependent exponential time courses, the
ground-state binding affinity for dCTP and kpol
for the wild-type and D276V polymerases were determined (Fig.
3; Table II). The amplitude of the
exponential time courses should be equivalent to the concentration of
DNA. Under the conditions of the assay, 95% of the DNA should be
enzyme-bound. For several determinations, the amplitude of the
rapid-exponential phase was 63 ± 8% and 37 ± 6% of the
extendable DNA for the wild-type and D276V polymerases, respectively.
This indicates that a population of these polymerases can bind in a nonproductive conformation that is sensitive to the valine substitution at Asp276. To determine whether this nonproductive DNA
binding form could isomerize to a productive complex without
dissociating from DNA, unlabeled DNA was added to trap enzyme that may
dissociate during the course of the reaction. The excess
non-radioactively labeled DNA trap had no effect on the amplitudes or
rates of the rapid or slow phases, indicating that for the wild-type
enzyme that the nonproductive DNA binding form can isomerize to the
productive form without dissociating from the DNA. Similar results were
observed with the mutant enzyme except that the slow phase was
partially sensitive to the added trap. A thorough kinetic and
thermodynamic analysis of these alternate binding modes is currently
under study.
The alternate binding modes result in a decreased population of
polymerase poised for nucleotide insertion. Suo and Johnson (57, 58)
observed similar kinetic behavior when HIV-1 reverse transcriptase
binds templates with secondary structure and suggested that the
template nucleic acid is blocking access to the pol dNTP binding
pocket. A similar proposal for pol
suggests that the DNA substrate
may bind at the polymerase active site such that the terminal
template-primer base pair is sitting where the nascent base pair would
form. In this conformation the template-primer is out of register for
DNA synthesis and would block an incoming nucleotide from entering the
polymerase active site. This conformation is observed in the binary
complex of pol
bound to nicked DNA. In addition, the ability of DNA
polymerases to catalyze pyrophosphorolysis requires that the terminal
primer nucleotide move into the polymerase active site to achieve
PPi addition. As the valine substitution at
Asp276 stabilizes the incoming nucleotide sitting in the
active site, it also may stabilize the terminal primer nucleotide in
the polymerase active site. This is consistent with the lower apparent
active concentration of the mutant polymerase.
In conclusion, the dNTP binding affinity is increased for pol
with
a one-nucleotide gapped DNA substrate, suggesting a role for the
amino-terminal lyase domain in nucleotide binding. Interestingly, altering the interactions with the incoming nucleotide and/or lyase
domain by site-directed mutagenesis of Asp276 also
increased the binding affinity of the correct incoming nucleotide. This
is the first example that we are aware of where strategic modification
of the dNTP binding pocket has resulted in an increased nucleotide binding affinity. It will be interesting to determine if the
observed changes in binding affinity are due to "ionic tethering"
(59) where Asp276 and Arg40 modulate domain
interactions to influence ligand binding. Finally, since this
interaction between Asp276 and the incoming dNTP is
observed only in the closed conformation of pol
, the increase in
the free energy in ground-state binding for the D276V mutant suggests
that the rate-limiting conformational change is not the open to closed
structural transition, but instead is triggered in the closed
polymerase conformation. Deoxynucleoside triphosphate binding,
therefore, occurs in at least three steps. First, there is an initial
nonspecific binding that occurs primarily through polymerase
interactions with the triphosphate/metal and sugar moieties of the
nucleotide. This step is weak so as to facilitate rapid sampling of the
nucleoside triphosphate pools and is kinetically silent. This sampling
is probably associated with the subdomain movements inferred from the
crystal structure differences between the binary pol-DNA and ternary
substrate complexes. The second step, ground-state binding, is
associated with template base recognition through hydrogen bonding
and/or steric complementarity. In turn, the degree of complementarity
influences the probability of triggering a rate-limiting conformational
change (i.e. step three) that leads to a catalytically
competent complex that induces chemistry.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Robert E. London and Thomas A. Darden for critical reading of the manuscript.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed. Tel.: 919-541-3267;
Fax: 919-541-2260; E-mail: wilson5@niehs.nih.gov.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M002884200
 |
ABBREVIATIONS |
The abbreviations used are:
BER, base excision
repair;
dRP, 5'-deoxyribose phosphate;
AP, apurinic/apyrimidinic;
HIV, human immunodeficiency virus;
pol, polymerase.
 |
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