From the Joseph Gottstein Memorial Cancer Laboratory, Departments of Pathology and Biological Structure, University of Washington School of Medicine, Seattle, Washington 98195
Received for publication, September 22, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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DNA polymerases contain active sites that are
structurally superimposable and conserved in amino acid sequence. To
probe the biochemical and structure-function relationship of DNA
polymerases, a large library (200,000 members) of mutant Thermus
aquaticus DNA polymerase I (Taq pol I) was created
containing random substitutions within a portion of the dNTP binding
site (Motif A; amino acids 605-617), and a fraction of all selected
active Taq pol I (291 out of 8000) was tested for base
pairing fidelity; seven unique mutants that efficiently misincorporate
bases and/or extend mismatched bases were identified and sequenced.
These mutants all contain substitutions of one specific amino acid,
Ile-614, which forms part of the hydrophobic pocket that binds the base
and ribose portions of the incoming nucleotide. Mutant Taq
pol Is containing hydrophilic substitution I614K exhibit 10-fold lower
base misincorporation fidelity, as well as a high propensity to extend
mispairs. In addition, these low fidelity mutants containing
hydrophilic substitution for Ile-614 can bypass damaged templates that
include an abasic site and vinyl chloride adduct ethenoA. During
polymerase chain reaction, Taq pol I mutant I614K exhibits
an error rate that is >20-fold higher relative to the wild-type enzyme
and efficiently catalyzes both transition and transversion errors.
These studies have generated polymerase chain reaction-proficient
mutant polymerases containing substitutions within the active site that
confers low base pairing fidelity and a high error rate.
Considering the structural and sequence conservation of Motif A,
it is likely that a similar substitution will yield active low fidelity
DNA polymerases that are mutagenic.
Prolonged survival of individual species depends on the accurate
transmission of genetic material from one generation to the next (1).
However, in times of stress, the propensity to mutate and to rapidly
create variants that can escape selection pressures facilitates
survival of a small fraction of the original population (2). Thus,
evolution may be characterized by periods of high fidelity DNA
replication, as well as by the presence of transient mutators, which
have a selective growth advantage during adverse conditions (3).
Identifying mechanisms of generating potential mutators is crucial
toward understanding the dynamic processes that govern evolution, as
well as toward devising effective chemotherapeutic strategies against
pathogenic bacteria (4, 5) and cells (6) that mutate at elevated rates.
Cells have evolved multistep mechanisms to guarantee the exceptionally
high fidelity of DNA replication that is required for the maintenance
of species. The genetic sequence of organisms is maintained over
prolonged evolution by the fidelity of DNA replication (7), the
efficiency of DNA repair processes (8), and the recombination-mediated
lateral transfer events (9). Quantitatively, nucleotide selection at
the active site of DNA polymerases is the most significant contributor
to the fidelity of DNA replication (10). Nucleotide selection includes
correct Watson-Crick base pair formation between complementary bases; further discrimination of base selection occurs by a conformational change at the active site during each nucleotide addition step (11) and
preferential extension of the correct base pair by the addition of the
next complementary nucleotide (12). Together, these processes
contribute ~100,000-fold to the overall accuracy of DNA replication
(one base change per 108-10 bases per generation (13)).
Inefficient extension of mispaired bases in vivo would
facilitate 3'-5' exonuclease removal of the nascent nucleotide.
Exonucleolytic (3'-5') proofreading activity of most DNA polymerases
occurs on a separate domain (alternatively, this activity could reside
in a separate protein) and contributes, on average, 10-fold to the
overall mutation rate (14). In addition, errors in catalysis by DNA
polymerases are subsequently corrected by a mismatch repair system,
which contributes an additional 2-3 orders of magnitude to the overall
accuracy of DNA replication (15). Disruption of either mismatch repair
system or polymerase 3'-5' exonuclease function within cells leads to a
mutator phenotype (16, 17). Mice harboring disruption in mismatch
repair (18) or in the 3'-5' exonuclease of DNA polymerase The structure of a DNA polymerase resembles the human right hand and
contains three distinct subdomains (finger, palm, and thumb (19)). High
resolution crystal structures of DNA polymerase within the pol
I2 family of enzymes indicate
that the base of the incoming nucleotide stacks with the hydrophobic
planar amino acids located in the fingers subdomain (Motif B), and the
triphosphate portion is bonded through metal cations by ionic
interactions with Asp-610 located in the palm subdomain (Motif A (20,
21)). During nucleotide incorporation, DNA polymerases undergo a
conformation change from open to a closed conformation, bringing the
fingers subdomain in close proximity to the palm subdomain (21). Planar
hydrophobic residues of the fingers subdomain sense the binding of a
properly templated incoming nucleotide, and this signal is transduced
to the catalytic residues of the palm subdomain (22). Among those residues that are located between the base and phosphate interacting amino acids and can participate in transducing this signal are the
highly conserved DYSQIELR Motif A residues (in Taq pol I, amino acids 605-617). The nucleotides encoding these amino acids are
conserved within DNA polymerase I of all prokaryotes and eubacteria sequenced (Fig. 1) (9). The amino acids
in this region are positioned to have a potentially important
contribution toward DNA polymerase fidelity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
develop
cancer in multiple organs with elevated
frequency.1 These studies
provide direct evidence linking deficits in the fidelity of DNA
synthesis with increased incidence of cancer.
View larger version (57K):
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Fig. 1.
Motif A sequence alignment. Sequence
alignment of 34 prokaryotic DNA polymerase I catalytic sites shows that
the Motif A DYSQIELR sequence has been maintained in most organisms.
The sequences for the alignments were obtained from
GenBankTM, and amino acid sequences for the entire
polA genes of individual organisms were aligned.
The high contribution of the polymerase active site to the overall
fidelity of DNA synthesis suggests that subtle alterations within the
catalytic site should lead to polymerases with lower fidelity. This is
especially true for polymerases lacking a 3'-5' proofreading
exonuclease activity that can excise misincorporated nucleotides.
Site-directed mutagenesis studies have identified a conserved tyrosine
located within the dNTP-binding pocket that, when substituted to
nonplanar amino acids, reduce the fidelity of the polymerase by
5-10-fold, but these mutant polymerases also exhibit markedly reduced
catalytic activity (23-25). Previously, we established a library of
~8000 different active Taq DNA polymerase mutants using
random sequence mutagenesis and stringent selection protocol. Each
mutant contained one or more substitutions within Motif A and
maintained 10-200% of the wild-type activity. In this study, we
screened 291 different mutant DNA polymerases containing substitutions
within the dNTP binding site for altered polymerase fidelity. Many of
the low fidelity mutants contained multiple substitutions; however,
each contained a substitution at a single position. Mutants containing
substitutions of one residue (Ile within the conserved DYSQIELR
sequence), which can be substituted to diverse amino acids, yield an
enzyme that introduces transition and transversion errors 20-fold more
efficiently than the WT Taq polymerase. Alterations at the
other sites did not have a significant effect on fidelity. Because this
residue is conserved in structure and sequence in polymerases from
prokaryotes, eukaryotes, and archea (26, 27), it represents a
potentially important target for the creation of mutator DNA polymerases.
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MATERIALS AND METHODS |
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Screen for Fidelity--
Three hundred fifty colonies containing
mutant Taq DNA polymerases (Taq pol Is) that
complemented E. coli containing a pol I
temperature-sensitive mutation at 37 °C were isolated and grown in
nutrient broth individually overnight at 30 °C. Each culture was
grown to A595 of 0.3 at 30 °C in 10 ml, Taq pol I expression was induced with 0.5 mM
isopropyl-1-thio--D-galactopyranoside, and
incubations were continued for 4 h. Taq DNA polymerases
were partially purified (total volume, 50 µl) using a modified
protocol of Ref. 28, which attains a purity of >50% purification of
Taq pol I while removing detectable endogenous polymerase
and nuclease activities. Each of the Taq pol Is that retain
at least 10% activity relative to WT enzyme at 72 °C (291 out of
350 total) were screened for fidelity using two different
primer·templates. Primer·template constructs were prepared by
hybridizing 5'-32P end-labeled 23-mer primer (5'-cgc gcc
gaa ttc ccg cta gca at) with 46-mer template (5'-gcg cgg aag ctt
ggc tgc aga ata ttg cta gcg gga att cgg cgc g) or 47-mer (3'-gcg cgg
ctt aag ggc gat cgt tat agc tta agg cct tta aag ggc cc-5'), using a
1:2 primer:template ratio. The primer·template (5 nM) was
incubated in a reaction mixture (10 µl) containing 50 mM
KCl, 10 mM Tris-HCl (pH 8), 0.1% Triton X-100, 2.5 mM MgCl2, 250 µM each of two or
three of the four dNTPs, and equal amounts of activity (0.01 or 0.05 units) using mutant and wild-type DNA polymerases. Reactions were
terminated after 30-min incubation at 55 °C with the addition of 2 µl of formamide containing stop solution (Amersham Pharmacia
Biotech). Products were analyzed by 14% denaturing polyacrylamide gel
electrophoresis as described (22, 29, 30).
Polymerase Purification--
Wild-type and mutant
(mutants 53, 75, 94, 212, 265, and 346) Taq pol Is
were purified to homogeneity using a modified procedure (31). Step 1: bacteria cultures (DH5 cells; 2 liters) harboring pTaq or selected mutant pTaqLIB plasmid were
harvested and lysed in the presence of Buffer A (30 mM
Tris-HCl (pH 7.9), 50 mM glucose, 1 mM EDTA,
0.5 mM phenylmethylsulfonyl fluoride, 0.5% Tween 20, 0.5%
Nonidet P-40) with lysozyme (4 mg/ml) by freezing and thawing at
70 °C and 70 °C. Step 2: Taq pol I was precipitated
by the addition of polyethyleneimine at a final concentration of 0.1%, recovered by centrifugation, and washed with buffer containing low salt
(0.025 M KCl) buffer C (20 mM HEPES, pH 7.9, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
0.5% Tween 20, 0.5% Nonidet P-40), and then solubilized in 0.15 M KCl buffer C. Step 3: the enzyme was diluted to 50 mM KCl and loaded onto a preequilibrated HiTrap Heparin
5-ml column at a flow rate of 1 ml/min. The column was washed with 10 volumes of Buffer C (50 mM KCl), and the protein was eluted
in 1-ml fractions using a linear gradient from 50 to 750 mM
KCl (60 ml). Aliquots of each fraction were assayed for polymerase
activity by measuring incorporation of [
-32P]dGTP at
70 °C using activated calf thymus DNA as a template with
Mg2+ and all four dNTPs including
[
-32P]dGTP. Peak fractions containing WT and mutant
enzymes consistently eluted at ~300 mM KCl and were
stored in 20% glycerol at
70 °C.
Kinetic Analysis of Misincorporation and Misextension Frequency-- A 47-mer template (3'-gcg cgg ctt aag ggc gat cgt tat agc tta agg cct tta aag ggc cc-5') was hybridized with primer 23-mer (5'-cgc gcc gaa ttc ccg cta gca at). The steady-state Michaelis-Menten parameters Vmax and Km were calculated by incubations with limiting amounts of Taq pol I in the presence of 5 nM primer·template and varying concentration of either the correct dNTP (dATP; 0.001-1 µM) or the incorrect nucleotides (dGTP, dCTP, and dTTP; 1-1000 µM) for 10 min at 55 °C as described (32). The fidelity of mutant 265 (I614N/L616I) relative to WT Taq pol I was determined on the identical template sequence primed with 25-mer (5'-cgc gcc gaa ttc ccg cta gca ata t) and varying concentrations of either dGTP or one of the other three incorrect nucleotides. The kinetic rate parameter kcat was calculated by dividing Vmax by the enzyme concentration. All products were analyzed by 14% polyacrylamide gel electrophoresis and quantified by phosphorimager analysis. Mismatch extension was determined by a similar protocol, except that the primer sequences were 24-mers (5'-cgc gcc gaa ttc ccg cta gca at X, where X is either a, g, c, or t). In these experiments, four sets of primer·templates were constructed such that each contained either a matched or one of the three mismatched 3' primer terminus. The efficiency of dTTP incorporation opposite template dA was measured for each primer·template construct as described (12).
Template Bypass-- A 36-mer template (3'-gcg cgg ctt aag ggc gat cgt tat aag acg Xcg gtt-5') was hybridized with primer 23-mer (5'-cgc gcc gaa ttc ccg cta gca at), where X is either ethenoA, abasic site, or dT residue. Incubations were done under standard conditions noted above with 250 µM each of the 4 dNTPs for 10 min at 55 °C in the presence of either WT Taq pol I and purified mutant 53, 265, 75, or 212 (at one of two concentrations: 2 or 20 fmol/µl). The site of the lesion is marked by an X.
Error Spectrum--
The error spectrum of WT and mutant
Taq pol I was determined following iterative replication of
a 1.3-kb target gene encoding human thymidylate synthase. PCR samples
contained 0.03 pmol of vector containing the target gene, 50 mM KCl, 10 mM Tris-HCl (pH 8), 0.1% Triton
X-100, 1.5 mM MgCl2, and 1 µl of purified
Taq pol Is (5 units) in 100-µl volumes in the presence of
250 µM of each of the dNTPs. All PCR samples were
incubated at 94 °C for 30 s, 50 °C for 30 s, and
72 °C for 3 min for 45 cycles. Products (1.3 kb) from these
amplification reactions were isolated by electrophoresis and cloned
into the Topo T:A cloning vector. The cloned vector was transformed
into bacteria, vectors from randomly chosen colonies were purified and
sequenced, and 630 identical bases were analyzed for each clone. The
error rate was determined by calculating the mutation frequency per
base per duplication. The mutation frequency of each polymerase was
determined by calculating the average number of mutations per
clone per bases sequenced (630) following PCR. The error rate was
determined by dividing the mutation frequency by the number replication
cycles (as determined after calculating the product yield after PCR).
The average number of replication cycles under PCR conditions were as
follows: 8 (reflecting ~250-fold DNA amplification) by WT and mutant
346, 6 for mutant 75, and 5 for mutant 53.
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RESULTS |
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Identification of Mutant Polymerases with Altered Fidelity-- DNA polymerases contain active sites that are conserved in three-dimensional structure and amino acid sequence. Previously, we used random sequence mutagenesis coupled with genetic selection to demonstrate all active site amino acids, except those directly involved in the catalytic mechanism and/or protein tertiary structure, can be substituted by diverse amino acids (9). These data suggest active sites are highly plastic and can accommodate numerous amino acid substitutions without compromising protein function and viability of the organism. To determine whether substitutions within Motif A can affect the fidelity of DNA synthesis, we analyzed all 291 selected Taq pol I for the ability to incorporate noncomplementary nucleotides. Briefly, this analysis involved incubation of Taq pol I with 32P-labeled oligonucleotide primer·templates in the absence of the next complementary nucleotide. Products were separated by polyacrylamide gel electrophoresis and analyzed by autoradiography. Primer elongation in the absence of 1 or 2 correct dNTPs provides an effective initial screen for mutant enzymes with alterations in the fidelity of DNA synthesis (29, 30, 33).
Each of the 291 selected active Taq pol Is, including
27 with wild-type amino acid sequence, was analyzed for the
distribution of products synthesized in at least 10 different
conditions (with combinations of 3 and 2 nucleotides using two separate
primer·template constructs). Representative results with purified
mutants (3 polymerases with low fidelity, 1 with high fidelity, 1 with
normal fidelity, and WT Taq pol I) are shown in Fig.
2. In the presence of four dNTPs, all
polymerases efficiently extend the primer to a position opposite the 5'
template terminus; this result is consistent with the ability of the
selected polymerases to complement E. coli polA12 strain and
function in vivo. In the absence of a single dNTP, WT
Taq pol I can misincorporate nucleotides and extend a mismatched primer to a limited extent. Both mismatch formation and
subsequent extension are less efficient at repetitive template sequences of single nucleotides (i.e. positions at which
Taq pol I must misincorporate opposite at least two
successive identical nucleotides). Similar results were obtained for
all 27 WT Taq pols containing silent mutations. A diverse
distribution of elongation products was observed for the active
mutants. Seven mutants consistently misincorporated and/or misextended
bases at a higher frequency and synthesized longer products relative to
WT Taq pol I in most conditions tested in which
complementary nucleotides were lacking and thus are presumed to exhibit
low fidelity. These low fidelity mutants can copy past sites at which
the complementary nucleotide is missing, whereas the WT enzyme pauses
at these sites. Several other polymerases synthesized a distribution of
products significantly shorter than the WT Taq pol I
(e.g. mutant 212) and thus potentially exhibit high
fidelity.
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All low fidelity mutants contain amino acid substitutions at a single position, Ile-614. This Ile-614 residue can be replaced with amino acids that differ in size, charge, shape, and hydrophilicity while maintaining near WT activity. Diverse amino acid substitutions at position 614, occurring alone or in concert with secondary substitutions, confer low fidelity (Fig. 2 and Table I). In contrast, a putative high fidelity polymerase with six substitutions, mutant 212 (L605R/L606M/V607K/A608S/L609I/S612R), does not contain a substitution at position 614.
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Measurement of Polymerase Fidelity--
DNA polymerase fidelity is
conferred by 1) the ability of the active site to incorporate the
proper templated nucleotide relative to noncomplementary nucleotides,
and 2) the ability to extend a properly matched relative to a
mismatched 3' primer termini (12). To investigate the efficiency of
misinsertion, we measured the kinetics of incorporation of a single
complementary and noncomplementary nucleotide using WT and mutant
Taq polymerases. Efficiency of nucleotide misinsertion was
determined opposite a DNA template dT residue primed with a
23-nucleotide oligomer that was labeled at the 5'-end with
32P. Apparent Michaelis constant (Km),
apparent maximum velocity (Vmax), and
relative insertion frequency were measured for each dNTP (Table
II). Each of the mutant polymerases
incorporates the complementary nucleotide with a maximum velocity and
catalytic efficiency
(Vmax/Km) similar to that of
the WT. These enzymes formed base pairs opposite template dT in the
following order: A:T G:T > T:T > C:T. The
catalytic efficiency for misincorporation of each of the
noncomplementary nucleotides by mutant 53 (I614K) is ~10 times
greater than that of the WT enzyme. This propensity by mutant 53 (I614K) to misincorporate nucleotides is largely due to lower
Km for improperly base paired dNTPs relative to the
WT enzyme. Mutant 346 (A608D/E615D) exhibits a "mixed" fidelity, with a high propensity to catalyze some mispairs
(e.g. dT:dC primer:template) and a lower tendency to
catalyze other errors. In contrast, mutant 212, containing 6 substitutions within the catalytic site, appears less error-prone
relative to WT enzyme. Mutant 212 (L605R/L606M/V607K/A608S/L609I/S612R)
catalyzes transversion errors by forming base pairs T:C or T:T five
times less efficiently relative to WT enzyme. At an independent site,
mutant 265 (I614N/L616I) also incorporates the complementary nucleotide
at a catalytic efficiency similar to that of the WT enzyme, and this
mutant introduces transition errors at >10-fold higher efficiency
relative to WT Taq pol I (Table
III). These results indicate that
hydrophilic substitution at position Ile-614 confers low base pairing
fidelity.
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To determine the efficiency of mispair extension, we constructed a series of primer(24-mer)·templates, each containing a 3' terminal A:T, G:T, C:T, or T:T base pairs, and measured the frequency of extension at increasing concentration of the next correct dNTP (dTTP) in incubations containing limiting amounts of WT or mutant enzymes. All plots of velocity relative to substrate (dTTP) concentration exhibited saturation kinetics. WT Taq pol I efficiently extended 3' matched and mismatched primer·template termini in the following order: A:T > G:T >C:T >T:T (Table IV). The Vmax values for misincorporations and misextensions for specific base pair by the WT enzyme are very similar (except T:T); in contrast, the Km values for misincorporation by the WT enzyme are significantly greater than that for misextension of specific base pairs. Mutant 53 (I614K) is up to 50 times more efficient at extending transversion errors relative to the WT Taq pol I. Mutant 346 (A608D/E615D) is especially adept at extending C:T transversions, whereas mutant 212 (L605R/L606M/V607K/A608S/L609I/S612R), the putative high fidelity mutant, extends C:T errors at a 10-fold lower frequency relative to the WT enzyme. The high misincorporation and misextension efficiencies of mutant 53 (I614K) indicate a 10-50 times lower fidelity relative to the WT enzyme. Furthermore, the kinetic data indicate mutant 53 (I614K) Taq pol I exhibits an elevated propensity to catalyze transition as well as transversion mutations.
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Template Bypass--
These low fidelity mutant Taq pol
Is misinsert and misextend nucleotides at efficiencies comparable to
known recently discovered polymerases, including those in the
UmuC group, which typically misinsert 1 per 1000 bases (34, 35). The
polymerases in this newly discovered group are characterized by
relatively low specific activity on normal templates but robust bypass
activity on templates with damaged bases. To determine whether the low
fidelity Taq polymerase can also bypass damaged templates,
polymerization across template containing either an abasic site or
vinyl chloride alkylation product ethenoA was studied (Fig.
3). In these reactions, the primer·templates were constructed such that the primer 3' terminus was 7 nucleotides from the damaged template site, and efficient bypass
of the lesion would be accompanied by addition of 5 complementary nucleotides past the site of damage. WT Taq pol I and
purified mutants were able to efficiently synthesize across undamaged
templates. With the WT enzyme, significant pausing was observed
opposite abasic residue in the template, and a nearly complete block
was observed at the site of ethenoA lesion. In contrast, mutant
Taq pol Is containing a hydrophilic substitution at position
614 exhibited either a reduction in pausing or no pausing at the abasic
site. Mutant 265 (I614N/L616I) exhibited no pausing at the abasic site and was able to bypass ethenoA lesion. Mutants 212 (L605R/L606M/V607K/A608S/L609I/S612R) and 75 (I614M) did not carry out
synthesis across from abasic sites or ethenoA adducts in identical
control incubations. These data suggest, at least with the mutants
tested, that there is a direct relationship between fidelity and the
ability to bypass template lesions.
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Error Spectrum--
The evolution of highly active polymerases
with low fidelity suggests that subtle alterations within the DNA
polymerase catalytic site can lead to enhanced error rates during DNA
synthesis. In vitro iterative replication by such a
polymerase could be useful in generating libraries containing multiple
mutations. We carried out repetitive replication by WT and mutant
Taq pol Is utilizing the 1.3-kb human thy gene
encoding thymidylate synthase as a template. Both WT and I614K were
able to amplify low amounts of the starting material to generate
abundant levels of a 1.3-kb PCR product, which was subsequently excised
from an agarose gel, purified, and cloned. A stretch of 630 bases of
thy gene was sequenced from randomly picked clones, and the
error rate was determined. WT Taq pol I exhibited an error
rate of 3.3 × 105. Mutant 53 (I614K)
exhibited an error rate of 8.0 × 10
4,
or >20-fold higher error rate during PCR relative to the WT enzyme.
Following PCR with wild-type or mutant 346 (A608D/E615D), most clones
lack any changes in DNA sequence. In contrast, clones synthesized by
mutant 53 (I614K) contained multiple single base substitutions, with an
average of 2-3 base changes per clone (Fig. 4).
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An analysis of the mutation spectrum by WT, mutant 346 (A608D/E615D),
and mutant 53 (I614K) shows that WT Taq pol I and mutant 346 are especially adept at producing the transition error A:T G:C,
whereas mutant I614K makes a significant number of the other transition
error G:C
A:T, as well as transversion error A:T
T:A (Table
V). The error rate by mutant 75, which
contains a nonhydrophilic substitution (I615M) and the WT enzyme are
similar, although their error spectrum differ (Table V). The error
spectrum of WT Taq pol I is consistent with published
results, which show that WT Taq pol I predominantly
catalyzed T
C transition errors (36), resulting from efficient
dGTP:dT (incoming nucleotide:template) mismatch (Table II).
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DISCUSSION |
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The accuracy of DNA replication is crucial for maintaining genomic stability from one generation to the next (37, 38). Nevertheless, in times of crisis, it is beneficial for cells to exhibit diversity and thus mutate at higher rates. The fidelity of DNA replication is largely determined at the DNA polymerase active site, which is responsible for 5-6 orders of magnitude of the overall mutation rate of cells (11). Thus far, the majority of the bacteria populations that mutate at high rates, which have been investigated, contain loss of specific DNA repair pathways (39). Studies with mutant DNA polymerases have mainly focused on analyzing the effects after the loss of 3'-5' exonucleolytic proofreading activity. E. coli and yeast harboring DNA polymerases with loss of exonucleolytic activity exhibit a 10-fold elevated mutation rate (14, 40). Development of additional mutants, particularly those containing substitutions within the active site of polymerases, coupled with high resolution crystal structures, should advance our understanding of the determinants of polymerase accuracy, as well as facilitating studies of the phenotypes associated with mutated polymerases exhibiting poor fidelity. However, current structure-based site-directed mutagenesis studies have not been successful at producing mutant polymerases with WT-like activity that exhibit low fidelity.
We have found, following random mutagenesis of a portion of the
polymerase active site and stringent selection, a single amino acid
residue (Ile-614) that, when substituted to a variety of hydrophilic
amino acids, reduces the fidelity by at least 10-fold. No other amino
acid within Motif A, when substituted, consistently exhibited such low
base pairing fidelity. In addition, nonhydrophilic substitutions,
including mutant I614M, did not alter the error rate during DNA
synthesis (Table V). In reactions containing 3 nucleotides (Fig. 2),
mutants containing substitutions at Ile-614 are able to misincorporate
a base opposite the template position for which there is not a
complementary dNTP. In addition, these mutant polymerases can also
extend nascent primers containing mismatched DNA termini more
efficiently than can WT Taq pol I. The ability for
polymerases containing hydrophilic substitutions at position 614 to
efficiently catalyze misincorporation was tested kinetically. Mutant
I614K was shown to misincorporate nucleotides 10-fold more efficiently
relative to WT enzyme; in addition, kinetic experiments showed that
I614K mutant is also efficient at forming transversion errors by
misextending pyrimidine-pyrimidine base pairs at higher rates relative
to WT. These kinetic data suggest the mutant I614K Taq pol I
should produce both transition and transversion errors, and WT
Taq pol I and mutant 53 (I614K) should exhibit unique error
spectrums following DNA replication of a specific sequence. We tested
these predictions by conducting PCR amplification of a homogeneous
sequence and measured the spectrum of mutations produced by mutant 53 (I614K). The results show that whereas WT Taq pol I and
mutant 346 (A608D/E615D) contain very similar distribution of errors
and that these errors mirror the published spectrum of WT
Taq pol I (36), mutant 53 (I614K) exhibits markedly elevated
transversion errors, especially A:T T:A and an error rate that is
20 times higher than WT Taq pol I. Interestingly, mutation spectrum of WT Taq pol I under mutagenic conditions
in the presence of Mn2+ resembles that of mutant 53 (I614K)
under normal conditions, although the error rate of mutant 53 is higher
than that of Mn2+ catalyzed WT
enzyme.3
The fidelity of mutant Taq polymerase containing a hydrophilic substitution at Ile-614 is comparable to UmuC class of polymerase (34, 35). This class of polymerases contains members with modest DNA polymerase activity, yet these polymerases are particularly adept at bypassing template lesions (for reviews, see Refs. 41 and 42). We find hydrophilic substitutions at Ile-614 result in highly active DNA polymerases that can also bypass damaged templates (abasic site and replication blocking vinyl chloride adduct ethenoA). In addition, hydrophilic substitutions at Ile-614 facilitate the incorporation of bulky fluorescent nucleotide analogs,3 and a wide variety of hydrophobic and hydrophilic substitutions at Ile-614 allow successive rNTP incorporation and synthesis of RNA (49). The low fidelity, the ability to bypass template lesions, and an error spectrum that parallels incubations in the presence of manganese suggest that hydrophilic substitutions for Ile-614 should lead to a "wider" active site.
Amino acid Ile-614 is located in a highly conserved DYSQIELR Motif A
sequence; this Ile residue is maintained in large majority of
prokaryotic pol I class of enzymes (Fig. 1). Interestingly, the Motif A
nucleotide sequence is highly conserved within individual prokaryotic
species of diverse genera (e.g. Thermus,
Rickettsia, and Mycobacteria). High resolution x-ray
crystal structure of Taq pol I complexed with DNA and an
incoming nucleotide triphosphate suggests that three hydrophobic amino
acids (Ile-614, Phe-667, and Tyr-671) pack near the ribose and base
portions of the incoming nucleotide (Fig.
5). Substitution of the homologous Tyr to
a nonplanar amino acid within E. coli pol I (23, 24) and
mammalian DNA pol (43) yields enzymes with 10-fold lower fidelity
(and reduced catalytic activity). In the closed Taq pol
I-DNA-ddNTP ternary form of the structure, this residue is not
in contact with the nucleotide, but instead hydrogen bonds to Glu-615.
In the open ternary form, this Tyr residue occupies the site of the
template base, opposing the incoming nucleotide and hydrogen bonded to it. Substitutions of Phe-667 to Tyr within Taq pol I yields
active enzymes capable of incorporating dideoxynucleotides (44). In the
closed form of Taq pol I, Phe-667 is >3.7 Å from the base, but in the open form, this residue packs near the ribose, the base, and
the middle phosphate oxygen of the incoming nucleotide. Ile-614 packs
against the ribose ring and the other free oxygen of the middle
phosphate in both the closed and open forms of the Taq pol
I-DNA-ddNTP ternary structure (21). We propose that diverse
substitutions for Ile-614, and especially hydrophilic substitutions,
lead to a more "open" pocket that can accommodate damaged
templates, non-Watson-Crick base pairs, and diverse nucleotide analogs.
This model proposes that stable stacking/packing interactions with the
base and ribose rings are crucial for polymerase fidelity and is
consistent with a model for nucleotide incorporation proposed for HIV-1
reverse transcriptase (45) and other polymerases (46).
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In summary, we have evolved a set of polymerases containing
substitutions at a single amino acid with low base pairing fidelity, the ability to bypass template lesions, and the ability to
incorporate nucleotide analogs. Substitution of Ile-614, an amino acid
that is structurally conserved in all DNA polymerases, produces
active DNA polymerase with very broad substrate specificity. These
findings are consistent with models of adaptive evolution that 1) in
times of stress, the inherent plasticity of enzyme active site
facilitates the generation of beneficial mutants with altered substrate
specificity, which could provide a selective advantage, and 2)
following successful survival through periods of adverse conditions, WT
nucleotide sequence (one that is fit and the most prevalent) can be
generated through recombination-mediated lateral transfer (9). This
study suggests that other populations of mutators may contain
substitutions within the polymerase catalytic site that confer low
fidelity. Considering the vital role of DNA polymerases during DNA
replication, repair, and recombination, it may be important to genotype
tumors characterized by elevated mutation rates for polymorphic
differences within the polymerase catalytic site.
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FOOTNOTES |
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* This work was supported by Medical Scientist Training Program Grant NIH NIGMS5T3207266 and Molecular Training Program in Cancer Research Grant CA09437 (to P. H. P.) and by National Cancer Institute Grants R35 CA39903 and CA78885 (to L. A. L.).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.: 206-543-6015;
Fax: 206-543-3967; E-mail: laloeb@u.washington.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008701200
1 B. Preston, personal communication.
3 P. H. Patel, H. Kawate, E. Adman, M. Ashbach, and L. A. Loeb, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: pol I, polymerase I; Taq, Thermus aquaticus; WT, wild-type; PCR, polymerase chain reaction; kb, kilobase(s).
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Welch, D. M.,
and Meselson, M.
(2000)
Science
288,
1211-1215 |
2. |
Radman, M.,
Matic, I.,
and Taddei, F.
(1999)
Ann. N. Y. Acad. Sci.
870,
146-155 |
3. | Mao, E. F., Lane, L., Lee, J., and Miller, J. H. (1997) J. Bacteriol. 179, 417-422[Abstract] |
4. |
Oliver, A.,
Canton, R.,
Campo, P.,
Baquero, F.,
and Blazquez, J.
(2000)
Science
288,
1251-1254 |
5. |
LeClerc, J. E.,
Li, B.,
Payne, W. L.,
and Cebula, T. A.
(1996)
Science
274,
1208-1211 |
6. | Loeb, L. A. (1996) in Genetic Instability in Cancer (Lindahl, T., ed), Vol. 28 , pp. 329-342, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
7. | Kornberg, A., and Baker, T. (1992) DNA Replication , 2nd ed , W. H. Freeman and Co., New York |
8. | Lindahl, T., and Nyberg, B. (1972) Biochemistry 11, 3610-3618[Medline] [Order article via Infotrieve] |
9. |
Patel, P. H.,
and Loeb, L. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5095-5100 |
10. | Kunkel, T. A., and Loeb, L. A. (1981) Science 213, 765-767[Medline] [Order article via Infotrieve] |
11. | Johnson, K. A. (1993) Annu. Rev. Biochem. 62, 685-713[CrossRef][Medline] [Order article via Infotrieve] |
12. | Perrino, F. W., Preston, B. D., Sandell, L. L., and Loeb, L. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8343-8347[Abstract] |
13. | Drake, J. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7160-7164[Abstract] |
14. | Echols, H., Lu, C., and Burgers, P. M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2189-2192[Abstract] |
15. |
Modrich, P.
(1989)
J. Biol. Chem.
264,
6597-6600 |
16. | Fishel, R., Lescoe, M. K., Rao, M. R. S., Copeland, N. G., Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R. (1993) Cell 75, 1027-1038[Medline] [Order article via Infotrieve] |
17. | Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G., Smith, L. G., Lescoe, M. K., Kane, M., Earabino, C., Lipford, J., Lindblom, A., Tannergard, P., R. J., B., Godwin, A. R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., and Liskay, R. M. (1994) Nature 368, 258-261[CrossRef][Medline] [Order article via Infotrieve] |
18. | Reitmair, A. H., Schmits, R., Ewel, A., Bapat, B., Redston, M., Mitri, A., Waterhouse, P., Mittrucker, H. W., Wakeham, A., Liu, B., Thomason, A., Griesser, H., Gallinger, S., Ballhausen, W. G., Fishel, R., and Mak, T. W. (1995) Nat. Genet. 11, 64-70[Medline] [Order article via Infotrieve] |
19. | Beese, L. S., Derbyshire, V., and Steitz, T. A. (1993) Science 260, 352-355[Medline] [Order article via Infotrieve] |
20. | Doublie, S., Tabor, S., Long, A. M., Richardson, C. C., and Ellenberger, T. (1998) Nature 391, 251-258[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Li, Y.,
Korolev, S.,
and Waksman, G.
(1998)
EMBO J.
17,
7514-7525 |
22. | Patel, P. H., and Preston, B. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 549-553[Abstract] |
23. | Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804-813[Medline] [Order article via Infotrieve] |
24. |
Joyce, C. M.,
Sun, X. C.,
and Grindley, N. D.
(1992)
J. Biol. Chem.
267,
24485-24500 |
25. | Desai, S. D., Pandey, V. N., and Modak, M. J. (1994) Biochemistry 11868-11874 |
26. | Delarue, M., Poch, O., Tordo, N., Moras, D., and Argos, P. (1990) Protein Eng. 3, 461-467[Abstract] |
27. | Wang, J., Sattar, A. K., Wang, C. C., Karam, J. D., Konigsberg, W. H., and Steitz, T. A. (1997) Cell 89, 1087-1099[Medline] [Order article via Infotrieve] |
28. | Grimm, E., and Arbuthnot, P. (1995) Nucleic Acids Res. 23, 4518-4519[Medline] [Order article via Infotrieve] |
29. | Preston, B. D., Poiesz, B. J., and Loeb, L. A. (1988) Science 242, 1168-1171[Medline] [Order article via Infotrieve] |
30. |
Suzuki, M.,
Avicola, A. K.,
Hood, L.,
and Loeb, L. A.
(1997)
J. Biol. Chem.
272,
11228-11235 |
31. | Engelke, D. R., Krikos, A., Bruck, M. E., and Ginsburg, D. (1990) Anal. Biochem. 191, 396-400[Medline] [Order article via Infotrieve] |
32. |
Boosalis, M. S.,
Petruska, J.,
and Goodman, M. F.
(1987)
J. Biol. Chem.
262,
14689-14699 |
33. | Kim, B., Hathaway, T. R., and Loeb, L. A. (1998) Biochemistry 37, 5831-5839[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Johnson, R. E.,
Washington, M. T.,
Prakash, S.,
and Prakash, L.
(2000)
J. Biol. Chem.
275,
7447-7450 |
35. |
Tang, M.,
Shen, X.,
Frank, E. G.,
O'Donnell, M.,
Woodgate, R.,
and Goodman, M. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
8919-8924 |
36. | Tindall, K. R., and Kunkel, T. A. (1988) Biochemistry 27, 6008-6013[Medline] [Order article via Infotrieve] |
37. | Echols, H. (1982) Biochimie (Paris) 64, 571-575[Medline] [Order article via Infotrieve] |
38. | Loeb, L. A., and Kunkel, T. A. (1982) Annu. Rev. Biochem. 51, 429-457[CrossRef][Medline] [Order article via Infotrieve] |
39. | Miller, J. H. (1998) Mutat. Res. 409, 99-106[Medline] [Order article via Infotrieve] |
40. |
Schaaper, R. M.
(1989)
Genetics
121,
205-212 |
41. | Friedberg, E. C., and Gerlach, V. L. (1999) Cell 98, 413-416[Medline] [Order article via Infotrieve] |
42. | Goodman, M. F. (2000) Trends Biochem Sci 25, 189-195[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Dong, Q.,
Copeland, W. C.,
and Wang, T. S.
(1993)
J. Biol. Chem.
268,
24163-24174 |
44. | Tabor, S., and Richardson, C. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6339-6343[Abstract] |
45. | Patel, P. H., Jacobo-Molina, A., Ding, J., Tantillo, C., Clark, A. D., Jr., Raag, R., Nanni, R. G., Hughes, S. H., and Arnold, E. (1995) Biochemistry 34, 5351-5363[Medline] [Order article via Infotrieve] |
46. |
Goodman, M. F.,
and Fygenson, K. D.
(1998)
Genetics
148,
1475-1482 |
47. | Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
48. | Merritt, E. A., and Murphy, M. E. P. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 869-873[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Patel, P. H.,
and Loeb, L. A.
(2000)
J. Biol. Chem.
275,
40266-40272 |