From the Laboratory of Molecular Genetics, NIEHS,
National Institutes of Health,
Research Triangle Park, North Carolina 27709 and the
¶ Department of Molecular Biophysics and Biochemistry, Yale
University, New Haven, Connecticut 06520
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ABSTRACT |
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To investigate the interactions that determine
DNA polymerase accuracy, we have measured the fidelity of 26 mutants
with amino acid substitutions in the polymerase domain of a
3'-5'-exonuclease-deficient Klenow fragment. Most of these mutant
polymerases synthesized DNA with an apparent fidelity similar to that
of the wild-type control, suggesting that fidelity at the polymerase
active site depends on highly specific enzyme-substrate interactions
and is not easily perturbed. In addition to the previously studied
Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side
chain located on the exposed surface of the polymerase cleft near the
polymerase active site. Analysis of base substitution errors at four
template positions indicated that each of the five mutator
polymerases has its own characteristic error specificity, suggesting
that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains
may contribute to polymerase fidelity in a variety of different
ways. We separated the contributions of the nucleotide insertion and
mismatch extension steps by using a novel fidelity assay that scores
base substitution errors during synthesis to fill a single nucleotide
gap (and hence does not require mismatch extension) and by measuring
the rates of polymerase-catalyzed mismatch extension reactions. The
R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at
the nucleotide insertion step, whereas R668A results in lower fidelity
in both nucleotide insertion and mismatch extension. Relative to wild
type, several Klenow fragment mutants showed substantially more
discrimination against extension of a T·G mismatch under the
conditions of the fidelity assay, providing one explanation for the
anti-mutator phenotypes of mutants such as R754A and Q849A.
Three steps are responsible for the high fidelity of DNA
replication; they are nucleotide selectivity and exonucleolytic
proofreading of errors by the polymerase and post-synthetic correction
of mismatches. Among these, the DNA polymerase itself usually provides
the greatest contribution to fidelity, producing on average only one
substitution error for every 104 to 105
nucleotides incorporated, depending on the polymerase (1). Polymerase
errors can be initiated either by misinsertion of a nucleotide or by
misalignment of the template-primer. Either initiating event can
ultimately result in base pair substitution, deletion, or addition.
Using as a model system the extremely well characterized Klenow
fragment of Escherichia coli DNA polymerase I, our approach for understanding the structural basis for polymerase fidelity has been
to screen a battery of amino acid substitutions directed to the active
site region, including side chains thought to be involved in nucleotide
binding and interactions with primer and template.
Polymerases from different families share fundamental similarities in
tertiary structure and catalytic mechanism (reviewed in Refs. 2-5).
The polymerase domains resemble a partially open right hand, with a
cleft formed by three subdomains that have been designated fingers,
palm, and thumb. The palm subdomain, which forms the base of the
polymerase cleft, contains important active site residues, in
particular the cluster of carboxylate side chains that coordinate the
pair of divalent metal ions that catalyze the polymerase reaction (6).
The fingers subdomain also contributes side chains to the active site,
especially those involved in nucleotide recognition (7, 8). The thumb
subdomain interacts with the template-primer duplex upstream of the
site of nucleotide addition (8-11). Many of the amino acid side chains that form the exposed surface of the polymerase cleft are highly conserved in their respective polymerase families.
Although the only structural information for Klenow fragment with
duplex DNA is an editing complex with the DNA primer terminus at the
3'-5'-exonuclease active site (12), co-crystal structures with DNA at
or close to the polymerase active site have been reported for other
polymerases of the pol I1
family. Binary polymerase·DNA complexes have been described for the
DNA polymerase I of Thermus aquaticus (Taq DNA
polymerase) (10) and for the Klenow fragment analog derived from
Bacillus stearothermophilus DNA polymerase I (11), and a
ternary complex (polymerase·DNA·ddNTP) has been solved with the DNA
polymerase of bacteriophage T7 (8). Because of the high degree of
structural and sequence similarity, information from these three
co-crystals can be used to interpret data obtained with Klenow fragment mutants.
Klenow fragment catalyzes DNA synthesis with high fidelity, having an
average base substitution error rate of Materials
Strains and Reagents--
Bacterial strains and reagents for
polymerase fidelity measurements have been described (18).
Oligonucleotides--
DNA oligonucleotides were from Research
Genetics. The 20-mer primers, 5'-GTAACGCCAGGGTTTTCTCA and
5'-GTAACGCCAGGGTTTTCTCG, when annealed to the 33-mer template,
(5')ACGTCGTGACTGAGAAAACCCTGGCGTTACCCA, give a correct (T·A) or an
incorrect (T·G) base pair, respectively, at the primer terminus. The
template sequence corresponds to the region surrounding the opal codon
introduced at positions 87-89 of the lacZ Mutant Polymerases--
Mutant derivatives of Klenow fragment
were constructed and purified as described (19, 20). Biochemical
properties of the majority of these proteins have been reported
previously (7, 15, 19, 21, 22); exceptions are N579A, S582A, S608A,
T609A, R631A, K635A, S670A, N675A, Q677A, N678A, S707A, and R835L. All polymerases in this study carried the D424A mutation, which eliminates the 3'-5'-exonuclease activity (23).
Methods
DNA Synthesis Reactions--
Reactions (25 µl) were performed
as described previously (18), with 150 ng of gapped M13mp2 DNA in 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol, and 1 mM each of dATP, dTTP, dGTP, and dCTP. Wild-type or mutant polymerase was added to initiate synthesis. The amounts (in picomoles) of each enzyme needed to fill the
gap completely, determined by electrophoresis of a portion of the
reaction on a 0.8% agarose gel, were as follows: wild-type (D424A),
0.17; N579A, 0.2; S582A, 0.53; S608A, 2; T609A, 0.5; R631A, 1.2; K635A,
6.8; R668A, 7.2; S670A, 0.4; N675A, 0.87; Q677A, 40; N678A, 0.88;
R682A, 0.48; S707A, 3.3; Q708A, 0.94; E710A, 20; H734A, 0.43; R754A,
1.6; K758A, 10; F762A, 0.95; Y766A, 9.6; R835L, 0.4; R841A, 1.3; N845A,
0.2; Q849A, 8.2; H881A, 0.49; and E883A, 1.8. Reactions were terminated
by adding EDTA to a final concentration of 15 mM. All
reactions whose products were analyzed for lacZ mutant
frequency yielded DNA that migrated with double-stranded nicked
circular molecules in an agarose gel (data not shown).
Fidelity Assays--
Reaction products were transfected into
E. coli and plated to score the color of M13 plaques, as
described (18). For the forward mutation assay, correct synthesis
yields blue plaques, whereas a variety of polymerization errors are
scored as light blue or colorless plaque phenotypes. Three reversion
substrates were also used, with errors scored as blue revertants of a
colorless plaque phenotype. Base substitutions were scored for eight of nine possible mispairs at a TGA codon at positions 87-89 in the lacZ Single Nucleotide (TG"A") Gap Fidelity Assay--
An M13mp2
substrate having a single nucleotide gap opposite the template A of a
TGA codon artificially introduced within the lacZ Measurement of Polymerase-catalyzed Terminal Mismatch
Extension--
The oligonucleotides described above were annealed so
as to give a 3'-terminal G opposite a template T (mismatch substrate) and a 3'-terminal A opposite the same template T (control substrate). In either case the primer oligonucleotide was 5'-labeled with 32P. Polymerase ( Mutant Derivatives of Klenow Fragment--
The Klenow fragment
mutations that we studied are indicated in Fig.
1. Wild-type residues were replaced by
Ala (or, in one case, Leu) in order to remove potentially important
side chain interactions. The majority of these 28 side chains are
highly conserved in the pol I family of polymerases (Fig.
2) and all reside on or near the exposed
surface of the cleft in the polymerase domain (Fig. 1). Most of the
mutant proteins came from earlier studies on the role of side chains
involved in catalysis and nucleotide binding within the polymerase
active site region on the palm and fingers subdomains (7, 19, 21).
Additional mutations were made in side chains, primarily in the thumb
subdomain, that were identified as contacts to the DNA duplex in the
Klenow fragment·DNA editing complex (12). In order to focus on
fidelity-determining processes at the polymerase active site without
complications due to exonucleolytic proofreading, all Klenow fragment
derivatives in this study carried the 3'-5'-exonuclease-deficient D424A
mutation (23). For simplicity, each protein is described by the
genotype of the polymerase domain; thus the D424A control is referred
to as wild-type, and E710A denotes the double mutation D424A,E710A.
Forward Mutation Assay--
We first measured the frequency of
errors produced by each mutant polymerase during gap-filling synthesis
across a lacZ target in phage M13mp2 (Fig. 2). The wild-type
polymerase gave lacZ mutants at a frequency of 57 × 10 Reversion Assays--
Because the forward mutation assay detects
many different types of errors in a variety of sequence contexts,
substantial changes in particular subsets of errors may not be apparent
unless large numbers of lacZ mutants are sequenced. For
example, although the forward mutant frequency for the Y766A derivative
was increased only 3.7-fold overall (Fig. 2), a detailed analysis of
error specificity in the lacZ forward mutant assay indicated
that this enzyme exhibited a 60-fold increase in misincorporation of
dTMP opposite template G and greater than 10-fold increases in several
other base substitution and deletion errors (16). In the present study
of 26 mutant polymerases, it was clearly impractical to sequence large
collections of lacZ mutants generated by each enzyme
(although this was done for a few enzymes, see legend to Fig. 2). We
therefore decided to use a series of reversion assays that monitor base
substitution or frameshift errors at specific template positions. These
assays, which are highly sensitive due to low background mutation
frequencies, were used to examine a number of different Klenow fragment
derivatives, including those having forward mutant frequencies greater
than the wild-type value.
Base Substitution Reversion--
The first reversion substrate was
an M13mp2 DNA with a 361-base gap containing a TGA codon at position
87-89 in the lacZ
The average reversion frequency obtained in the TGA89 assay
for the wild-type Klenow fragment was 17 × 10
The DNA sequences of revertants generated by the four new mutator
polymerases were compared with those generated by the wild-type enzyme,
in order to determine error rates for each of the eight detectable
mispairs at the TGA codon (Table II).
Remarkably, 127 of 136 revertants recovered after gap-filling by the
E710A mutant polymerase had the dark blue plaque phenotype
characteristic of misinsertion of dCMP opposite template adenine,
making the E710A polymerase a 70-fold mutator for this particular error
at the opal codon site. Revertants produced by the R668A, R682A, and N845A mutant polymerases, on the other hand, gave predominantly light blue plaques and were characterized further by DNA sequencing (see below).
Detection of a base substitution error in a gap-filling assay requires
two steps, insertion of an incorrect nucleotide and extension of the
resulting mispaired primer terminus. Difficulty in extending particular
mispairs will lead to their underrepresentation in the spectrum of
mutations recovered from the assay. Moreover, if a mutant polymerase is
more discriminating than the wild-type enzyme in mismatch extension
under the assay conditions (as are several of the Klenow fragment
mutants we have studied (see below)), then this may either result in an
anti-mutator phenotype or limit the ability to detect reduced fidelity
in the insertion step. This reasoning could certainly apply to the
R754A and Q849A derivatives, which behaved as anti-mutators in both the
forward mutation assay and the TGA89 reversion assay (Fig.
2 and Table I). To separate the effects of nucleotide insertion
selectivity and mismatch extension, we developed the TG"A"
reversion substrate that requires only insertion of a single nucleotide
and does not depend on mismatch extension to score an error. With this
substrate, all three possible misinsertions opposite the template A in
the gap are scored as revertants. In the TG"A" assay, wild-type
Klenow fragment gave an average reversion frequency of 50 × 10
Sequence analysis of the revertants obtained from the four newly
identified mutator polymerases showed that each mutator has its own
characteristic error specificity and that the results of the
TGA89 and TG"A" assays can differ in informative ways
(Table II). As indicated above, A·dCTP errors dominated the
revertants generated by the E710A protein; this contrasts with
wild-type Klenow fragment and the other mutator derivatives, which make predominantly T·dGTP errors. From the small number of light blue plaques obtained with E710A in the TGA89 assay, we can
infer that the frequency of T·dGTP errors must actually be lower than
the wild-type value. The R668A, R682A, and N845A mutators all gave
The errors recovered opposite the template A positions in the two
reversion assays gave information about misinsertion specificity and,
by comparing the two assays, about mispair extension. Thus, N845A was a
stronger mutator in the TG"A" single nucleotide insertion assay
than in the TGA89 assay, because this enzyme tends to make A·dATP insertion errors (
The two anti-mutators examined in the single nucleotide assay gave
slightly different results from one another. The reversion frequency
obtained with the R754A polymerase in the TG"A" assay was similar
to the value for the wild-type enzyme, suggesting that the anti-mutator
phenotype of this protein in the gap-filling assays is due to increased
discrimination at the mismatch extension step. The reversion frequency
obtained with the Q849A derivative was about 3-fold lower than the
wild-type value in the TG"A" assay, whereas the value in the
TGA89 gap-filling assay was barely above the assay
background (Table I). These results suggest that the anti-mutator
phenotype of Q849A is largely determined by mismatch extension,
although there may also be a small contribution from increased
selectivity at the misinsertion step.
Frameshift Reversion Substrates--
We examined some of the
mutant Klenow fragment derivatives for single base frameshift fidelity,
using a pair of frameshift reversion substrates containing a run of 5 template thymidine residues in the lacZ Polymerase-catalyzed Terminal Mismatch Extension--
To
understand further the contribution of mismatch extension to the error
rates obtained in our experiments, we compared the single turnover
rates for nucleotide addition to a correctly paired (T·A) and a
mispaired (T·G) primer terminus by a subset of the mutant Klenow
fragment derivatives. Reaction conditions were chosen so as to mimic
those used for primed synthesis in the fidelity assays, and high
concentrations of enzyme were used to maximize binding of the DNA
substrate. The proteins tested showed a wide range of behavior (Table
IV). Of the mutator alleles identified in
this and previous studies, two (R668A and Y766S) were less discriminating than wild-type in extension of the T·G mispair, one
(N845A) was similar to wild type, and three (R682A,
The processes that take place at the polymerase active site of
Klenow fragment are extremely accurate, allowing on average only one
error for every 104 to 105 nucleotides
incorporated. Which parts of the protein are responsible for
maintaining this high fidelity? Our analysis of 26 mutations in the
polymerase domain of Klenow fragment has identified four novel base
substitution mutators (R668A, R682A, E710A, and N845A) to add to the
Y766A and Y766S mutators, previously described (15, 16). It has also
identified several mutations in conserved residues that result in an
anti-mutator phenotype.
Mutations Having No Apparent Effect on Polymerase
Fidelity--
The majority of the Klenow fragment mutations in our
study had little or no observable effect on polymerase fidelity.
Although nucleotide binding and catalysis might be considered crucial
to nucleotide misinsertion, several mutations that affect these
processes (e.g. F762A, K758A, and E883A, Refs. 7 and 21) did
not influence fidelity. We also did not detect any substantial
frameshift mutators, even among polymerases having mutations in regions
that we had expected to play a role in controlling
misalignment-mediated errors.
The small number of mutator polymerases revealed by our survey suggests
that fidelity is determined by a limited number of interactions.
However, it is possible that some interesting mutator polymerases may
have escaped detection, even with the use of multiple assays, since
there are several factors that can conceal a mutator effect. One factor
is the broad error specificity of the M13mp2 forward mutation assay
used here (Fig. 2), which means that a mutator effect at a subset of
errors will be averaged over a large target and may be hard to detect
simply by measuring the forward mutant frequency. Additionally, a
mutator polymerase could increase the frequency of some errors while
decreasing the frequency of others, giving little net change in the
overall forward mutant frequency.5 The
TGA89 reversion assay addresses some of these concerns by focusing on a smaller number of errors at a smaller target. It has the
potential to amplify the signal from base substitution mutators
(compare Fig. 2 with Table I), but it will fail to detect a putative
mutator polymerase that has an error specificity that does not result
in a large number of mutations at the opal codon site. Even in the
reversion assays, frequencies are dominated by the most common errors,
so that a dramatic increase in a rare error may not be apparent until
the revertants are sequenced; consider, for example, the quantitatively
rather small contributions of the T·dCTP errors made by R682A and
N845A in the TGA89 assay and the A·dGTP errors made by
R668A in the TG"A" assay (Tables I and II).
The single nucleotide (TG"A") gap assay is an important addition to
the repertoire of fidelity assays because it does not require mispair
extension, which could limit detection of a mutator effect in the
gap-filling fidelity assays. As expected, this assay allowed a greater
recovery of errors resulting from purine·purine mispairs, which are
usually refractory to extension (28), and revealed features of the
error specificity (for both wild-type and mutant Klenow fragment
derivatives) that would not have been suspected from the results of the
two gap-filling assays.6
Anti-mutators and the Role of Mismatch Extension--
A few Klenow
fragment derivatives, particularly R754A, Q849A, and H881A, gave mutant
frequencies in the fidelity assays that were substantially lower than
the values for the wild-type enzyme. Several mutations, including these
three, result in increased discrimination (relative to wild-type)
against extension of a T·G mispair under conditions similar to those
used in the fidelity assays (Table
IV).7 Therefore,
it seems likely that the anti-mutator behavior of these proteins is due
largely to inefficient mispair extension, and this is consistent with
the data for R754A and Q849A in the opal codon reversion assay (which
requires mispair extension) and the single gap TG"A" assay (which
does not) (Table I).
Of a representative sample of 13 Klenow fragment mutants, eight were
more discriminating than wild type in T·G mispair extension (Table
IV), resulting either in an anti-mutator phenotype (R754A and Q849A) or
in diminishing the apparent magnitude of a mutator phenotype (E710A).
The only two proteins that were significantly less discriminating than
wild-type were both mutators (R668A and Y766S). Why do so many
mutations cause increased discrimination in extension of this mispair?
There is no obvious correlation with biochemical properties since the
eight mutant proteins listed in Table IV cover a wide range of
biochemical phenotypes (7, 17, 19, 21). It is also hard to know how to
view the role of a side chain whose removal makes the polymerase
more accurate. Perhaps the loss of an interaction with the
primer terminus has a more significant effect on a mismatched DNA,
which is already somewhat destabilized. This explanation could apply to
Q849A and H881A, where the mutated side chains contact the primer
terminus, but not to R754A, since Arg-754 contacts the dNTP phosphate
tail (7, 8). An alternative possibility, which does not depend on the
mutated side chains having a specific role in fidelity, is that an
apparent increase in fidelity in the mispair extension reaction may
correspond to a shift in rate-limiting step to one that is inherently
more discriminating.
Base Substitution Mutators--
From this and our previous study
(16) we have identified a total of at least five side chains (Arg-668,
Arg-682, Glu-710, Tyr-766, and Asn-845) that play an important role in
preventing base substitution errors. An additional possibility is
Asn-678, since N678A gave a small increase in mutant frequency in the
forward assay as well as marginal effects in three of the four
reversion assays (Tables I and III). The mutator Klenow fragment
derivatives have a variety of biochemical phenotypes (7, 19, 21, 22, 31); relative to wild-type Klenow fragment, R668A and E710A show
substantial decreases in reaction rate, R668A, R682A, and E710A have
decreased DNA binding affinity, and all show moderate changes in
Km(dNTP). Not only is it hard to discern
any correlation of mutator phenotype with a particular subset of
biochemical properties, but one can also find other Klenow fragment
mutants with similar biochemical properties but no mutator phenotype. The obvious attributes these five side chains have in common are that
they are all highly conserved in the pol I family and that they are
clustered very near the polymerase active site.
Each of the four base substitution mutators we describe here has its
own distinct error specificity. For E710A, the most frequent error is
the A·dCTP mispair (Table II), an error not frequently made by
wild-type Klenow fragment. By contrast, the frequency of T·dGTP
errors is reduced (relative to wild-type), presumably because of the
difficulty the E710A protein has in extending the T·G mispair (Table
IV). R668A appears to favor errors involving insertion of dGTP; R682A
and N845A both gave an increase in T·dCTP errors, and N845A also gave
an increase in A·dATP errors. (For comparison, the previously
characterized mutators with substitutions at Tyr-766 (Y766A and Y766S)
gave increases in several types of errors, particularly transition
mispairs (16).) Although these error specificities provide an
interesting basis for consideration of fidelity mechanisms, an
important caveat is that the data in Table II are derived from
mutations recovered at two small targets. Some types of errors cannot
be detected at these targets, and the possible effects of the
surrounding sequence have not been determined. A more comprehensive
description of the error specificity of the four mutator polymerases
identified in the present study must therefore await a full analysis of
lacZ mutations recovered in the forward mutational assay, as
was carried out previously for the Y766A and Y766S mutants (16).
Whereas it seems likely that the errors made by all these mutator
polymerases reflect a loss in fidelity in the insertion step, our data
provide relatively little information on the way in which mispair
extension might augment or diminish the observed error rates. The data
of Table II suggest the need for caution in generalizing the kinetic
data obtained with the T·G mispair (Table IV) to other mispairs.
Thus, the R668A mutant protein, which is the least discriminating in
T·G extension (Table IV), may be particularly bad at extending A·Pu
mispairs. Conversely, R682A discriminates more against T·G mispairs
but, perhaps, less against A·A. Given the different geometrical
constraints posed by individual mispairs, one might expect to see
mispair-specific effects in the extension reaction, and we are
currently investigating this issue.
Fidelity Mutants in Relation to Polymerase Structures--
What is
the structural basis for the decrease in fidelity resulting from
mutations at Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845?
Co-crystal data for polymerases related to Klenow fragment, especially
the ternary complex structure of T7 DNA polymerase (8), provide an
excellent starting point to explore this
issue.8 A
limitation, however, is that we can only guess at the structural changes that may take place when an incorrect incoming dNTP or a
mispaired primer terminus is bound at the polymerase site. Structural information on complexes containing mismatches will be crucial for a
full understanding of polymerase fidelity mechanisms.
Three general mechanisms (which are not mutually exclusive) have been
proposed to account for polymerase fidelity; they are water exclusion
from the active site, steric complementarity between the active site
binding pocket and a nascent Watson-Crick base pair, and hydrogen
bonding to the minor groove of the template-primer duplex and the
nascent base pair. Water exclusion provides a possible mechanism for
the polymerase to magnify the free energy difference between correct
and incorrect base pairs in the context of the enzyme active site (32).
Given that all the mutator mutants we have identified involve
substitution of a smaller side chain, they could act by allowing access
of water to the active site. To account for the observed error
specificities, however, each alanine substitution should allow water
access to a restricted and specific part of the active site so that all
mispairs are not equally affected. A mutation that allows general water
access, reducing selectivity throughout the active site, might be
expected to increase the frequency of all polymerase errors; thus far
we have not identified any mutator mutants with this phenotype.
Polymerase co-crystal structures support the idea that selectivity
could depend on a precise fit of the binding pocket to a correct
nascent base pair at the active site. In the T7 DNA polymerase ternary
complex, the incoming nucleotide is sandwiched tightly between the
primer terminus and conserved side chains on the O-helix,
with no flexibility to accommodate mismatched base pairs (8). The side
chains corresponding to our mutator mutants are clustered around this
binding region, so that replacement of one of these side chains by Ala
might compromise the tight fit of the binding site. To account for the
observed error specificity, the changes in the binding site in each
mutator polymerase must be such that binding of only a subset of
nascent mispairs is enhanced. In general terms, we can imagine how this
might work for the E710A mutant. The Glu-710 side chain is known to be
close to the 2' position of the deoxyribose of the incoming nucleotide
(8, 31). When Glu-710 is replaced by Ala, the additional space could therefore accommodate any aberrant base pair that deviates from normal
geometry in a way that would place extra bulk in this part of the
active site.
In the binary enzyme·DNA complex of B. stearothermophilus
Klenow fragment, a tight pocket is seen surrounding the terminal base
pair of the DNA duplex (11). All five of the side chains implicated by
our mutator mutants, as well as the homologs of Gln-849 and His-881,
are part of this pocket. These co-crystals are able to carry out
nucleotide addition to the bound DNA, indicating (at a minimum) that
the complex can achieve a catalytically competent conformation;
however, the relationship of the observed primer terminus position to
the position of the incoming nucleotide remains to be established.
Although many similar interactions are seen in the polymerase binary
and ternary complexes, the data for the invariant tyrosine,
corresponding to Tyr-766 of Klenow fragment, is puzzling. In both
Taq and B. stearothermophilus binary complexes with DNA, the Tyr-766 homolog is stacked against the template base at
the end of the DNA duplex (10, 11), consistent with the idea that this
side chain could play a role in template positioning (and consequently
in fidelity) (7). In the T7 DNA polymerase ternary complex, however,
the corresponding Tyr is hydrogen-bonded to the homolog of Glu-710
(8).9 An
intriguing possibility is that this side chain might be actively involved in the movement of the O-helix that appears to
accompany ternary complex formation.
It has long been recognized that polymerases should be able to
distinguish correct from incorrect base pairs by hydrogen bonding to
the minor groove of the template-primer duplex (33, 34). The positions
of the two minor groove hydrogen bond acceptors (N-3 of purines and O-2
of pyrimidines) are similar in Watson-Crick base pairs (35) but are not
the same in mismatched base pairs. In all three polymerase co-crystals
the A-like conformation of the DNA duplex close to the active site
creates a wider and shallower minor groove, facilitating interactions
of the minor groove hydrogen bond acceptors with several of the side
chains shown to be involved in fidelity in the present study (8, 10,
11). In both the T7 DNA polymerase ternary complex and the B. stearothermophilus binary complex, the side chain equivalent to
Arg-668 interacts with the acceptor on the primer strand, and the
Gln-849 homolog interacts with the acceptor on the template strand, of
the terminal base pair. The Taq DNA polymerase binary
complex is slightly different, perhaps because the primer terminus is
further from the active site; the Gln-849 homolog (as part of a
hydrogen-bonding network with residues equivalent to the Arg-668
homolog and Glu-710) interacts with the primer strand, whereas the
residue equivalent to Arg-841 forms a hydrogen-bonded network with the
acceptor on the template strand and the Asn-845 homolog. In the T7 DNA
polymerase ternary complex, the Arg-668 homolog also interacts with the
sugar of the incoming nucleotide. The interactions of Arg-668 with both primer terminus and incoming nucleotide might perhaps correlate with
the effect of the R668A mutation on both misinsertion and mispair
extension fidelity. For the other side chains discussed above, we have
to postulate that interactions with the template-primer duplex can
influence selection of the incoming dNTP. This seems intuitively
reasonable since the loss of a binding contact to template or primer
strand could allow more flexibility in positioning the primer 3'-OH and
an increased tolerance of geometrically aberrant nascent mispairs. The
side chains equivalent to Arg-682 and Tyr-766 are not involved in
hydrogen-bonding interactions with the minor groove in any of the
co-crystal structures. This does not rule out an indirect contribution
of these side chains to fidelity via this mechanism since
removal of a side chain close to the primer terminus could modify DNA
binding geometry and thus influence minor groove recognition.
In conclusion, we have found that mutations that alter base
substitution fidelity are clustered around the primer terminus at the
polymerase active site. This contrasts with mutations that affect
errors resulting from strand slippage that are located further from the
primer terminus in the duplex DNA-binding region. Based on the
available structural data for complexes of DNA polymerases of the pol I
family, plausible scenarios can be envisaged that account in general
terms for the observed mutator phenotypes. Our data do not exclude any
of the mechanisms that have previously been proposed to explain
nucleotide insertion fidelity, although the need to account for the
observed error specificities will place quite stringent demands on any
structural explanation that emerges. It is conceivable that DNA
polymerases may use a combination of strategies to ensure fidelity and
that the different mutator polymerases may act in quite distinct ways.
For example, the important attribute of the E710A mutation could be
that it diminishes steric complementarity between the active site and
the nascent base pair, whereas R668A could affect fidelity because it
removes an important hydrogen bond donor to the minor groove at the
primer terminus. Structural studies on polymerase complexes containing
mismatches should lead to a more complete understanding of polymerase
fidelity, including a full explanation for the characteristic error
specificities of the mutator polymerases described here.
INTRODUCTION
Top
Abstract
Introduction
References
2 × 10
5
and an average frameshift error rate of
5 × 10
6,
even in the absence of exonucleolytic proofreading (13). This polymerase discriminates against nucleotide misinsertion by having a
lower binding affinity for the incorrect dNTP and a slower catalytic rate for misinsertion than for correct insertion (14); although the
precise values vary from mispair to mispair, the rate discrimination is
quantitatively larger than the nucleotide binding discrimination (15).
To date, two mutation sites that influence fidelity in Klenow fragment
have been described. Substitution of Ser or Ala for Tyr-766, at the
carboxyl terminus of the long O-helix in the fingers
subdomain (see Fig. 1), results in decreased nucleotide selectivity
during polymerization (15) and a consequent increase in base
substitution errors and frameshift errors at non-reiterated sequences
(16). A Klenow fragment mutant that lacks 24 amino acids at the tip of
the thumb subdomain has decreased DNA binding affinity and processivity
and an increased rate of frameshift errors at homopolymeric sequences
(17). These properties are consistent with the interactions of the
thumb tip with the minor groove of the template-primer duplex that have
been observed crystallographically (8, 10, 12). In this study we have
screened a large number of mutations in the polymerase cleft region of
an exonuclease-deficient Klenow fragment, for effects on polymerase
fidelity, and have identified four new mutator DNA polymerases,
each with its own distinct error specificity.
EXPERIMENTAL PROCEDURES
sequence, such
that the primer terminal base is paired with the T of the TGA codon.
sequence (24). Single base deletions were detected
using a substrate containing an additional T in a TTTT run at positions 70-73. Plus one or
2 frameshifts were monitored in a substrate containing a run of 5 Ts at the same position but in the
1 reading frame (25).
coding
sequence was constructed and purified as described by Osheroff et
al. (26). DNA synthesis conditions were based on a slightly
different short gap fidelity assay (27). Reactions (20 µl) contained
20 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, 10 mM MgCl2, 1 mM ATP, 150 fmol of
gapped DNA, 1 mM each dATP, dCTP, dGTP, and dTTP, and 400 units of T4 DNA ligase. DNA synthesis was initiated by addition of
wild-type or mutant Klenow fragment derivatives in the amounts
described above. After incubation at 37 °C for 60 min, reactions
were stopped by addition of EDTA to a final concentration of 15 mM. Reaction products were fractionated on a 0.8% agarose
gel, and covalently closed circular products were isolated by
electroelution from the agarose gel for transfection into E. coli strain MC1061 as above.
2 µM) and DNA substrate
(
20 nM) were allowed to equilibrate in 20 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, and the reaction
was initiated by addition of an equal volume of 2 mM dGTP
(complementary to the next template nucleotide) in the same buffer.
Samples were removed, either manually or using a rapid quench-flow
instrument, at appropriate time intervals, quenched by addition of
excess EDTA, and analyzed as described (22) to give the apparent
first-order rate constant for nucleotide addition.
RESULTS
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Fig. 1.
Positions of amino acid side chains altered
in this study. A stereo view of the -carbon backbone of the
polymerase domain (amino acids 548-928) of Klenow fragment (12). The
palm subdomain (residues 648-717 and 848-928) is colored
red, the fingers (residues 718-847) blue, and
the thumb (residues 548-647) green. Residues 601-608, at
the tip of the thumb subdomain, are missing because they were
disordered in the co-crystal. The side chains shown are those mutated
for this study. Mutations of the gray residues gave no
change in fidelity (relative to wild-type Klenow fragment), mutations
of the purple residues had an anti-mutator phenotype, and
mutations of the green residues had a mutator phenotype. The
carboxylate triad, Asp-705, Asp-882, and Glu-883, at the polymerase
active site is shown in yellow. This figure was generated
using Ribbons (36).
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Fig. 2.
Amino acid conservation and forward mutant
frequencies for mutant derivatives of Klenow fragment. The
approximate positions of the Klenow fragment mutations studied are
indicated relative to the linear sequence of the polymerase domain,
together with the corresponding forward mutant frequencies for DNA
synthesis across the lacZ target (n.s., no
synthesis.). Where measurements were made in duplicate, both values are
listed; results from three or more determinations are given as
mean ± S.D., with the number of determinations in
parentheses. The background frequency (not subtracted) for
uncopied DNA is 5 to 7 × 10
4. Additional
information was obtained by sequencing the lacZ mutations
recovered from three experiments; these were from R631A (47 mutations),
H734A (48 mutations), and R841A (100 mutations). In all three cases the
error distribution was not significantly different from that of
wild-type Klenow fragment. Also shown is the extent to which each
mutated side chain is conserved in the pol I family of DNA
polymerases; where the side chain is not invariant, conservative
substitutions are listed in parentheses. For residues from
Asp-705 to the carboxyl terminus the sequence conservation information
is taken from a partial alignment of the sequences of 23 bacterial, 7 bacteriophage, 8 mitochondrial, and 2 eukaryotic DNA polymerases
(J. Jäger and C. M. Joyce, unpublished work). For residues
amino-terminal to Asp-705, the information is derived from the 23 bacterial sequences since the sequence motifs in this part of the
protein are less well conserved and are therefore harder to identify
with confidence in the more distantly related polymerase
sequences.
4 (Fig. 2), based on data from this and previous
studies (13, 17). The D705A and D882A derivatives of Klenow fragment,
which have very low catalytic activity, were unable to perform
gap-filling synthesis even when a vast excess of polymerase was present
in the reaction and were not investigated further. Of the remaining 26 proteins, the majority gave forward mutant frequencies similar to that
of wild-type Klenow fragment. Several mutant derivatives, particularly
R754A, Q849A, and H881A, gave lacZ mutants at frequencies lower than that observed for the wild-type enzyme. The R668A and Y766A
polymerases gave mutants at 2.6- and 3.7-fold higher frequencies, respectively, than wild-type. The Y766A polymerase had previously been
demonstrated to be a strong base substitution mutator (15, 16). The
N678A, R682A, and E710A polymerases also gave smaller but reproducible
increases in the frequency of lacZ mutants.
mutation target (TGA89
assay). Base substitution errors resulting from eight of the nine
possible mispairs are detectable as blue plaque revertants (24). Of
these eight, reversion to the wild-type coding sequence via an A·dCTP
mispair results in a dark blue plaque phenotype that is easily
distinguished on indicator plates (24). The other seven mispairs all
yield revertants having a similar light blue plaque color,
necessitating sequence analysis or hybridization probing to define
error specificity for these mispairs.
5
(Table I). A 6-fold increase was observed
with the known base substitution mutator, Y766A (16), serving as a
positive control. Four other mutant derivatives (R668A, R682A, E710A,
and N845A) also showed an increase in base substitution errors relative
to the wild-type enzyme. The R754A and Q849A enzymes gave reversion frequencies lower than the wild-type value. The reversion frequencies obtained with the rest of the mutant enzymes that were tested were not
significantly different from that of wild-type Klenow fragment (Table
I).
lacZ revertants generated during synthesis by wild-type and mutant
derivatives of Klenow fragment
Base substitution error specificity for mutator polymerases in two opal
codon reversion assays
5 (Table I), which corresponds to an increase of
>16-fold over the frequency of errors recovered at the template A
position in the TGA89 gap-filling assay. The substantial
increase in recovery of A and G misinsertions opposite the template A
(Table II) was as expected since purine-purine mispairs are
particularly poor substrates for extension by Klenow fragment (28).
Removing the requirement for mismatch extension also unmasked a strong
preference of wild-type Klenow fragment for A·dATP insertion errors;
again, this was as expected from earlier biochemical studies (28).
2-3-fold increases in T·dGTP errors; because T·dGTP is such a common error, these changes were largely responsible for the increased reversion frequency in the TGA89 assay. However, other
changes, which had less effect on the overall reversion frequency, are probably more interesting mechanistically because the change relative to wild-type was greater. Thus, R682A and N845A both gave
10-fold increases in T·dCTP errors. Intriguingly, R668A, R682A, and N845A changed the distribution of errors opposite the template G in the
TGA89 opal codon; all three preferentially formed G·dGTP
mispairs rather than G·dATP mispairs, whereas the opposite was true
for wild-type Klenow fragment.
6-fold increase over wild type) but is
very inefficient at extending the resulting A·A mispairs. R668A, on
the other hand, does not appear to be a mutator in the TG"A" assay
(Table I) because the most dramatic increase is in a relatively uncommon error (A·dGTP), and there is little change in the frequency of the dominant A·dATP error (Table II). In contrast with the results
of the TG"A" assay, the R668A mutant polymerase gave no A·Pu
errors in the gap-filling TGA89 assay, suggesting that this protein has particular difficulty extending A·Pu mispairs. The R682A
mutation caused an increase in A·dATP errors in both assays; the increase in the TGA89 assay may indicate an improvement
in extension of A·A mispairs.
target. These
assays score primarily single nucleotide deletion or addition errors,
respectively. We focused mainly on Klenow fragment derivatives having
mutations in the thumb subdomain because previous experiments with
Klenow fragment and human immunodeficiency virus-1 reverse
transcriptase have suggested that contacts between this region of the
polymerase and the template-primer duplex are important in controlling
frameshift fidelity (17, 29). Although the
(590-613) mutant of
Klenow fragment, lacking 24 residues of the thumb tip, is a frameshift mutator (Table III; Ref. 17), single
alanine substitutions at highly conserved amino acids in the thumb tip
(Asn-579, Ser-582, Ser-608, and Thr-609) had little effect on
frameshift fidelity with either substrate (Table III). Since there are
extensive sequence-independent interactions between the thumb tip and
the sugar phosphate backbone of the duplex template-primer (8, 10),
removal of a single interaction may not be sufficient to give a
detectable phenotype.2 Small
increases in frameshift error rates resulted from some mutations in
highly conserved side chains in the thumb subdomain and the neighboring
portion of the palm as follows: K635A (
1 frameshifts), N678A, and
R682A (+1/
2 frameshifts); these effects were smaller than those
observed for mutations in a similar region of human immunodeficiency
virus-1 reverse transcriptase (29, 30).3 Aside from
R682A, none of the base substitution mutators gave an increase in
frameshift errors. In contrast, several polymerases, most notably
R668A, E710A, and Q849A, caused a substantial decrease in frameshift
mutagenesis (Table III).
Frameshift reversion frequencies for Klenow fragment mutants
(590-613),4 and E710A)
were more discriminating than wild type. Several of the enzymes that
behaved as anti-mutators in the fidelity assays, e.g. R754A,
Q849A, and H881A, were particularly compromised in T·G mispair
extension.
Rates of extension of matched and mismatched primer termini
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are grateful to Xiaojun Chen Sun for purifying many of the proteins used in this study and to Jim Kiefer for help with Fig. 1. We thank Nigel Grindley and Joann Sweasy for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by funds from the Intramural AIDS Targeted Antiviral Program (to NIEHS) and National Institutes of Health Grant GM-28550 (to Yale).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.
§ Present address: Paradigm Genetics Inc., Research Triangle Park, NC 27709.
Supported by National Institutes of Health National Research
Service Award 5-F31 GM-18508.
** Present address: Life Technologies Inc., Rockville, MD 28050.
To whom correspondence should be addressed: Dept. of Molecular
Biophysics and Biochemistry, Yale University, 266 Whitney Ave., P. O.
Box 208114, New Haven, CT 06520-8114. Tel.: 203-432-8992; Fax:
203-432-9782; E-mail: catherine.joyce{at}yale.edu.
The abbreviations used are: pol I, DNA polymerase I; Pu, purine.
2 Since the mutated side chains interact with a region about 7 to 8 base pairs upstream of the primer terminus, it is possible that the corresponding mutant proteins might be frameshift mutators in homopolymeric runs longer than the T5 tract tested.
3 The relevant side chains in human immunodeficiency virus-1 reverse transcriptase are suggested to contact the primer-template duplex 2-6 bases upstream of the primer terminus via contacts to the minor groove and the phosphodiester backbone of the primer strand (29, 30). Based on co-crystal structures, Arg-631, Lys-635, Asn-675, and Asn-678 of Klenow fragment seem the most likely candidates for contacting a similar region of the DNA (8, 10).
4
Consistent with this result, the (590-613)
Klenow fragment is an anti-mutator for base substitutions although it
is a mutator for frameshifts (17).
5 A good illustration of these factors is provided by our data for the E710A derivative; this protein gave a lacZ mutant frequency 2-fold above wild type in the forward mutation assay (Fig. 2), yet it is an 80-fold mutator for insertion of dCMP opposite the template A in the opal codon (Table II), a strong anti-mutator for frameshift errors (Table III), and a possible anti-mutator for T·dGTP insertion errors (Tables II and IV).
6 Interestingly, the recovery of A·dCTP errors using the single nucleotide gap substrate was similar to the frequency in the opal codon reversion assay, suggesting, perhaps, that the reaction conditions used for gap filling (long reaction times, high dNTP concentrations, and exonuclease-deficient enzymes) allow extension of all but the most refractory mismatches.
7 An anti-mutator must affect T·dGTP, the most common error made by wild-type Klenow fragment, in order to have a detectable phenotype.
8 For simplicity, amino acid side chains are described in terms of the Klenow fragment numbering.
9 As pointed out elsewhere (31), this interaction is hard to reconcile with the biochemical consequences of mutations at Tyr-766 and Glu-710, particularly with the negligible effect of the Y766F mutation on polymerase fidelity and other biochemical properties (15, 16, 22).
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REFERENCES |
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