From the Laboratory of Molecular Genetics and
¶ Laboratory of Structural Biology, NIEHS, National Institutes of
Health, Research Triangle Park, North Carolina 27709 and the
§ Division of Mutagenesis, National Institute of Safety
Research, 5 Nokbundong, Eunpyungku, Seoul, Korea
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
During base excision repair, DNA polymerase DNA polymerase Early studies of the fidelity of DNA synthesis by pol Given that perhaps as many as one million damaged nucleotides per cell
per day may be repaired by BER (21), any low fidelity DNA synthesis
during BER could potentially introduce a number of mismatches into the
eukaryotic genome. Thus, it is important to understand the fidelity of
the pol Since the processive gap-filling reaction may be more relevant to the
fidelity of BER than the earlier studies of distributive primer
extension, we have developed in the present study three short gap
substrates with which to measure fidelity. One substrate is a model for
the predominant role of pol The results reveal that pol Materials--
Bacterial strains, M13mp2 bacteriophage
derivatives, and the preparation of the gapped substrate for the
forward mutation assay have been described (37). DNA polymerase Forward Mutation Assay--
The forward assay scores errors in
the lacZ Construction of Short Gapped Substrates for Reversion
Assays--
Short gapped DNA substrates were constructed in which the
gap contains a portion of the lacZ
For the 1ntGap substrate, oligonucleotide-directed mutagenesis was
employed to construct an SphI site at position 58 of the lacZ Short Gap Fidelity Assays--
Fidelity was determined in
reaction mixtures (20 µl) containing 20 mM Tris-HCl, pH
8.0, 2 mM dithiothreitol, 25 mM NaCl, 10 mM MgCl2, 5% glycerol, 1 mM ATP,
150 ng (32 fmol) of gapped DNA, 500 µM each dATP, dCTP,
dGTP, and dTTP, 400 units of T4 DNA ligase and pol Fidelity of Recombinant Pol
For purposes of direct comparison to results obtained during short
gap-filling synthesis (below), we also determined the base substitution
fidelity of recombinant human pol Base Substitution Fidelity during Short Gap-filling
Synthesis--
The base substitution error rate of recombinant human
pol Base Substitution Fidelity during Simple BER Synthesis--
The
base substitution error rate of recombinant human pol
Surprisingly, 19 of 47 revertants generated by human pol Frameshift Fidelity during Short Gap-filling Synthesis--
The
single-base deletion error rate of recombinant human pol DNA polymerase In mammalian cells, several base excision repair pathways have been
identified, involving filling of a single nucleotide gap and a gap of 2 to 6 nucleotides. Evidence indicates that pol Recombinant human pol fills 1-6-nucleotide gaps processively, reflecting a contribution of
both its 8- and 31-kDa domains to DNA binding. Here we report the
fidelity of pol
during synthesis to fill gaps of 1, 5, 6, or >300
nucleotides. Error rates during distributive synthesis by recombinant
rat and human polymerase (pol)
with a 390-base gap are similar to
each other and to previous values with pol
purified from tissues. The base substitution fidelity of human pol
when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at
least 60-fold higher than for distributive synthesis. Base substitution
fidelity when filling a 1-nucleotide gap is higher than when filling a
5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the
dNTP binding pocket and/or a difference in base stacking or DNA
structure imposed by pol
. Nonetheless, 1-nucleotide gap filling is
inaccurate, even generating complex substitution-addition errors.
Finally, the single-base deletion error rate during processive
synthesis to fill a 6-nucleotide gap is indistinguishable from that of
distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism
of processivity by pol
does not allow the enzyme to suppress
template misalignments.
INTRODUCTION
Top
Abstract
Introduction
References
(pol
)1 is the smallest of the
mammalian cellular DNA polymerases. Evidence suggests that it can
participate in several DNA transactions in vivo, including
DNA replication (1, 2), recombination (3, 4), and base excision DNA repair (reviewed in Ref. 5). Base excision repair is needed to replace
damaged nucleotides that are depurinated, deaminated, oxidized, or
methylated as a result of normal cellular metabolism or environmental
insult (reviewed in Ref. 6). There are at least two forms of base
excision repair (BER) in mammalian cells (5). The predominant form is
"simple" BER (7), involving excision of a single damaged nucleotide
and its replacement catalyzed primarily by pol
(8, 9).
Alternatively, BER may occur by excision of 2 to 6 nucleotides
involving proliferating cell nuclear antigen and flap endonuclease 1, and DNA synthesis may be catalyzed by pol
(10, 11) or by pol
or
pol
(12, 13). This pathway is referred to as "alternate" BER.
employed a
long single-stranded template within a 390-nucleotide gap (14). The
average pol
error rate for the 12 possible base substitution errors
was 7 × 10
4 (15), while the average error rate for
single-base deletion errors was 3-9 × 10
4 (16).
These rates are substantially higher than those measured for the other
cellular DNA polymerases that perform the bulk of genomic DNA
replication (17). These higher error rates seem consistent with the
fact that pol
lacks an intrinsic 3'
5' proofreading exonuclease
activity. The observations that pol
has low frameshift fidelity and
synthesizes DNA in a distributive manner during primer extension to
copy long single-stranded templates suggested that deletion errors
within mononucleotide template runs may result from strand slippage
initiated during the dissociation-reassociation phase of a
polymerization reaction (18). This hypothesis has been supported by
numerous subsequent observations (e.g. Ref. 19 and
references therein; reviewed in Ref. 20) and is central to the present
study (see below).
synthetic reaction. However, recent studies suggest that
pol
synthesis to fill a short gap may differ significantly from
that during a distributive primer extension reaction. Pol
contains
a polymerase domain that binds to duplex template-primer DNA and a
unique 8-kDa processivity domain (22) that contains the active site for
AP lyase activity (23-26). This 8-kDa domain binds to the 5'-phosphate
in short gap substrates (27) and to single-stranded DNA (28), such that DNA synthesis by pol
to fill gaps of 2-6 nucleotides is processive rather than distributive (29). The crystal structure of a pol
·DNA·ddNTP complex revealed that the 5'-terminal phosphate in a
single nucleotide gap is bound near the AP lyase active site (25, 26,
30). A 90° kink in the DNA template is also observed, precisely at
the 5'-phosphodiester linkage of the template base in the polymerase
active site. This sharp bend exposes the base pair on the downstream
side of the active site, allowing that template-strand base to stack
with a histidine residue in the 8-kDa domain rather than with its
neighboring base in the active site. This alteration in base stacking
interactions has been suggested to be relevant to the fidelity of a
single-nucleotide gap-filling reaction (31). Moreover, cross-linking
studies suggested that the 8-kDa domain also contributes to dNTP
binding in the polymerase active site (32, 33). Thus, unlike
distributive synthesis on a long single-stranded template, pol
fills gaps of up to 6 nucleotides in a processive manner, and the 8-kDa
domain participates in this reaction in multiple ways.
in simple BER. This substrate contains
a single template adenine in a single-nucleotide gap, similar to the
gapped substrates used in recent kinetic studies of misinsertion by rat
pol
(34, 35). The other two gapped DNA substrates are models for
synthesis in the alternate BER pathway; one contains a 5-base gap and a
target for scoring eight different base substitution errors, including
the same three misincorporations opposite template adenine as in the
1-base gap (this substrate was used earlier to identify pol
mutants
with strongly reduced base substitution fidelity (36)); the other
substrate contains a 6-nucleotide target to monitor the single-base
deletions within a TTTT run during processive synthesis.
has low fidelity when filling short
gaps, and generates errors not previously observed during distributive
synthesis, including clustered multiple substitutions and complex
additions. Base substitution error rates are similar during filling of
a >300-nucleotide gap and a 5-nucleotide gap, but are lower for three
mispairs during synthesis to fill a single-nucleotide gap. Single-base
deletion error rates in a homopolymeric run are similar during
distributive and processive synthesis. These data are discussed in
light of a model wherein the 8-kDa domain of pol
influences
fidelity during the filling of short gaps.
EXPERIMENTAL PROCEDURES
from rat Novikoff hepatoma, homogenous fraction VI (38), was generously
provided by Dale W. Mosbaugh, Oregon State University, Corvallis, OR.
Recombinant wild-type rat and human pols
were expressed in
Escherichia coli and purified as described (39). ATP and
dNTPs were from Amersham-Pharmacia Biotech, Inc. T4 DNA ligase,
SphI, and EcoRI were from New England Biolabs.
Sequenase version 2.0 and sequencing reagents were from U. S. Biochemical Corp. (Cleveland, OH). Synthetic oligonucleotides were from
Genosys Biotechnologies, Inc. (The Woodlands, TX).
gene in M13mp2 (37). Correct polymerization to
fill a 390-nucleotide gap produces DNA that yields blue plaques upon
introduction into an E. coli
-complementation strain and
plating on indicator plates. Errors are scored as light blue or
colorless plaques. Sequence analysis of DNA extracted from mutants
defines the lacZ mutations.
-complementation
sequence of M13mp2 which has been modified by the introduction of an
in-frame opal codon (Fig. 2, 1ntGap and
5ntGap) or a template TTTT sequence (Fig. 2,
6ntGap). Construction of the 5ntGap and 6ntGap reversion substrates began with addition, by oligonucleotide-directed mutagenesis (40), of a single T at position 59 (where position 1 is the first
transcribed base of the gene). This disrupts the reading frame to yield
a colorless plaque phenotype. Following alkaline lysis and Qiagen
(Chatsworth, CA) purification, the RF I DNAs were purified by CsCl
equilibrium density gradient centrifugation to ensure that they were
free of single-strand DNA that could compete with the exogenously added
single-strand DNA template for hybridization to the primer during
formation of gapped substrates. The replicative form DNA was then
linearized with EcoRI to generate the primer strand DNA for
construction of the 5ntGap and 6ntGap substrates. Single-stranded DNA
with the T nucleotide inserted at position 59 was used as the template
for additional oligonucleotide-directed mutagenesis to generate the
template strand DNAs for the 5- and 6-nucleotide gapped substrates. For
the 5ntGap substrate, 5 nucleotides were inserted at position 58 of the
lacZ
coding region, restoring the reading frame and
introducing an in-frame opal codon
(5'-GTTGA-3'). Template DNA for the 6ntGap substrate was
constructed by the addition of 6 nucleotides at the same position,
maintaining the +1 reading frame and introducing a short homopolymeric
run (5'-GTTTTA-3'). Gapped substrates were formed from the
linear primer and the single-stranded circular plus-strand DNAs as
described (37). Substrates were separated from excess primer and
single-strand DNA by preparative agarose gel electrophoresis.
complementation gene. This disrupts the gene with an
overall +2 nucleotide insertion. Replicative form DNA was purified as described above and linearized with SphI to generate primer
DNA for the 1ntGap reversion substrate. Further mutagenesis was carried out on single-strand DNA containing the SphI site to insert
an A nucleotide at position 2 of the SphI site. This
generates a template in the normal reading frame but containing an
opal codon. Gapped substrate was formed and purified as
above. These substrate DNAs each have a colorless plaque phenotype. In
the 5ntGap and 1ntGap assays, base substitution errors that restore
-complementation result in a blue plaque phenotype. With
the 6ntGap substrate, deletions (
1,
4) or insertions (+2) that
restore the reading frame result in blue plaques.
. Human DNA
polymerase
was used at a 25:1 molar ratio of enzyme to DNA.
Following incubation at 37 °C for 60 min, reactions were stopped by
adding EDTA to 15 mM and the products were separated on
agarose gel. Gel slices containing covalently closed circular DNA
products were electroeluted from gel slices and concentrated. DNA
samples were introduced into E. coli MC1061 by
electroporation and plated as described (37). After scoring revertant
and total plaques, revertants were replated to confirm the phenotype
and then reversion frequencies were calculated. Sequence analysis of
revertants was performed to define the sequence responsible for the
blue plaque phenotype.
RESULTS
during Distributive DNA
Synthesis--
Previous studies had established the error rate and
specificity during synthesis to fill a 390-base gap by pol
from rat hepatoma and chick embryo (14-16). We began this study by examining the fidelity of recombinant rat and human polymerases
during distributive synthesis to fill this long gap where the polymerases exhibited lacZ total mutation frequencies of 250 × 10
4 and 240 × 10
4, respectively
(Table I). Error specificity was defined
by sequence analysis of greater than 100 mutants. The error spectra for
the two enzymes are summarized in Fig. 1
and Table I. As was found for the native rat and chick enzymes (14),
about two-thirds of the errors by the recombinant enzymes are
1 base
frameshifts (Table I), with half occurring at a previously identified
pol
hot spot, the template TTTT residues at positions +70 through +73 (Fig. 1). As before,
1 base frameshifts are generated much more
often than are plus 1-base frameshifts. Also as before, a T
G hot
spot is observed at position +103. Evidence suggests that this error
results from a dislocation mechanism (41, 42). This single T
G
transversion accounts for nearly 30% of all the base substitutions in
the spectra, even though 241 different substitution errors can be
scored in the 250-nucleotide target sequence (37). Both lacZ
mutant collections contained deletions of 317 base pairs (Table I).
These occur between a 5-base direct repeat sequence and are
characteristic of the two previous pol
error spectra (14). The
mutation frequencies for a variety of different errors are shown in
Table I. Overall, the results reveal that the rat and human polymerases
have error rates similar to each other and to earlier
determinations with the native rat and chick polymerases
. This
lends confidence that the recombinant polymerases are acceptable models
for the enzymes isolated from eukaryotic tissues.
Frequency of various classes of observed mutations for DNA polymerases
while conducting distributive DNA synthesis
View larger version (18K):
[in a new window]
Fig. 1.
Spectrum of single mutations produced by pol
during long gap-filling synthesis. Four lines of primary
wild-type DNA sequence are shown. The upper two lines of
primary DNA sequence (of the viral (+)-template strand) are the
regulatory regions for the lacZ
gene carried in M13mp2.
Position +1 is the first transcribed base. The lower two
lines are the first 129 bases (43 codons) of the
lacZ
gene. This figure presents mutations produced by
recombinant rat pol
shown above each line of
wild-type sequence and mutations generated by recombinant
human pol
shown below each line. The
letters used for base substitutions indicate the new base
found in the viral template strand DNA sequence, and is indicated
directly above or below the wild-type base. For
frameshift events, the loss of a base is indicated by an open
triangle (
) directly above or below the
base lost, while the addition of a base is indicated by a filled
triangle (
). When a frameshift occurs in a run of 2 or more of
the same base, it is not possible to assign the event to an individual
base. Therefore, the symbol is centered above or
below the run. For both the rat and human enzymes, there
were mutants with widely separated sequence changes in the same
molecule. Such linked mutations are not shown in this figure.
for distributive synthesis
opposite template TGA opal codons within a 361-nucleotide gap. When a TGA codon at template nucleotides 87-89 was used (43), the
average error rate was 5.5 × 10
4. When a
361-nucleotide gap substrate was used that contained the same TGA codon
used to construct the 5-nucleotide gap (Fig. 2), an error rate of 5.5 × 10
4 was again observed.
View larger version (23K):
[in a new window]
Fig. 2.
Substrates for short gap-filling reversion
assays. The reversion targets and surrounding sequences are shown
for the 5ntGap and 1ntGap base substitution assays and the 6ntGap
frameshift assay. The inserts are located near the amino terminus of
the wild-type M13mp2 lacZ complementation gene;
numbers below the template sequence (lower
strand) indicate nucleotide position in the wild-type M13mp2
sequence (where +1 is the transcription start). The direction of DNA
synthesis is indicated by the arrows. The template
nucleotides to be copied by pol
are larger. The position of the
A nucleotide in the 5- and 1-nt targets is the same; it is
the first nucleotide synthesized.
was determined during synthesis to fill the 5ntGap substrate
(Fig. 2). The reversion frequency for the DNA products of this
synthesis2 was 2600 × 10
6 (Table II), at least
460-fold higher than for the uncopied template DNA. Thus, human pol
generates synthesis errors at easily detectable rates during
gap-filling DNA synthesis. Sequence analyses of 49 lacZ
mutants recovered from filling the 5-base gap indicated that pol
produced substitution errors at each of the three phenotypically detectable target nucleotides (Table
III), involving seven of the eight
detectable mispairs. The overall average error rate for all
substitutions at the TGA target was 13 × 10
4.
However, substitutions did not occur randomly; no revertants consistent
with a T·dTMP mispair were observed while 29 of 49 revertants were
consistent with a T·dGMP mispair. Thus, rates for specific errors
ranged from
0.9 × 10
4 for two transversion
mispairs (T·dTMP and A·dAMP) to 29 × 10
4 for
the T·dGMP mispair (Table III). An unexpected result was the recovery
of lacZ mutants containing two closely-spaced
misincorporations (Fig. 3). These
represented 13% (6/49) of the collection. In contrast, such
substitutions have not been observed during synthesis by pol
(or
any other polymerase) using the long-gap forward mutation assay and
undamaged DNA (14, 41, 42, the present study). The error rate for these
closely-spaced double misincorporations during synthesis to fill a
390-base versus a 5-base gap differs by at least 60-fold
(Table IV, compare 180 × 10
6 to
3.1 × 10
6).
Mutation frequencies for human pol while conducting short
gap-filling DNA synthesis
Base substitution specificity for human pol while conducting short
gap-filling DNA synthesis
View larger version (20K):
[in a new window]
Fig. 3.
Multiple mutations and nucleotide additions
occurring during shorter gap-filling DNA synthesis. A,
the template (large type) and surrounding sequence in the 5-nt
gap-filling substrate is shown on the upper line; the
reversion target, TGA, is indicated. The letters
on the lines below indicate new bases found in
the template strand DNA sequence of the revertants. In 6 of 49 revertants, there were multiple DNA synthesis errors made by pol while copying the 5-nt target. B, the template/target
nucleotide, A, and surrounding sequence in the 1-nt
gap-filling substrate is shown on the upper line. On the
lines below is shown the template strand DNA sequence for 19 of the 47 mutants analyzed. The sequence of these revertants indicates
that while copying the 1-nt template, pol
frequently makes complex
base substitution/addition errors. The single A nucleotide
in the template has been replaced by four nucleotides.
Comparative error rates for DNA polymerases long versus short
gap-filling DNA synthesis
was next
examined during synthesis to fill the gap containing a single template
adenine (Fig. 2). As above, the reversion frequency for the DNA
products of this synthesis was well above the background value for the
uncopied template DNA (Table II). However, the reversion frequency was
8-fold lower than for synthesis with the 5-base gap (Table II).
Sequence analysis revealed that 19 of 47 revertants contained
substitutions resulting from misinsertions opposite the template
adenine, corresponding to an average base substitution error rate of
2.2 × 10
4. Again, the rates for the three mispairs
that could be scored differed (Table III), being highest for the
A·dCMP transition mispair. A comparison of error rates for these
three mispairs during synthesis to fill the 1-base and 5-base gap
reveals consistently higher apparent fidelity for 1-base gap-filling;
the differences are 2.2-fold for the A·dCMP mispair, 9.1-fold for the
A·dGMP mispair, and 3.8-fold for the A·dAMP mispair (Table
III).
during
synthesis to fill the single-base gap contained 4 nucleotides in place
of the template adenine (Fig. 3). Such errors have not been observed
during synthesis by pol
(or any other polymerase) using the
long-gap forward mutation assay (14, 41, 42, the present study) or
during synthesis to fill the 5-base gap. Thus, the origin of these
errors is unique to filling a single-base
gap.3
was
examined during synthesis to fill the 6-base gap (Fig. 2). The 5'
-GTTTTA-3' template sequence corresponds to that of the hot spot for
1 base frameshift errors by pol
in the forward mutation assay
(Fig. 1). Processive DNA synthesis by pol
to fill this 6ntGap
results in a reversion frequency of 37 × 10
4, more
than 1000-fold higher than that of the control uncopied template (Table
II). Sequence analysis of 31 revertants revealed that all had lost one
T in the TTTT run. This yields a error rate of one deletion for every
670 template T residues copied (Table IV). This result is similar to
the deletion error rate when the TTTT run at nucleotides 70-73 in the
lacZ coding sequence is copied by rat or human pol
during distributive synthesis to fill the 390-base gap in the forward
assay (Table I, Table IV).
DISCUSSION
plays a key role in mammalian base excision
repair. Pol
is a small nuclear DNA polymerase with a host of biochemical properties that make this enzyme well suited for its role
in base excision repair. Pol
carries both gap-tailoring (23) and
gap-filling enzymatic activities (29, 44), and it physically interacts
with at least 3 other base excision repair proteins: AP endonuclease
(45), DNA ligase I (46, 47), and XRCC1 (48, 49). Structure-function
analysis revealed that the enzyme has a processivity domain which
functions only on short-gapped substrates (27). In recent years,
additional roles of pol
have been suggested, most involving
synthesis to fill relatively short-gapped intermediates in
recombination (3, 4) and replication (1, 2). A noteworthy feature of
pol
is that it does not have an intrinsic proofreading exonuclease
activity (50).
performs gap-filling
in both of these BER pathways (8, 10, 11), and that synthesis to fill
gaps of 2 to 6 nucleotides can be processive (29). To better understand
the fidelity of these base excision repair processes, as well as
fidelity during primer extension to copy long single-stranded templates
that may be relevant to pol
's participation in some types of
replication (1) and recombination events (3, 4), we describe here the
base substitution and single-base deletion fidelity of recombinant pol
using substrates containing gaps of varying sizes. Data obtained
with the 390-base gapped substrate (Table I, Fig. 1) demonstrate that
error rates and error specificities during distributive synthesis by
the recombinant rat and human enzymes are similar to earlier studies of
native pol
purified from several eukaryotic sources (14-16, 18).
The error specificities of all the pols
tested to date include the same hot spots for single-base deletions and for dislocation base substitution errors. These data indicate that the recombinant polymerases
are valid models for understanding principles that govern the fidelity of the native polymerases. The error rates of the
recombinant rat and human enzymes are also similar to each other (Table
I). Although there are 14 amino acid differences between the rat and
human polymerases
, all are located on the surface of the enzymes in
the crystal structures (31), and the data in Table I and Fig. 1
indicate that the differences do not influence fidelity when these
enzymes synthesize DNA in the absence of other BER proteins.
generates 1 base substitution error for every
13,000 nucleotides polymerized during synthesis to fill a
390-nucleotide gap. This average rate is for the 12 possible mispairs
that can be scored at 125 detectable template nucleotides in the
lacZ target. The error rate varies markedly by mispair and
position (Fig. 1). During synthesis to fill gaps of >300 nucleotides, pol
is less accurate than average at either of two opal
codons, generating 1 base substitution error for every 1,800 nucleotides polymerized (Table IV). With all three long gaps, no
downstream 5' terminus is adjacent to the site at which errors are
scored, and synthesis is distributive. The average single-base
substitution error rate at the opal codon during synthesis
to fill a 5-nucleotide gap is even higher, 1/770 (13 × 10
4, Table IV). This value is similar to the misinsertion
rates for G·dTMP and G·dAMP mispairs determined by Chagovetz
et al. (34) using a 6-nucleotide gap. In our study with a
5-nucleotide gap with the template nucleotides for scoring errors only
2, 3, or 4 nucleotides distant from the 5' terminus (Fig. 1), we also
observe multiple base substitution errors (Fig. 3) at a rate much
higher than during distributive filling of longer gaps (Table III).
Closely-spaced substitution errors have not been observed in the
spectra of errors generated in vitro by other DNA
polymerases. Key to considering their origins and the generally low
fidelity of pol
are biochemical (36) and structural information
suggesting that the ability of a polymerase to form hydrogen bonds or
van der Waal contacts with atoms in the minor groove of the nucleic
acid is important. In the structures of the Pol I family polymerases,
the DNA near the polymerase active site is A-form-like with a wide,
shallow minor groove, and these polymerases form hydrogen bonds with
minor groove O-2 and N-3 atoms (51-53). However, in the ternary
complex of pol
with a single nucleotide gap, the duplex DNA is
B-form and there are fewer hydrogen bonds between the polymerase and the duplex primer stem upstream of the active site (30). If the DNA is
also B-form when pol
is bound to a 5-nucleotide gap, it may be less
able than other polymerases to sense correct base pairing geometry in
the active site and/or at the primer terminus. This might account for
the overall low base substitution fidelity of pol
and for
generation of multiple mutations during 5-nucleotide gap filling. The
ability of pol
to generate multiple, closely-spaced substitutions
is also consistent with a model (Fig.
4A), wherein the 8-kDa domain
binds to the downstream 5' terminus to promote processive extension of
misinserted nucleotides that cannot be proofread because pol
lacks
3'-exonuclease activity. Consistent with this idea, the kinetic
constant for DNA synthesis during filling of short gaps is greater than
that for product release (35).
View larger version (25K):
[in a new window]
Fig. 4.
Model depicting the base substitution and
frameshift infidelity intermediates of DNA polymerase during short
gap-filling synthesis. A, during shorter gap-filling,
the 8-kDa domain of pol
is bound to the 5'-phosphate moiety at the
3'-end of the gap. Because the polymerase domain is tethered to the
substrate, synthesis by pol
proceeds in a processive manner
(dashed arrow). Analysis of revertants from shorter
gap-filling synthesis by pol
demonstrates that pol
can
misincorporate a nucleotide and then continue synthesis from the
mispaired template-primer terminus. Multiple misincorporations are
facilitated due to processive synthesis by DNA polymerase
resulting
in tandem and clustered base substitution errors. B,
misalignment of the template and primer strands during DNA synthesis
can lead to frameshift errors resulting in the addition or deletion of
one or more nucleotides. During distributive synthesis, pol
binds
to the DNA substrate, conducts synthesis, dissociates, and then rebinds
(solid arrows). When the polymerase is not bound,
template-primer misalignment can occur, leading to frameshift errors.
In the presence of 5'-phosphorylated downstream DNA, pol
will
remain tethered to the substrate and synthesis will be processive
(dashed arrow). The data presented here indicate that the
rate of frameshift errors during distributive and processive synthesis
by pol
is nearly the same. This suggests that either unpaired bases
can migrate through the run while the polymerase is bound to the
template-primer or the polymerase domain dissociates from the DNA to
allow misalignment as illustrated.
In experiments in which UV-irradiated shuttle vectors containing a
bacterial suppressor tRNA gene were employed as a mutagenic target
(54-57), a significant number of multiple substitution mutations were
observed. The data presented here suggest that these errors could have
resulted from gap-filling synthesis in vivo by DNA polymerase . More recently, we have found that the spectrum of somatic hypermutations within an immunoglobulin gene in mismatch repair-deficient pms2(
/
) mice is characterized by a high
proportion of tandem double-base substitutions that could result from
error-prone filling of short gaps by pol
(58).
During synthesis to fill a 1-nucleotide gap, human pol makes one
mistake for every 4,500 nucleotides polymerized at the opal
codon (Table IV). This average rate is about 6-fold lower than for
filling a 5-nucleotide gap (Table IV), with rates for individual
misincorporations opposite template adenine being 2-9-fold lower than
during 5-nucleotide gap filling (Table III). These differences could at
least partly be explained by decreased T4 DNA ligase activity when
mispairs are present at the nick. The relatively low base substitution
fidelity of pol
and the small differences in fidelity for filling a
1-nucleotide gap versus a longer gap are consistent with
kinetic determinations of misinsertion fidelity reported by Ahn
et al. (35). They found (see values in parentheses in Table
III) that pol
was not highly accurate for the same three mispairs
(albeit in a different sequence context), and that the fidelity of pol
for single-nucleotide gap-filling varied no more than 10-fold from
that observed during synthesis with a non-gapped template-primer.
However, our results are in contrast to observations by Chagovetz
et al. (34), who report much higher fidelity for filling a
single-nucleotide gap. As one example, they reported that pol
misinserts TMP opposite template guanine (typically one of the most
frequent errors by DNA polymerases) at a rate of only 1 × 10
5, a rate 180 times lower than during synthesis to fill
the 6-nucleotide gap (34). There are several possible explanations for
the difference between this study and ours, including the fact that
different mispairs were examined and these were in different
neighboring sequence contexts. Nonetheless, we conclude from the
present study, and that of Ahn et al. (35), that the average
base substitution fidelity of pol
is low during single-nucleotide
gap filling. The fidelity of BER in vivo may still be higher
since other BER proteins could enhance pol
selectivity or
inefficient ligation of mispairs could allow removal of misinsertions
by a 3'-exonuclease not intrinsic to pol
.
This study and the Ahn et al. (35) and Chagovetz et
al. (34) reports describe one or more examples of higher fidelity for filling a one-nucleotide gap than for synthesis with substrates having distant or no downstream 5' termini. A possible explanation is
that the 8-kDa domain, which binds to the downstream 5' terminus, contributes to polymerase selectivity during synthesis of the final
nucleotide in a gap. Biochemical evidence for this comes from
cross-linking studies which suggest that the 8-kDa domain contributes
to dNTP binding in the polymerase active site (32, 33). The crystal
structure of a pol ·DNA·ddNTP complex (30) reveals a 90° kink
in the DNA at the 5'-phosphodiester linkage of the template residue in
the polymerase active site. This exposes the (n+1) base pair on the
downstream side of the active site, which stacks with His-34 in the
8-kDa domain rather than with the active site template base (Fig.
5A). Elimination of base-base stacking could prevent nonspecific stabilization of incorrect incoming
dNTPs, thus providing a selective advantage for correct incorporation.
Stacking of the (n+1) base with His-34 is not observed in the structure
of a rat pol
·DNA·ddNTP complex which lacks a downstream primer
(59) (Fig. 5B). Moreover, the latter structure reveals
minimal interactions of the 8-kDa domain with the polymerase active
site, such that it may not contribute to dNTP binding (Fig. 5B).
|
Synthesis to fill a single-nucleotide gap also generated
lacZ mutants containing four nucleotides in place of the
template adenine (Fig. 3). The number of additional nucleotides
observed in the lacZ revertants (3 rather than 1, 2, 4, 5, etc.) is partly defined by the requirement to maintain the correct
reading frame for blue plaque phenotype. Thus, other complex errors may
be generated in this reaction but remained undetected. Examination of
the sequence surrounding the template adenine did not reveal an obvious
sequence that could be used to template, by simple strand displacement, the three examples of this type of error that were observed (Fig. 3).
Such errors have not been found in previous studies of other polymerases, during synthesis by pol to fill long gaps (14, 41,
42), or using any other gapped substrate in this study. Moreover, no
such lacZ mutants were generated during single-nucleotide gap-filling by exonuclease-deficient Klenow polymerase or by several mutant derivatives of this enzyme (63). Thus, to date, these complex
errors are unique to single-nucleotide gap-filling by pol
. Their
origin may eventually be explained by the unique features of the pol
DNA synthesis reaction, involving a second DNA binding site in the
8-kDa domain and strong reconfiguration of the single-nucleotide gapped
substrate (30).
Previous studies have shown that during distributive primer extension
without a downstream 5' terminus, pol generates single-base frameshift errors at a relatively high rate (14, 16, 18, 41). These and
subsequent studies of other DNA polymerases, especially HIV-1
reverse transcriptase (19, 60-62), have revealed a correlation between
processivity and the rate of single-base frameshift errors in
homopolymeric runs. This correlation led to the suggestion that these
errors may be initiated by strand slippage that occurs during
polymerase dissociation-reassociation with the template-primer. The
observation that pol
fills short gaps processively (29) offered an
opportunity to examine this hypothesis. When frameshift fidelity was
monitored in a 6-nucleotide gap containing a template TTTT run, pol
was highly error prone (Table II). This represents the first reported
measurement of the frameshift fidelity of a short-gap filling
polymerization reaction and is consistent with the possibility that the
infidelity of BER may contribute to microsatellite instability. This
low frameshift fidelity is also consistent with the structural data discussed above (and reviewed in Ref. 53), that reveals limited minor
groove contacts of the enzyme with the duplex template-primer stem. The
pol
single-base deletion error rate per detectable nucleotide
polymerized to fill the 6-nucleotide gap was not different than
for distributive primer extension without a 5' terminus adjacent to the
run (Table IV). One possible explanation is that pol
remains fully
engaged with the template-primer during processive synthesis, and an
unpaired template base can migrate through the run to form the
misaligned intermediate. Alternatively, we favor the model shown in
Fig. 4B, in which the polymerase domain dissociates from the
DNA allowing misalignment, but pol
remains tethered to the DNA for
processive synthesis through interactions of the 8-kDa domain with the
adjacent 5' terminus. This concept of error-prone synthesis initiated
by strand slippage due to polymerase dissociation-reassociation and
promoted by virtue of a second DNA binding site conferring processivity
to the reaction is supported by a recent study of the frameshift
fidelity of T4 DNA polymerase with and without its processivity factor,
the gp45 sliding clamp (20). In that study, the error rate for
1-nucleotide deletions in a homopolymeric run was similar for the T4
polymerase alone or with its accessory proteins. This led to the idea
that the polymerase could dissociate from the template-primer to allow
misalignment but remained tethered to the DNA for processive synthesis
through polymerase interactions with the topologically bound sliding clamp.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Brian J. Vande Berg and Phuong Pham for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
919-541-2644; Fax: 919-541-7613; E-mail: kunkel{at}niehs.nih.gov.
The abbreviations used are:
pol , DNA
polymerase
; BER, base excision repair; nt, nucleotide.
2
These data are taken from Beard et
al. (36). They are shown here for direct comparison to the results
with the other assays, and to emphasize the recovery of the
"closely-spaced" double mutations, which were only briefly
mentioned in the previous article that focused on pol mutator polymerases.
3 Eight of the nine remaining revertants had a base substitution mutation at one of the other two positions of the TGA codon. The origin of these revertants is uncertain, since these nucleotides were not included as single-stranded template bases for DNA synthesis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|