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INTRODUCTION |
Mammalian DNA polymerase 
(pol
)1 has quickly become one
of the best studied polymerases because the gene for the enzyme was
cloned (1, 2). The availability of multiple crystal structures of human
and rat pol , including those of the enzyme complexed with both of
its substrates and the metal cofactor, has aided the investigation of
the structure-function relationships of this enzyme (3-8).
pol is a 39-kDa protein with both nucleotidyltransferase and
5'-deoxyribose phosphodiesterase activities (9, 10). Evidence has been
provided for a role for pol in both base excision repair and
meiosis (11-13). There is no evidence that pol functions in
replication of the mammalian genome, but pol has been shown to
participate in DNA replication in Escherichia coli in the
absence of DNA polymerase I (14). Mice that are completely deficient in
pol die at 18 days post-conception due to massive apoptosis of
post-mitotic neurons, suggesting that pol is essential for embryonic development (15, 16). The physiological DNA substrate for pol
is believed to be a small gap because it has been shown that pol
is processive on gaps of 6 bases or less and that the activity and
fidelity of pol are highest on a 1-bp gap with a 5'-phosphate (17,
18).
DNA polymerase has a modular organization with an 8-kDa N-terminal
domain connected to the 31-kDa C-terminal domain by a protease-hypersensitive hinge region. The N-terminal 8-kDa domain was
originally characterized as a single-stranded DNA binding domain (19,
20). Subsequently, it was found to interact most efficiently with the
5'-phosphate of the downstream primer of the gapped DNA (21, 22). This
interaction is mediated by a helix-hairpin-helix motif (HhH), which
is found in several other DNA repair enzymes (4, 23, 24).
Matsumoto and Kim (9) later demonstrated that pol catalyzes removal
of dRP from AP endonuclease-incised AP sites via -elimination, as
opposed to hydrolysis, and that this dRP lyase activity resides in the
N-terminal 8-kDa domain of pol . The DNA polymerase active site is
found in the 31-kDa domain (25). pol does not possess proofreading activity.
We have developed a genetic screen to identify the amino acid residues
of pol that are critical for fidelity (26, 27). Our screen is based
upon the discovery that rat pol substitutes for E. coli
DNA polymerase I in DNA replication (14). We constructed a library of
pol mutants in which only the 8-kDa domain of the protein was
mutated. By using our genetic screen, we isolated several mutator
mutants of pol , including one that is altered from Thr to Ser at
position 79 (T79S). The T79S alteration is located in helix IV of the
N-terminal 8-kDa domain of pol and appears to be distant from the
catalytic active site, having no contact with the substrate during
catalysis, as shown in Fig. 1. Thr-79 is positioned directly between
two HhH motifs, HhH 1 and 2. HhH motif 1 interacts with the downstream
oligonucleotide, and HhH 2 interacts with the primer strand in single
nucleotide gapped DNA.
To elucidate the role of the Thr-79 residue in maintaining pol fidelity, we have employed a transient state kinetic approach. The
catalytic pathway of pol is shown in Scheme
1. First, pol binds to the DNA
substrate, followed by binding to the dNTP substrate. After formation
of the ternary complex of pol , DNA, and dNTP, a conformational
change occurs to produce an active complex (*) that can catalyze DNA
primer extension. After phosphodiester bond formation, pyrophosphate is
released. Finally, pol dissociates from the DNA substrate during
the rate-limiting step. Mutator mutant proteins have been shown to
alter kinetically the steps in the pathway of DNA polymerization (28,
29). This often leads to a decrease in fidelity. Our transient state
kinetic results demonstrate that the interaction of the HhH domains of
pol with the downstream DNA and the primer of the DNA substrate is
important for accurate DNA synthesis by pol . Our findings suggest
that movements of the HhH motifs are mediated by Thr-79. This movement results in presentation of the template within the active site of pol
and stabilization of the primer. We suggest that the placement of
the template within the pol active site and stabilization of the
primer strand are critical determinants of DNA synthesis fidelity.
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Scheme 1.
Minimal kinetic pathway of DNA
polymerization. , DNA polymerase ;
D, DNA; N, dNTP; P,
pyrophosphate.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Media--
The strain BL21 DE3 was used
for protein expression and has genotype F ompT
hsdSB(rBmB) gal dcm
(DE3). The strain FT334 is an HB101 derivative
and was used to detect thymidine kinase mutations in the forward
mutation assay. Its genotype is recA13 upp tdk (30).
E. coli strains CSH50 F'((pro-lac),
ara, thi, strA, F(proAB, lacIQZM15, traD36)) (31)
and MC1061 ((hsdR, hsdM+,
araD (ara, leu), (lacIPOZY), galU,
galK, strA)) were used in the M13 fidelity assays. Strain SC18-12
is derived from E. coli B/r and has the genotype
recA718 polA12 uvrA155 trpE65 lon-11 sulA1 (27). The SC18-12
strain was used in screening of a cDNA library of pol mutants
(27).
ET medium was E salts (32) supplemented with 0.4% glucose and 20 µg/ml tryptophan. Eglu medium is ET without tryptophan. Transformants
were selected on Luria-Bertani agar (33) supplemented with 30 µg/ml
chloramphenicol and 12 µg/ml tetracycline. Nutrient broth was
prepared according to the manufacturer's directions (Difco). Minimal
agar and soft agar used in M13 assays was as described by Bebenek and
Kunkel (31). HSV-tk selection medium is described by Eckert
and Drinkwater (30).
Reagents--
Deoxynucleoside triphosphates, adenine
triphosphate, and [
-32P]ATP (>5000 Ci/mmol, 10 mCi/ml) were purchased from New England Biolabs, Sigma, and Amersham
Biosciences, respectively.
5-Bromo-4-chloro-3-indolyl-D-galactoside (X-gal) and
5-fluoro-2'-deoxyuridine were purchased from Sigma. The
oligonucleotides used in the preparation of the DNA substrates were
synthesized at the Keck Molecular Biology Center at Yale University.
Expression and Purification of Mutant Enzymes--
The cDNAs
of pol -wt and T79S were subcloned into the pET28a vector (Novagen),
3' to a hexahistidine tag, resulting in fusion of the tag to the N
termini of the proteins. These enzymes were expressed and purified as
described previously (34), using a fast protein liquid
chromatography-driven Ni2+ column (Hi-Trap chelating resin,
Amersham Biosciences). The protein was eluted with an imidazole
gradient. This was followed by Hi-Trap SP column (Amersham Biosciences)
where the protein was eluted with a NaCl salt gradient. Proteins were
greater than 90% homogenous based on a Coomassie Blue-stained
SDS-PAGE. Concentrations of pol proteins were based on an
280 = 21,200 M
1
cm1 and a molecular mass of 40 kDa for His-tagged
pol .
Identification of Mutator Mutants Using the Trp+
Reversion Assay--
The T79S mutant was identified in a genetic
screen developed in our laboratory to isolate pol mutator mutants
(27). We used the Trp+ reversion screen to identify the
mutator mutants from a library of random mutants constructed between
nucleotides 1 and 300; this fragment encodes amino acids 1-100 of pol
. The library was constructed by mutagenic PCR and subcloning, as
described (27). In our genetic screen, mutator mutants induce
significantly more Trp+ revertants than cultures containing
the pol wild-type gene. The mutation of each pol mutant was
identified by the dideoxy DNA sequencing method using Sequenase 2.0 (United States Biochemical Corp.) according to the manufacturer's directions.
Spontaneous Mutation Frequency of pol -wt and T79S in the
Trp+ Reversion Assay--
To confirm that T79S was a
mutator mutant, we compared the spontaneous mutation frequencies of pol
-wt and the T79S mutant in E. coli as described (27). The
mutation frequency was calculated by dividing the number of
Trp+ colonies by the total number of colonies (27, 35).
DNA Substrates--
The DNA substrates employed in the
biochemical assays described below are displayed in Fig. 2. The primer
oligonucleotides were gel-purified as described (34) and were
radiolabeled at the 5' end by standard methods using T4 polynucleotide
kinase (New England Biolabs) and [-32P]ATP. The
oligonucleotides were annealed at a primer:template molar ratio of
1:1.2 in 50 mM Tris, pH 8.0, 250 mM NaCl. The
mixture was incubated sequentially at 95 °C for 5 min, slow cooled
to 50 °C for 30 min, and 50 °C for 20 min and then immediately
transferred to ice. Annealing of primer was confirmed on an 18%
polyacrylamide native gel followed by autoradiography as described
(28).
Pre-steady-state Analysis--
Reactions were performed in which
radiolabeled gapped DNA (300 nM 45X-22-22) was in 3-fold
excess relative to pol (100 nM). These reactions are
referred to as burst experiments (29). The burst experiment was
performed at saturating concentrations of dNTP while minimizing any
enzyme inhibition, which may occur with excess dNTP. Reactions were
initiated by rapid mixing of the pol ·DNA and Mg·dNTP solutions
(final concentration of MgCl2 = 10 mM). At
selected time intervals, the reactions were quenched with 0.3 M EDTA. The reaction products were separated as described below.
Single Turnover Misincorporation Assays--
To elucidate the
relative ability of the T79S enzyme compared with pol -wt to
incorporate correct and incorrect dNTPs into a primer-template, we
determined the equilibrium dissociation constant for dNTP binding,
Kd, and the maximum rate of polymerization,
kpol, for correct and incorrect dNTPs for each enzyme. For both pre-steady-state and single turnover condition assays,
reactions were conducted in buffer (50 mM Tris-Cl, pH 8.0)
containing 2 mM dithiothreitol, 20 mM NaCl, and
10% glycerol. All concentrations given refer to the final
concentrations after mixing. The kinetics of correct dNTP incorporation
were determined under single turnover conditions using rapid chemical
quench performed on a KinTek Instruments model RQF-3 rapid quench-flow
apparatus (36) thermostatted at 37 °C. Single turnover kinetic
experiments were performed under conditions where the enzyme
concentration greatly exceeds the Kd value for
gapped DNA. Single turnover conditions were determined empirically to
be a ratio of enzyme to DNA of 15:1 (data not shown) for T79S. These
conditions allow binding of greater than 95% of the DNA substrate by
pol and thereby measure the rate of a single catalytic turnover of
the enzyme. Reactions were conducted at 37 °C in 50 mM
Tris, pH 8.0, 10 mM MgCl2, 20 mM
NaCl, 2 mM dithiothreitol, 10% glycerol, 50 nM
32P-end-labeled primer-template (45X-22-22) and 750 nM enzyme (both pol -wt and T79S). Typically,
experiments were carried out by loading 15 µl of the pol
-primer-template complex in buffer in one sample loop and 15 µl of
a single dNTP in the second sample loop. Reactions were initiated by
rapid mixing of the two reactant solutions and were quenched at various
times with 0.5 M EDTA. For correct incorporation reactions,
substrate concentrations were typically 0-200 µM for
single base gap or 0-1 mM for 3'-recessed DNA substrate,
and reaction times were 0-5 s.
The kinetics of misincorporation were determined manually under the
above single turnover conditions. Reactions were performed by
preincubating 750 nM enzyme with 50 nM
primer-template at 37 °C for 1 min. Reactions were initiated by the
addition of substrate, incubated for the indicated reaction times, and
stopped by the addition of 0.5 M EDTA. For incorrect
incorporations, substrate concentrations were typically 0-2
mM, and reaction times were 0-2700 s.
The reactions resulted in the addition of one dNTP onto the primer. The
n (unextended) and n + 1 (extended by one
nucleotide) DNA products were resolved on a 20% Sequel NE (American
Bioanalytical) polyacrylamide gel. The bands were quantified by an
Amersham Biosciences Storm 840 PhosphorImager to measure product
formation as a function of time.
Data Analysis--
The data were fit by nonlinear regression
using the program Sigmaplot version 4.14 (Jandel Scientific). The data
from burst experiments were fit to the equation [product] = A × (1 
exp(kobst)) + ksst, where A is the
amplitude of the burst; kobs is observed
first-order rate constant for dNTP incorporation, and kss is the observed steady-state rate constant.
Single turnover kinetic data were fit to the single exponential
equation [product] = A(1 exp(kobst)), where A is
the amplitude, and kobs is the observed
first-order rate constant for dNTP incorporation. To obtain
Kd, the equilibrium dissociation constant, and
kpol, the maximum rate of polymerization, the
data were fit to the hyperbolic equation kobs = kpol[dNTP]/(Kd + [dNTP]). Fidelity values were calculated using the following
equation, fidelity = ((kpol/Kd)correct + (kpol/Kd)incorrect)/(kpol/Kd)incorrect.
Single Turnover Mispair Extension Assays--
These assays were
performed as described above, in single turnover conditions, except the
primer-template contained mispaired termini (35T-20G). The insertion of
the next correct nucleotide, dGTP, was measured.
M13mp2-based Reversion Assays--
The fidelity of pol -wt
and T79S was measured using two M13mp2-derived templates, each of which
contain a 390-bp gap opposite the lacZ gene and were
constructed as described (31). For gap-filling DNA synthesis, 0.1 pmol
of gapped DNA was incubated with 20 pmol of rat pol -wt or T79S in
30 µl of buffer containing 50 mM Tris-Cl, pH 8.0, 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 200 µg/ml bovine serum albumin, and
500 µM dNTPs at 37 °C for 1 h at 37 °C, and
the reactions were terminated by the addition of EDTA to a final
concentration of 15 mM. An aliquot of each reaction was
analyzed on a 0.8% agarose gel containing ethidium bromide to be
certain that the gap was filled completely (31). In each case, the
reaction products comigrated with a double-stranded nicked molecular
size standard, indicating that the gap had been filled to completion
within our limits of detection (90%). Aliquots of each reaction were
then transfected into the MC1061 strain, and the polymerase-induced
mutation frequency was calculated from the ratios of mutant (blue) and
nonmutant (colorless) plaques on CHS50 F' indicator E. coli
as described (31).
HSV-tk Forward Mutational Assay--
The T79S polymerase was
used in the HSV-tk forward mutational assay as reported
previously (37, 38). The M13-tk template contains a portion
of the HSV1-thymidine kinase gene. Products of DNA synthesis were
purified, and the polymerase-synthesized strand was rescued by
hybridization to a gapped heteroduplex molecule. Mutation frequency
was calculated as described by Eckert et al. (37) after
transfection into the FT334 strain. To ensure independence of selected
mutant colonies for the mutational spectra, FT334 cells were aliquoted
into multiple tubes containing VBA broth immediately after
electroporation as described previously (37, 38). Cultures were
incubated at 37 °C for 2 h and then plated separately on
selective media. To determine mutation specificities, the DNA sequence
of the MluI-EcoRV target region of each mutant was obtained at the Keck Molecular Biology Center at Yale University. Mutants selected from the same tube that contained identical mutations were not considered independent and were counted as one mutant. The
HSV-tk mutant frequency was defined as the number of
colonies resistant to both 40 µM 5-fluoro-2'-deoxyuridine
and 50 µg/ml chloramphenicol divided by the total number of
chloramphenicol-resistant colonies and was calculated as described
(37). We found that some tk sequences contained more than
one mutation. We considered mutational events to be single errors only
if the mutations were greater than 15 bases apart. Multiple errors
occurring within 15 bases were scored as one mutation, and frequencies
were calculated as described (37).
DNA Binding Assay--
The dissociation constant
KD (DNA) was measured using a gel mobility shift
assay (39). One nucleotide gapped (45X-22-22) and 3'-recessed (45X-22)
oligonucleotides were prepared and annealed as described above. Fifteen
protein concentrations ranging from 4 µM to 0.25 nM, expected to bracket the KD, were
incubated with 0.1 nM DNA that had been radiolabeled in
buffer containing 10 mM Tris, pH 7.5, 6 mM
MgCl2, 100 mM NaCl, 10% glycerol, and 0.1%
Nonidet P-40. After a 10-min incubation at 20 °C, samples were
loaded onto a 6% acrylamide nondenaturing gel with the current running
at 300 V. After loading, voltage was reduced to 150 V, and the gel was
run for 1 h. Fractions bound were determined by PhosphorImager
quantitation of the gel using an Amersham Biosciences Storm 840 PhosphorImager. The apparent dissociation constant, KD, was derived from Sigmaplot fitting of the
fraction bound versus protein concentration with the
equation, Y = ((m1 · x)/(x + KD)) + m3, where m1 is a scaling factor, and m3 is
the apparent minimum Y value (39).
dRP Lyase Assay--
Three oligonucleotides Oligo 1, Oligo 2, and Oligo 3 (Fig. 2) were annealed and labeled with
[-32P]dATP (3000 Ci/mmol) and reverse transcriptase.
After precipitation in ethanol, the labeled oligonucleotides were
dissolved in 10 mM HEPES, pH 7.5, and treated with
uracil-DNA-glycosylase immediately before use. The
uracil-DNA-glycosylase-treated substrate (10 pmol/assay) was incubated
in 10 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2 with indicated amounts of polymerases
at 25 °C for 15 min. The reaction was terminated by the addition of
SDS (final 0.5%), and the unexcised dRP was stabilized by 100 mM NaBH4. After addition of an equal volume of
the formamide/dye solution, the samples were resolved by
electrophoresis in a 8 M urea-containing 20% polyacrylamide gel. The gel was subjected to autoradiography with an
x-ray film and also scanned with a Fuji BAS PhosphorImager for quantitation.
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RESULTS |
T79S Confers a Mutator Phenotype to the SC18-12 E. coli
Strain--
To confirm that the candidate T79S mutant (Fig.
1) we identified in our genetic screen
possessed a true mutator phenotype, we compared the spontaneous
mutation frequencies of this mutant of the 8-kDa domain of pol with
that of the pol -wt strain using the Trp+ reversion
assay (27). We found that the T79S mutant has a spontaneous mutation
frequency that is 8-fold higher than the pol -wt strain.
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Fig. 1.
The Thr-79 residue functions in the
control of the helix-hairpin-helix motifs of pol .
Blue shows the HhH motifs, and gray and
red show the Thr-79 residue. The template is in
pink and the primer and downstream oligonucleotide is in
yellow. The two green magnesium ions are shown
along with the dNTP in pink. The magenta ball is
a sodium ion. T79S does not contact the DNA or dNTP substrates of pol
.
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T79S Shows a Rapid Burst of Product Formation--
We purified the
protein as described under "Experimental Procedures" (28). A
pre-steady-state burst experiment to monitor dTTP incorporation was
performed under conditions where
45X-22-22 DNA (Fig. 2) was in 3-fold
excess of pol . Fig. 3 demonstrates insertion of dTTP opposite A by T79S at 37 °C occurs via an initial fast phase (kobs = 3.782 s1)
followed by a slower, linear phase with a rate constant of 0.2536 s1. The biphasic nature of T79S is similar to WT pol (29) and indicates that the rate-limiting step of the catalytic cycle
occurs after phosphodiester bond formation.
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Fig. 2.
Primer-templates for gel mobility shift,
misincorporation, mispair extension, and dRP lyase assays.
Templating bases are underlined. Mispaired bases are in
boldface italic. The X position in the template
contained A, T, G, or C in four different substrates. tk
refers to the oligonucleotides constructed with HSV-tk DNA
sequence.
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Fig. 3.
T79S exhibits burst kinetics. Insertion
of dTTP into a gapped DNA substrate was measured using the chemical
quench-flow apparatus at 37 °C. A preincubated solution of 100 nM T79S ( ) and gapped DNA (300 nM) was mixed
with a solution of dTTP (100 µM) containing 10 mM MgCl2. The reactions were quenched and
monitored as described under "Experimental Procedures." The data
were fit to the burst equation with a kobs = 3.782 ± 1.179 s1 and a steady-state rate constant
of 0.253 s1.
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T79S Has An Intrinsic Antimutator Activity in Vitro--
The
in vivo data suggested that T79S confers a mutator phenotype
to the SC18-12 E. coli strain. One interpretation of these data is that the mutator phenotype is caused by T79S incorporating incorrect nucleotide substrates, as it substitutes for pol I in DNA
replication. To test this hypothesis (28), we compared the fidelities
of DNA synthesis of T79S and pol -wt.
First, a single turnover kinetic assay (28, 29, 39) was used to
quantify the relative rates of nucleotide misincorporation catalyzed by
the T79S and pol -wt proteins opposite templates C, G, A, and T
using a synthetic 5'-32P-end-labeled single base gapped
template (45X-22-22) as we have described previously (28, 29, 39).
Single gapped substrate was chosen because it was shown to be the most
likely physiological substrate for pol (17, 18). In this assay the
enzyme is in vast excess of DNA, thereby minimizing DNA binding
effects. Fig. 4 shows a plot of the
observed rate constants (kobs) versus the concentration of dGTP, enabling us to obtain the maximum rate of
polymerization, kpol, and the equilibrium
dissociation constant, Kd, for T79S. The
kpol and Kd rate constants were used to calculate the fidelities for pol -wt and T79S. As shown
in Table I, we observed that T79S
possessed an increased fidelity compared with pol -wt of 7-, 5-, and
6-fold when we tried incorporating the incorrect dCTP, dTTP, and dATP
substrates, respectively, opposite template C. The increased
discrimination of substrates exhibited by T79S mainly occurred at the
level of ground state binding (Kd). For
misincorporation of dTMP and dGMP opposite template T,
misincorporations of dGMP, dAMP, dTMP opposite template G and dAMP,
dGMP, dCMP opposite template A, no significant differences in the
fidelity of DNA synthesis were observed between T79S and pol -wt
(data not shown). These data suggested that T79S exhibited antimutator
activity during DNA synthesis opposite template C.
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Fig. 4.
Single turnover experiments of correct
nucleotide incorporation opposite cytosine. A preincubated
solution containing enzyme (750 nM) and gapped DNA (50 nM) was mixed with MgCl2 (10 nM)
and various concentrations of dGTP as described previously (28,
29, 34, 39). The reactions were terminated by EDTA, and the product, a
23-mer, was resolved by denaturing sequencing gel electrophoresis. The
data were fit to the single exponential equation to obtain
kobs. This figure depicts the secondary kinetic
plot of kobs versus the dGTP
concentration for T79S (). The data were fit to a hyperbolic
equation as described under "Experimental Procedures." The
solid line represents the best fit of the data to the
hyperbolic equation.
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T79S Is Not a Frameshift Mutator at a Run of T
Residues--
Although T79S was identified as a mutator in our
in vivo screen, we found it to be an antimutator opposite
template C, in the in vitro misincorporation assay. To
determine whether T79S was a mutator for frameshifts, we employed a
reversion assay that detects minus 1-base frameshifts at a TTTTT
sequence or at 36 other non-repetitive sites within in the
lacZ gene (40). We obtained a spontaneous mutation frequency
of 4.84 × 103 for the T79S protein in this
frameshift reversion assay, which is 1.4-fold less than that of the pol
-wt enzyme which has a spontaneous mutation frequency of 6.7 × 103. This demonstrates that the T79S protein is not a
mutator for frameshift errors in a run of T residues.
Forward Mutation Spectrum--
Next, we generated a forward
mutation spectrum to assess all possible errors committed by the T79S
protein. We employed the HSV-tk assay (37) that detects all
types of errors including large addition and deletions and multiple
mutations, in addition to frameshift and base substitution mutations.
This assay employs a primed single-stranded DNA substrate for DNA
synthesis. The mutation spectrum for T79S is shown in Fig.
5, and a summary of the data appears in
Table II. The major difference between
T79S and pol -wt in this assay is that T79S produces 8-fold more
multiple mutations than pol -wt protein. This suggests that DNA
synthesis by T79S results in multiple mutations, defined here as
mutations within 15 bases of each other and assumed to be the result of one pol binding event. The increased propensity of T79S to produce multiple mutations may account for its mutator phenotype in
vivo.
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Fig. 5.
Mutational spectrum of T79S determined by
using the HSV-tk gap filling assay. Frameshift
mutations, including 1-base additions ( ), 2-base additions ( ),
1-base deletions ( ), 2-base deletions ( ), and big deletions (224 with numbers inside) are shown below the HSV-tk target
sequence. Base substitutions are shown above this sequence.
Tandem multiple mutations, defined as being separated by 15 bases or
less, are not shown. Circled bases are part of multiple
mutational events.
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The T79S mutation spectrum displays several unique characteristics
relative to pol -wt. The most frequent base substitution mutation
observed for T79S is G to T, which is rarely observed for pol -wt.
The pol -wt enzyme (37) produces mainly A to G mutations. A putative
hot spot of T to C base substitution mutations occurs at position 203 of the HSV-tk target sequence. As shown in Fig. 5, two
single T to C mutations were produced at position 203, and two other T
to C substitutions also occurred at this site as part of two tandem
multiple mutations (data not shown). T to C base substitutions have not
been observed at position 203 for pol -wt. A hot spot of 1-base
deletion mutations occurs at position 147 after DNA synthesis by T79S;
pol -wt sometimes produces 1-base deletions at this site but not at
the frequency of T79S.2
A remarkable aspect of the T79S spectrum is the increase in 1-base
insertion mutations, some of which occur at non-repetitive sequences.
In analyzing the rates of 1-base frameshift mutations, we determined
that in general the rate increases as the length of the homonucleotide
run increases for both T79S and pol -wt (37), as shown in Table
III. However, the 1-base frameshift error rate for pol -wt increased 4-fold as the repeat length increased from three to four nucleotides, although this was not the case for
T79S; the highest rate of 1-base frameshift mutation was at nucleotide
runs of three for this mutant. In fact, the T79S mutant commits 1-base
frameshifts predominantly at runs of three Cs, having an 11.9-fold
increased frequency over pol -wt at these runs. The major hot spot
of mutation at a run of three Cs is at position 147 of the
HSV-tk target sequence. The slippage mechanism is usually
responsible for 1-base frameshift mutations at homonucleotide runs, and
this mechanism most likely operates in the case of T79S. However,
because the majority of 1-base frameshifts at a run of three Cs occurs
mainly at position 147, it is possible that the mechanism of
mutagenesis at this site could be more complex than misalignment of the
template. The fact that the run of C residues at position 147 is part
of a pseudorepeat sequence lends support to this hypothesis. T79S
produces a 3:1 ratio of 1-base deletions to insertions, whereas with
pol -wt the ratio of 1-base deletions to insertions is 10:1. A
putative hot spot for insertion mutations is at position 199, where C
is inserted within the run of Cs at this position. The most likely
mechanism to account for this 1-base insertion is slippage. Other
1-base insertions were detected at other positions of the tk
target. However, most of these insertion mutations were most likely not
produced by the slippage mechanism, because these occur in non-runs and
because the nucleotide that is inserted is not templated by the next
base in the template. What is intriguing is that each of these
insertions is preceded by the primer sequence 5'-CG.
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Table III
The error rate of 1-base frameshifts produced by T79S increases with
the length of the homonucleotide run
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The T79S Mutant Is a Misincorporation Mutator at Position 203 of
the HSV-tk Sequence--
We detected a putative hot spot of mutation
of T to C at position 203 of the HSV-tk target, as described
above. As shown in Fig. 5, two single T to C mutations were produced at
position 203, and two other T to C substitutions also occurred at this site as part of a tandem multiple mutation (data not shown). The T to C
mutation could result predominantly from direct misincorporation of
dGTP opposite template T or from slippage because there is a C after
template T at position 203. Therefore, we tested the hypothesis that
the T79S protein misincorporates dGMP opposite template T at this site,
using a single turnover kinetic assay with the 35T-19 (tk)
primer-template, which corresponds to the exact DNA sequence at
position 203. As shown in Table IV, T79S has a 5-fold decreased fidelity of misincorporation of dGMP opposite T
at position 203, in the single turnover assay. The mechanistic basis
for the decreased fidelity is mainly during the ground state binding,
Kd, of the dGTP substrate. The T79S mutant has a low
affinity for both correct (A) and incorrect (G) substrates at this
site. On the other hand, T79S was found to have the same fidelity as
the WT protein when misincorporating dGMP opposite T with the single
base gapped substrate, 35T-19-15 (tk), having the same
sequence as the gene at position 203, as shown in Table V. These data show that T79S has
difficulty in discriminating dGTP versus dATP opposite
template T when utilizing a 3'-recessed DNA substrate. The template
utilized in by pol in our E. coli genetic screen is most
likely similar to a 3'-recessed template.
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Table V
The T79S mutant misincorporates dGTP opposite template T with similar
fidelity as WT in a single base-gapped substrate
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Next, we asked whether the misincorporation event of dGTP opposite
template T at position 203 of HSV-tk sequence was due to a
slippage-mediated mechanism in the 3'-recessed primer-template (35T-19)
because there is a C after the template T. So we altered the C after
template T at position 203 to a G and performed the misincorporation
experiment with dCTP opposite T using 35TGG-19 as primer-template. In
this scenario, T, the templating base, would significantly "slip"
out of the active site and allow G to become the templating base. dCTP
would then be incorporated opposite G. As shown in Fig.
6, T79S produces significantly less n + 1 products compared with pol -wt, which suggests that
the misincorporation event at position 203 by T79S was not due to a
slippage-mediated mechanism but was the result of a direct
misincorporation event.
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Fig. 6.
T79S does not produce a slippage-mediated
error at position 203 of HSV-tk sequence.
35TGG-19 primer-template was employed in this experiment.
Misincorporation of dCTP opposite template T at position 203 was
performed under single turnover conditions as described earlier.
Reactions were performed for 7 different reaction times (0, 2, 4, 8, 12, 20, and 30 min) with each of the 5 different concentrations of
dCTP. The 1st lane is the 0-time point for each set of
reactions. pol -wt was found to produce significantly more
n + 1 products than T79S, which are most likely due to
slippage-mediated mechanism.
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T79S acts as a mutator using 3'-recessed primer-template as a substrate
but behaves like an antimutator when the 3' downstream sequence is
present. To determine whether misincorporation by T79S is sequence
context-specific, we repeated the experiment of misincorporation of
dGMP opposite T with T79S in a totally different sequence context
(45T-22). T79S showed a 38-fold decreased fidelity compared with pol
-wt, in misincorporating dGMP opposite template T, as shown in Table
VI. Here also the mechanistic basis of
decreased fidelity is mainly due to less discrimination than pol -wt
during ground state binding (Kd) of the dGTP substrate. However, in this new sequence context, T79S has a higher affinity for dGTP versus dATP when the templating base is T. These data suggest that T79S behaves as a low fidelity polymerase
specifically when utilizing 3'-recessed DNA substrate. On the other
hand, T79S was found to exhibit an antimutator phenotype with a 4-fold
higher fidelity than pol -wt using a single base gapped DNA
substrate (45T-22-22) as shown in Table
VII.
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Table VII
The T79S mutant misincorporates dGTP opposite template T with 4.4-fold
higher fidelity as WT in a single base gapped substrate of a
different sequence
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T79S Does Not Extend a Mispair--
We also compared the ability
of T79S to extend a dGMP:T mispair within the sequence context of
position 203 of the HSV-tk target, using single turnover
conditions with primer-templates 35T-20G and 35T-20A, respectively. As
shown in Table VIII, the overall catalytic efficiency for the extension of the dGMP:T mispair is the
same for the T79S and pol -wt enzymes. However, the mechanism governing the extension of the mispair differs for T79S and pol -wt.
The T79S polymerase discriminates against mispair extension 10 times
more than pol -wt at the level of kpol and
about 10 times less at the level of Kd. This
indicates that the molecular basis for discrimination against the
extension of mispairs is significantly altered for the T79S enzyme.
The Affinity of T79S for Gapped DNA Is Slightly Decreased Compared
with pol -wt--
A gel mobility shift assay was conducted to
estimate the affinity of pol for single base gapped DNA and
3'-recessed DNA (data not shown). The results of 5 experiments were
averaged and demonstrated that the T79S mutant had a 4-fold lower
estimated affinity for 1-base gapped DNA (45X-22-22) than pol -wt.
We also found that the affinity of T79S was 4-fold lower than that of pol -wt for 3'-recessed primer-template (45X-22) in each of two experiments. This indicates that alteration of Thr to Ser at position 79 results in the protein having a slightly decreased affinity for DNA.
dRP Lyase Assay--
Because T79S is located in the 8-kDa domain
of pol and this domain possesses dRP lyase activity (9, 41), we
wanted to see whether the mutation of threonine to serine at position 79 affected the dRP lyase activity (9) of the polymerase. We found no
change in dRP lyase activity as shown in Fig.
7, suggesting that Thr-79 is not
essential for the dRP lyase activity of pol .
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Fig. 7.
T79S retains dRP lyase activity that is
comparable with the wild-type enzyme. The experiments were
conducted as described under "Experimental Procedures." UDG,
uracil-DNA-glycosylase.
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DISCUSSION |
The T79S mutator mutant was identified by a genetic screen
developed in our laboratory. T79S has an 8-fold increased
Trp+ reversion frequency compared with pol -wt. We
purified the T79S protein and examined its ability to misincorporate
nucleotides in vitro, using a 1-bp gapped DNA substrate. In
these assays, T79S appeared to be an antimutator polymerase when
incorporating nucleotides opposite templates C and T within different
sequence contexts, and was as accurate as pol -wt opposite templates
A and G, respectively. These data indicate that the mutator phenotype of T79S we detected in our E. coli screen was not a result
of direct misincorporation by T79S in a single nucleotide gap. To clarify whether T79S possessed inherent mutator activity, we employed the HSV-tk forward mutation assay. T79S was found to induce
an 8-fold higher frequency of multiple mutations than pol -wt in the
forward assay, which was consistent with the mutator phenotype we
detected originally using our genetic screen. Upon close inspection of
the mutation spectrum obtained from copying the HSV-tk
target with T79S, we found that T to C transitions were frequent at
position 203 of this target, suggesting that T79S frequently
misincorporated dGMP opposite template T at position 203. To determine
whether this was the case, we prepared a 3'-recessed primer-template
with the identical sequence context present at position 203 of the tk target, and we performed a misincorporation assay. We
used a 3'-recessed primer-template because this type of DNA substrate best mimics the single-stranded DNA template used in the
HSV-tk assay. In accordance with the mutation spectrum of
T79S, we found that this enzyme has a 5-fold lower fidelity of DNA
synthesis for insertion of dGMP opposite T using a DNA substrate with
the sequence context of position 203 of the tk gene (35T-19
(tk)). Next, we asked if T79S was able to misinsert dGMP
opposite T using a 1-base gapped DNA primer template with the identical
sequence context of position 203 of the tk target. We found
that when using the single nucleotide gapped DNA, T79S had a fidelity
similar to pol -wt. Our data suggested that T79S misinserted dGMP
opposite T using a 3'-recessed DNA substrate and that perhaps sequence context was important for the misincorporation to occur. To test this
hypothesis, we examined misincorporation of dGMP opposite T using a
3'-recessed DNA primer-template with a sequence context that was
unrelated to position 203 of the tk target sequence, and we
found that T79S had a 38-fold lower fidelity than pol -wt. However,
when utilizing a single nucleotide gapped DNA substrate, T79S had a
higher fidelity than pol -wt. Therefore, we conclude that the T79S
polymerase variant is a misincorporation mutator when catalyzing DNA
synthesis in the presence of a 3'-recessed DNA substrate. The
tk mutation spectrum also revealed that T79S produces a much
higher amount of 1-base insertion mutations than pol -wt. The
majority of these mutations occur at a run of C residues, suggesting
that the slippage mechanism can account for these mutations. These data
show that when Thr-79 is altered to Ser, the primer DNA has acquired a
propensity to slip within the active site. Taken together, our results
show that the nature of the interaction of pol with the DNA is
important for accurate DNA synthesis. Our results also demonstrate that
the HhH domains of pol are critical for accurate DNA synthesis.
The Rate-limiting Step of T79S Is after Phosphodiester Bond
Formation--
Pre-steady-state burst analysis for T79S protein shows
a biphasic kinetic profile (Fig. 3) just like pol -wt (29)
indicating that the rate-limiting step occurs after phosphodiester bond
formation and is not changed due to its mutation to Ser.
T79S Has an Intrinsic Antimutator Activity When Using Single
Nucleotide Gapped DNA--
Single base gapped substrate is a preferred
physiological substrate for the pol enzyme. We used single turnover
kinetics to characterize all possible misincorporation events opposite A, T, C, and G in single nucleotide gapped DNA. The T79S mutant exhibited fidelity similar to pol -wt protein opposite A and G,
respectively, but showed a surprisingly higher fidelity than pol -wt
when incorporating nucleotides opposite templates C and T (Tables I and
VII). For incorporation opposite template C in single nucleotide gapped
DNA, the molecular basis of discrimination appears to be more dependent
on ground state binding (Kd) than on the
precatalytic conformation change or chemical steps of the reaction
(kpol). The pattern of higher
Kd(dNTP) values for the incorrect
incorporations opposite template C tested is striking (Table I). This
suggests that the mutant enzyme is capable of discriminating correct
versus incorrect nucleotides at the level of ground state
binding. Because Thr-79 is far away from the active site and has no
direct contact with the substrate or the DNA, this finding raises the
possibility that remote residues, like Thr-79, may be involved directly
or indirectly in the recognition and binding of nucleotide substrates.
T79S Is Altered Mainly in Its Discrimination Ability during Ground
State Binding on 3'-Recessed DNA--
T79S appeared to induce a higher
frequency of T to C transitions at position 203 of the
HSV-tk gene, suggesting that it frequently misinserted dGMP
opposite T at this position. Using a 3'-recessed template that was
identical to position 203, we showed that T79S misincorporated dGMP
opposite T at this site more often than pol -wt. It appeared that
this was a direct misincorporation event and was not due to a
slippage-mediated mechanism (Fig. 6). We also demonstrated that T79S
misincorporated dGMP opposite T using a 3'-recessed DNA sequence
unrelated to the tk sequence at position 203. In both cases,
T79S was less able than pol -wt to discriminate between the correct
and incorrect dNTP substrates during ground state binding. In fact,
when catalyzing DNA synthesis in the presence of the 3'-recessed DNA
substrate unrelated to position 203, T79S preferred to insert dGMP
versus dAMP opposite template T. We conclude that when
Thr-79 is altered to Ser, the fidelity of DNA synthesis is dependent
upon the structure of the DNA. This suggests that the geometry of the
nucleotide binding pocket and/or the active site of the T79S enzyme in
the presence of 3'-recessed DNA is different from that of pol
-wt.
T79S Commits 1-Base Insertion Mutations--
The ratio of single
base deletions to insertions is much lower for T79S than the WT enzyme.
Most of the insertions produced by T79S are at a run of three C
residues in the tk target, suggesting that they occur by
slippage of the primer. This suggests that alteration of Thr-79 to Ser
results in an inability of the enzyme to stabilize the primer within
the active site. The other insertions cannot be accounted for by the
slippage mechanism, because they occur in non-repetitive sequences and
are not templated. This indicates that the non-run insertions may be
mediated by some type of non-templated addition. What is curious is
that the primer sequence preceding the insertion is in every case
5'-CG. We suggest that the 5'-CG might form a structure that is
conducive to a non-templated addition of a nucleotide, especially in
the absence of stabilization of the primer strand within the T79S
active site.
Position of T79S in the 8-kDa Domain May Affect Catalysis--
The
HhH is a widespread motif involved in non-sequence-specific DNA
binding. Most of the HhH motifs function as DNA-binding modules.
However, some of them mediate protein-protein interactions or have
acquired enzymatic activity by incorporating catalytic residues. For
example, this motif in pol has dRP lyase activity. The sequence and
structural studies of different HhH-containing proteins show that most
of the HhH motifs are integrated as a part of a five-helical domain. It
typically consists of two consecutive HhH motifs that are linked by a
connector helix and that display pseudo-2-fold symmetry. Most HhH
domains are found to possess a conserved hydrophobic core and show
clear structural integrity to be recognized as a distinct protein fold.
DNA polymerase has two HhH motifs. One HhH motif is present within
the 8-kDa domain of pol , is composed of residues 1-81, and
interacts with the 5'-phosphate of the downstream primer of a single
nucleotide gapped DNA substrate. This interaction is important for the
catalytic efficiency and fidelity of pol (18, 42). The second HhH
motif interacts with the primer. T79S is located within the hinge
region of helix 4 of the N-terminal 8-kDa domain of pol , and in
this location it is directly between both of the HhH motifs of pol .
The hinge region in which Thr-79 is located appears to participate in
the movement of the two HhH motifs.
pol undergoes a conformational change from an open to a closed form
after the binding of the dNTP substrate and before phosphodiester bond
formation. This change results in the alignment of the primer, template, dNTP substrate, and catalytic residues of the protein in an
optimum geometric configuration for phosphodiester bond formation.
Amino acid residues important for catalysis and fidelity, including
Arg-283, Met-282, and Phe-272, are not poised for catalysis in the open
conformation of the enzyme. However, once the enzyme assumes the closed
conformation, these residues are brought into proper geometry for
catalysis and for monitoring the fidelity of the chemical step (29,
43). Residues Arg-283 and Phe-272 form hydrogen bonds and van der Waals
contacts with the templating nucleotide in the closed conformation. For
T79S, our data indicate that, like WT, once the closed conformation is
assumed in the presence of incorrect substrate, the rate of catalysis
of nucleotidyl transfer is quite slow. Thus, T79S is a mutator when
catalyzing DNA synthesis from a 3'-recessed primer-template mainly
because it cannot discriminate correct from incorrect substrates during ground state binding.
Examination of the crystal structure of pol complexed with DNA and
dideoxy-CTP (Protein Data Bank Code 1BPY) shows that in the
closed conformation the DNA is bent nearly 90o. In
interacting with DNA, the HhH motifs of pol in the 8-kDa and thumb
domains appear to push the templating base in the direction of the
nucleotide binding pocket and active site, as shown in Fig. 1. This
bent conformation of the DNA most likely results in template
presentation and primer stabilization that is required for accurate
discrimination of dNTP substrates during their ground state binding.
The HhH motifs also stabilize the sharp bend formed in the pol
-gapped DNA structure.
We suggest that Thr-79 is a residue that participates in the movement
of both HhH motifs and/or functions in the stabilization of these
motifs in the closed conformation. Our data show that when Thr-79 is
altered to Ser, the enzyme is much less accurate than pol -wt in the
absence of the downstream primer and produces 1-base insertion
mutations much more frequently than pol -wt. The mechanistic basis
for the decreased fidelity during misincorporation is loss of the
ability to discriminate correct from incorrect dNTP substrates during
the initial binding of these substrates to the polymerase. These data
are consistent with the interpretation that in the absence of the
downstream primer, the HhH motifs are unable to function to position
properly the template and primer within the active site. Thr-79 O-1
forms two hydrogen bonds with the main chain oxygens of Glu-75 and
Lys-81, which belong to helices D and E of pol as shown in Fig.
8. These interactions may help to
stabilize the orientation or relative angle between the two 
-helices
and, in turn, the width of the active site cleft. The N-terminal end of
the helix D carries residues (e.g. Lys-68) that form
important interactions with the upstream DNA primer. A serine in
position 79 may have slightly different rotamer preferences than a Thr,
and the position for O-1 seen in the wild-type enzyme may not be
equally populated in the mutant. We suggest that improper template
presentation results in active site geometry that can more easily
accommodate the incorrect dNTP substrate.
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Fig. 8.
The Thr-79 residue interacts with Glu-75 and
Lys-71 of pol . Residues are labeled in
green. The hydrogen bonds are depicted as lines
connecting one residue to another.
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Conclusions--
In summary, we have identified a mutator mutant
of pol that is altered at residue 79, from Thr to Ser and has no
direct contact with either the DNA or dNTPs. This suggests a view of enzyme function in which the residues outside the immediate areas of
substrate binding and catalytic activity are responsible for the
fine-tuning of polymerase function, including substrate specificity. Our data suggest that the precise positioning of the DNA template and
primer into the active site is critical for maintaining the fidelity of DNA synthesis. Our data also suggest that Thr-79
participates in a hydrogen bonding network that acts to position the
DNA within the active site and that this positioning is critical for
the fidelity of pol .
by Helping to Position the DNA within the Active
Site*
,
,
TOP
ABSTRACT
INTRODUCTION
