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INTRODUCTION |
The human mitochondrial genome
(mtDNA)1 encodes 37 genes
required for oxidative phosphorylation or mitochondrial protein
synthesis (1). Loss of these essential gene functions clearly induces a
multitude of severe metabolic disorders, and mutation of mtDNA is the
cause of inheritable mitochondrial diseases (2-4). Early reports
comparing nucleotide substitutions in mtDNA from somatic tissues of
different primates revealed a 10-fold higher rate of evolution for
mtDNA relative to the nuclear genome (5, 6), implying a relatively high
mutation rate for mtDNA. More recently, the accumulation of deletions
in mtDNA has been shown to correlate with increasing age (7), and a
current longitudinal study strongly supports the
age-dependent accumulation of non-inherited point mutations
in human mtDNA (8). Recent data suggest that mutations in mtDNA can
suppress apoptosis, a situation that would favor the growth of tumor
cells (9). Additionally, human somatic cancer cells can acquire a
homoplasmic mutant mtDNA genotype, presumably by mitotic segregation of
mutant mitochondria during proliferation of tumors (10). The prevalence
of mtDNA mutations in a variety of human cancers may be more than a
passive association (11). Thilly and co-workers (12, 13) have developed
sensitive methods to examine the spectrum of mtDNA mutations that form
in human cells in vivo. Molecular genetic analyses such as
these are the starting point for studying the biochemical mechanisms of
mutagenesis of the mitochondrial genome.
Mutations in mtDNA arise from several sources, all of which involve DNA
synthesis by the mitochondrial DNA polymerase, pol 
. Spontaneous
replication errors produce mismatches, and replication through
unrepaired mismatches can mutate mtDNA. Although Saccharomyces cerevisiae possesses the mismatch repair homolog Msh1 that can stabilize yeast mtDNA (14, 15), evidence for mitochondrial mismatch
repair in higher eukaryotes is currently lacking (16). mtDNA chemically
damaged by hydrolysis, reactive oxygen species, or environmental
mutagens contains non-coding or mis-coding lesions (17, 18). Evidence
for base excision repair of damaged mtDNA is abundant (17, 19-25), and
pol has a well established role in mitochondrial base excision
repair in vitro (26-28). Replication of DNA templates
damaged by platinum adducts may also lead to mutations in mtDNA (29).
Because pol is a component common to each mode of mutagenesis,
knowledge of its biosynthetic fidelity is critical to understanding
mitochondrial mutagenesis.
pol purified from chicken embryos or from pig liver mitochondria is
accurate in vitro, with these enzymes exhibiting error frequencies at a 3-nucleotide mutational target of <3.8 × 10
6 per nucleotide and <2.0 × 106
per nucleotide, respectively (30, 31). Both enzymes contain intrinsic
3'- to 5'-exonuclease activities that prefer mispaired 3' termini.
Partial inhibition of these exonuclease activities with 20 mM dGMP increases the frequency of errors, suggesting the
exonucleases proofread replication errors (30, 31). pol derived
from chicken, pig, Drosophila melanogaster, Xenopus laevis, Saccharomyces cerevisiae, and human sources
copurifies with 3'- to 5'-exonuclease activity (30-37), and the genes
for all known mitochondrial DNA polymerases possess three highly
conserved exonuclease motifs common to family A DNA polymerases
(38-41). Several lines of evidence show the exonuclease contributes to replication fidelity in vivo. Disruption of the exonuclease
motifs in the yeast MIP1 gene generates a mutator
phenotype, as exhibited by a several hundred-fold increase in the
spontaneous frequency of forming mitochondrial erythromycin-resistant
mutants (42). Expression of exonuclease-deficient pol fusion
proteins in cultured human cells also resulted in the accumulation of
point mutations in mitochondrial DNA (43). Also, the loss of
exonuclease function of pol in transgenic mice resulted in the
rapid accumulation of point mutations and deletions in cardiac mtDNA,
and the mutagenesis was accompanied by cardiomyopathy (44).
We and others (39, 45, 46) have cloned and overexpressed the human gene
encoding the catalytic subunit of DNA polymerase . In addition, we
have constructed an exonuclease-deficient human DNA polymerase by
replacing the conserved Glu and Asp residues in exonuclease region I of
the catalytic subunit (45). The full-length human cDNA for the
55-kDa accessory subunit has been isolated and overexpressed (47-49),
and the three-dimensional crystal structure of this protein has been
determined (50). When associated with the catalytic subunit, the
accessory subunit confers high processivity to pol through enhanced
DNA binding (47, 48). However, the fidelity of DNA synthesis by
structurally defined human pol with and without the accessory
subunit or exonucleolytic proofreading is not known. In this report we
present a comprehensive study of the fidelity of DNA synthesis by human
DNA polymerase in vitro. We reconstituted the various
forms of human pol and measured error rates during replication of
natural DNA substrates. We also performed steady state kinetic analysis
of base selection and mispair extension to determine the relative
contributions of exonucleolytic proofreading and the accessory subunit
to overall fidelity of replication.
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EXPERIMENTAL PROCEDURES |
Proteins--
The recombinant catalytic subunit of human DNA
polymerase (p140), the exonuclease-deficient form (p140
Exo), and their His6-tagged derivatives were
purified to homogeneity as described (45). The His6-tagged
accessory subunit (p55) was purified to homogeneity, and the
heterodimeric forms of the polymerase (p140·p55 and p140
Exo·p55) were reconstituted as described (47). Protein
concentration was determined relative to a BSA standard by quantitative
digital imaging of protein bands that had been resolved by
SDS-polyacrylamide gel electrophoresis and stained with Coomassie
Brilliant Blue R-250 (Bio-Rad).
Fidelity Assays--
The accuracy of DNA synthesis by human pol
was measured by copying a single-stranded region of bacteriophage
M13 DNA encoding the 
-peptide region of the 
-galactosidase gene.
Replication errors were scored by transfection and plating on
chromogenic indicator plates to score plaque colors. The necessary
bacterial strains, bacteriophage M13mp2 derivatives, and all procedures related to fidelity assays have been described (51-53). Gap-filling reaction mixtures (30 µl) contained 25 mM HEPES·KOH (pH
7.6), 2 mM dithiothreitol, 1 mM each dATP,
dCTP, dGTP, and TTP, 4 mM MgCl2, 50 µg/ml
acetylated BSA, ~150 ng of gapped M13mp2 DNA, and 16-220 ng p140 or
p140 Exo, as needed to fill gaps within 60 min at
37 °C. Reactions utilizing p140·p55 were supplemented with 0.1 M NaCl to achieve optimal in vitro activity
(47). Uncomplexed p140 was not active under these conditions (47).
Reaction products were isolated by phenol extraction and ethanol
precipitation, and closure of the gaps was confirmed by agarose gel
electrophoresis prior to transfection. Mutation frequencies were
calculated as described, and specific nucleotide changes in mutant DNAs
were determined by DNA sequencing.
Nucleotide Insertion Kinetics--
The fidelity of nucleotide
selection by pol was determined with a polyacrylamide gel-based,
single nucleotide extension assay utilizing synthetic
oligodeoxyribonucleotides (54-56). Oligonucleotides were obtained from
Oligos, Etc. (Wilsonville, OR), purified by denaturing polyacrylamide
gel electrophoresis and ethanol precipitation, and 5'-end-labeled with
T4 polynucleotide kinase (New England Biolabs) and
[-32P]ATP (Amersham Pharmacia Biotech). Labeled primer
(18-mer) and unlabeled template (36-mer) were hybridized (ratio = 1:1.1) to form the following primer-template with a recessed 3'-end: G
primer, 5'-TGA CCA TGT ACA TCA GAG-3'; C template, 3'-ACT GGT ACA TGT AGT CTC AGC CTG CAT ATA GTC ACT-5'.
Reaction mixtures (10 µl) contained 25 mM HEPES·KOH (pH
7.6), 2 mM 2-mercaptoethanol, 0.1 mM EDTA, 50 µg/ml acetylated BSA, 5 mM MgCl2, 1 pmol of
primer-template, 10-30 fmol of exonuclease-deficient p140 or
p140·p55, and one of the four common deoxynucleoside
triphosphates. As the next correct nucleotide, TTP was varied from 0 to
0.2 µM, whereas the three incorrect nucleotides were
varied from 0 to 1 mM. Following incubation at 37 °C for
5 min, reactions were terminated by the addition (10 µl) of 95%
deionized formamide and 10 mM EDTA. Samples (2 µl) were
boiled for 5 min and resolved by electrophoresis on 15% polyacrylamide
gels containing 7 M urea. Radioactive bands were detected
with a Storm 860 PhosphorImager (Molecular Dynamics) and quantified
with NIH Image software (version 1.62). To remain within the
limitations of steady state analysis, only values from reactions in
which less than 25% of the primer had been extended were used to
calculate initial reaction velocities. Kinetic constants were estimated
by linear least squares curve fitting of double-reciprocal plots.
Mispair Extension Kinetics--
Four additional DNA substrates
were constructed for mispair extension assays. Oligonucleotides were
purified, labeled, and hybridized as before to generate
primer-templates with recessed 3'-ends and A·C, T·C, C·C, or
G·G mispairs at their 3' termini as follows: A primer, 5'-TGA CCA TGT
ACA TCA GAA-3', and C template, 3'-ACT GGT ACA TGT AGT CTC
AGC CTG CAT ATA GTC ACT-5'; T primer 5'-TGA CCA TGT ACA TCA
GAT-3', and C template, 3'-ACT GGT ACA TGT AGT CTC AGC CTG
CAT ATA GTC ACT-5'; C primer, 5'-TGA CCA TGT ACA TCA GAC-3', and C
template, 3'-ACT GGT ACA TGT AGT CTC AGC CTG CAT ATA GTC
ACT-5'; G primer 5'-TGA CCA TGT ACA TCA GAG-3', and G template 3'-ACT
GGT ACA TGT AGT CTG AGC CTG CAT ATA GTC ACT-5'.
The composition of reactions was the same as for nucleotide insertion,
except the next correct nucleotide (TTP) was varied from 0 to 1 mM. Products were analyzed, and kinetic constants were
determined as before.
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RESULTS |
Average Fidelity of Human pol --
The accuracy of DNA
synthesis by human pol was measured in vitro by copying
a single-stranded region of bacteriophage M13mp2 DNA encoding a
407-nucleotide section of the -galactosidase gene. Correct
polymerization produces DNA that yields blue M13 plaques, whereas
errors are scored as light blue or colorless plaques. A broad view of
replication fidelity is provided by this forward mutation assay, which
scores all 12 possible single-base substitutions, each in a variety of
sequence contexts, as well as additions and deletions of 199 different
template nucleotides present as non-iterated or repeated sequences. We
measured the fidelity of polymerization reactions catalyzed by the
wild-type catalytic p140 subunit of human pol either alone or in
the presence of its p55 accessory subunit. To determine the
contribution of exonucleolytic proofreading to replication fidelity, we
also measured the fidelity of exonuclease-deficient pol either
alone or with p55. The frequencies of lacZ mutant plaques
generated in the four different polymerization reactions are shown in
Table I (top section). The wild-type
polymerase reactions generated products whose lacZ mutant
frequencies were only slightly above the background mutant frequency of
the uncopied control DNA (7 × 104). Reactions
catalyzed by the exonuclease-deficient polymerase had 1.8-fold higher
lacZ mutant frequencies, indicating that this polymerase was
less accurate and that the majority of the lacZ mutants
contained errors made during DNA synthesis in vitro.
Inclusion of the p55 accessory subunit further increased the mutant
frequency for p140 Exo, but proofreading was able
to balance this apparent mild reduction in fidelity caused by p55. DNA
extracted from collections of independent lacZ mutants were
sequenced to classify the types of errors made by each form of the
enzyme and to permit calculation of specific error rates (51).
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Table I
Frequency and classes of errors made by pol in the presence and
absence of exonucleolytic proofreading and the accessory subunit
Fidelity determinations were performed as described under
"Experimental Procedures" and "Results." The mutant frequency
values shown here are for single determinations, but previous studies
have shown that standard deviations are about 20% of mean values when
multiple determinations are performed. The numbers in parentheses are
the number of template nucleotides (sites) in the lacZ
template at which each type of error can be scored. Error rates were
calculated as described (51). Error rates listed as  fall within
2-fold of previously published values for uncopied DNA and therefore
are unlikely to reflect errors made during gap-filling DNA synthesis.
The background mutant frequency for uncopied control DNA was 7 × 104.
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Single-base substitutions were found in the majority of lacZ
mutants recovered from all four copying reactions (Table I). In the
case of wild-type pol without p55, the average substitution error
rate when considering all mispairs (8 × 106) and the rates for individual mismatches
(T·dGMP and A·dAMP listed as an example, the other 10 mismatches not shown) are all "less than or equal to" values, since
these values are similar to those for uncopied DNA. This indicates that
the exonuclease-proficient catalytic subunit has relatively high base
substitution fidelity. As expected, the exonuclease-deficient enzyme is
less accurate, e.g. by 
30-fold for the
T·dGMP mismatch and by 14-fold for the A·dAMP mismatch
(Table I). This suggests that the majority of misinsertions made by the
catalytic subunit are proofread by its intrinsic exonuclease.
Proofreading is also implied by the lower base substitution error rates
of the wild-type p140 as compared with the exonuclease-deficient p140
when p55 is present (four columns on right side of Table I). Somewhat
surprisingly, however, the substitution fidelity of the catalytic
subunit is lower in the presence of the p55 subunit. For example, the
exonuclease-deficient polymerase is 6-fold less accurate for the
A·dAMP mismatch in the presence of p55, and overall average
substitution fidelity is also somewhat lower. A similar trend toward
reduced fidelity in the presence of p55 is also apparent with wild-type
pol . For example, p55 raised the frequency of T·dGMP
errors from 0.7 × 106 to 6.2 × 106 (Table I). Note that to detect an error in
gap-filling assays, the polymerase must first make a mistake and then
extend it to prevent removal upon introduction of the copied M13mp2 DNA
into the E. coli host cell. Thus, p55 may increase the error
rate in gap-filling assays by promoting either misinsertion, mismatch extension, or both. These possibilities are tested below.
A number of the lacZ mutants recovered from the four copying
reactions contained single-base deletion or addition mutations (Table
I). Detailed knowledge of which deletions and additions result in a
detectable color change (51) permits calculation of frameshift error
rates that are corrected for differences in target size
(e.g. the lengths of homopolymeric runs). Frameshift error
rates calculated for wild-type p140 are again "less than or equal
to" values, since they are similar to the background values for
uncopied DNA. The only exception is the rate of 5.7 × 106 for deletions in homopolymeric runs of 4 or 5 nucleotides. This rate is well above the background value, indicating
that p140 generates deletions in these runs despite the presence of a
proofreading exonuclease that can excise terminal base·base
mismatches. The exonuclease-deficient p140 is clearly less accurate,
generating single-base additions and single-base deletions in a variety
of sequence contexts at rates that are consistently higher than those observed with wild-type p140 (Table I). This implies that the majority
of frameshift mutational intermediates made by wild-type p140 are
proofread by its intrinsic exonuclease. Note that the single-base
deletion error rate of exonuclease-deficient p140 increases from an
average value of 1.0 × 106 for the 97 non-iterated
nucleotides in the template sequence to an error rate of 26 × 106 for the 17 template nucleotides present in
homopolymeric runs of 4/5. This 26-fold increase in error rate with
increasing run length suggests that most of the frameshifts in
repetitive sequences result from misalignments formed by DNA strand
slippage (57), recently reviewed in Ref. 58.
The p55 subunit has no apparent effect on the frameshift fidelity of
wild-type p140. In contrast, p55 increases the frameshift error rates
of exonuclease-deficient p140 but in a sequence-dependent manner. Thus, p55 increases the deletion rate by 9-fold for
non-iterated nucleotides and by 3-fold for nucleotides present in
homopolymeric runs of two bases. However, it does not affect the rate
of deletions in runs of 3-5 bases. These data suggest that p55 may
promote extension of misaligned primer-templates in which the unpaired base is at or 1 base pair upstream of the 3' terminus but has little
effect when two correct terminal base pairs are possible in the
misaligned intermediate. A comparison of wild-type p140·p55 to
exonuclease-deficient p140·p55 also suggests that even when p55 is
present proofreading still corrects most of addition misalignments that
have an extra base in the primer strand. Proofreading is also apparent
for intermediates that have the extra base in template strand and lead
to deletions of non-iterated nucleotides or nucleotides present in 2- and 3-base homopolymeric runs.
Dislocation Mutagenesis--
Hypothetically, strand slippage in
repetitive sequences may also result in substitution errors by a
dislocation mechanism. This model suggests that strand slippage in a
template run is followed by incorporation of the next correct
nucleotide. Continued DNA synthesis results in a 
1 frameshift
mutation, and realignment followed by DNA synthesis results in a base
substitution error. In previous studies of mammalian DNA polymerase (59-61), this transient misalignment mechanism explained a hot spot
for T
G substitutions at position 70 (underlined) in the sequence
5'-GTTTT in the lacZ template. For three of the
four reactions catalyzed by pol in the present study, the spectra
of errors (not shown) revealed that position 70 is again a hot spot for
TG substitutions. The error rate for this particular substitution is
much higher than is the average rate for TG substitutions at the
other 22 sites in the template where this substitution can be scored
(Table II). Consistent with the
dislocation model and previous studies, pol also generated single T
deletions at a high rate in the TTTT run (Table II). A comparison of
rates for the wild-type and exonuclease-deficient p140 reactions
(19 × 106 versus 96 × 106) suggests that TG substitutions at position 70 are
proofread. A parallel comparison in the presence of p55 (84 × 106 versus 150 × 106)
suggests that p55 suppresses the efficiency of proofreading, perhaps by
promoting extension of a mismatch. For the exonuclease-deficient pol
reactions, the presence of p55 reduces the rate of deletions in the
TTTT run by 4-fold while enhancing the rate of TG substitutions at
position 70 by about 2-fold (Table II). This implies that p55 shifts
the balance from extension of the initial misalignment to extension of
a terminal mismatch after realignment.
Fidelity Measurements with Reversion Assays--
To reinforce and
extend the results of the forward mutation assay, we measured the rates
of base substitution and frameshift errors at specific template
positions using more sensitive (i.e. lower background)
reversion assays. In this approach, replication errors are scored as
blue plaque revertants of preexisting substitution or frameshift
mutations with colorless plaque phenotypes. Substrates previously
developed to score base substitutions, additions, and deletions in
repetitive sequences were employed to monitor the fidelity of the four
different pol DNA synthesis reactions studied above. The first
substrate monitors eight different single-base mismatches that revert a
TGA termination codon at positions 87-89 of the lacZ gene.
Wild-type p140 did not generate errors above the background reversion
frequency of uncopied DNA (0.5 × 105), yielding an
average substitution rate at the TGA codon of 1.7 × 106. The exonuclease-deficient mutant was at least
20-fold less accurate (Table III, top
line), implying that >95% of polymerase misinsertions within this
opal codon are proofread. As observed in the forward mutation assay,
p55 raised substitution error rates for both forms of p140, and
proofreading was still apparent. By comparison, heterodimeric D. melanogaster pol displayed a reversion frequency of 0.18 × 105 in the 
X174am3-based single
nucleotide reversion assay (62).
Three sets of frameshift reversion substrates were used. One set
contains runs of either four, six, or seven consecutive template T
residues in the 1 reading frame. Theoretically, blue plaque revertants can result from restoration of the correct reading frame by
additions (e.g. +1, +4, etc.) or by deletions
(e.g. 2, 5). However, the results of the forward
mutation assay demonstrate that the rate of single-base additions far
exceeds the rate of the other events that might yield blue plaques,
implying that results obtained with this set of reversion substrates
can be interpreted in terms of single-base addition error rates.
Synthesis by wild-type p140 failed to generate revertants above the
background reversion frequency of the control DNA with any of the three
substrates tested (2nd to 4th lines in Table III). This is consistent
with results from the forward mutation assay and extends the
observation of high base addition fidelity by wild-type p140 to include
a homopolymeric run of seven Ts. The exonuclease-deficient p140 was
substantially less accurate in all three assays, suggesting effective
proofreading of misaligned addition intermediates by the catalytic
subunit in runs of four, six, or seven Ts. In the presence of p55,
addition fidelity due to efficient proofreading is again observed for
runs of four and six Ts (2nd and 3rd lines in Table III). However, p55
increases the addition error rate of wild-type p140 in the run of seven
Ts (compare 6 × 105 to 55 × 105
in 4th line of Table III), while having little effect on the fidelity of exonuclease-deficient p140. This suggests that p55 reduces the
ability of wild-type p140 to proofread misalignments when the extra
primer-strand nucleotide can reside as many as 7 base pairs upstream of
the polymerase active site. These results are reminiscent of the
previous demonstration that porcine pol could not efficiently
recognize and excise pre-formed mispairs positioned more than four
nucleotides from the 3' terminus (34). Interestingly, Fan and Kaguni
(63) have recently described a possible physical interaction between
the accessory subunit and the exonuclease domain of pol .
The second set of frameshift substrates contains runs of template Ts in
the +1 reading frame. Based on the results of the forward assay,
revertants primarily result from single nucleotide deletions. Synthesis
by wild-type p140 ± p55 was accurate with the T3 run,
and the exonuclease-deficient p140 was substantially less accurate (5th
line in Table III). This pattern changes as the run length increases.
For example, with the T4 run (6th line), p140 is at least
20-fold more accurate than the exonuclease-deficient p140, but only
8-fold more accurate when p55 is present. In the T5 run
even wild-type p140 is inaccurate, such that inactivation of the
exonuclease and the presence of p55 only enhance the error rate for
single-base deletions by a factor of 2 or 3. This trend of decreasing
fidelity together with decreasing effects for the exonuclease or p55
continues as the length of the run increases, such that fidelity within
the T8 run is similar for all four polymerization reactions
(10th line in Table III). The lacZ reversion frequencies in
Table III were corrected for differences in run length to calculate error rates per detectable nucleotide polymerized. The results (Fig.
1) illustrate a quantitative relationship
between increasing run length and increasing error rate that is
predicted by the strand slippage model for formation of frameshift
errors. The results in Table III also reveal that the wild-type
p140·p55 complex deletes A·T base pairs in the T8 run
at a rate of 3800 × 106, a value 380-3200-fold
higher than the base substitution error rates of this complex (range of
1.2 to 10 × 106).
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Fig. 1.
Deletion error rates of wild-type and
exonuclease-deficient p140 within homonucleotide runs of increasing
length. A series of gapped M13mp2 DNA substrates containing
oligo(dT) tracts three to eight nucleotides in length were utilized in
in vitro mutational assays as described under
"Experimental Procedures." Gap-filling reactions utilized
exonuclease-proficient (open bars) or exonuclease-deficient
(closed bars) forms of human p140. Error rates were
calculated as described (51), using the data shown in Table III.
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A similar pattern of error rates as a function of protein composition
and run length emerged when we used a third set of frameshift substrates involving runs of template Cs in the +1 reading frame. However, in these runs, frameshift fidelity is substantially higher than with the T run substrates, illustrating the well known but poorly
understood effects of run composition and location on frameshift fidelity that have been seen before with other DNA polymerases (reviewed in Ref. 64).
Misincorporation by pol --
Base substitutions generated by
pol require selection and insertion of an incorrect nucleotide,
failure to proofread the misinserted nucleotide, and extension of the
resulting mispair. Possession of the exonuclease-proficient and
-deficient forms of pol allows an examination of these steps with
proofreading uncoupled from extension. To evaluate the selectivity of
nucleotide insertion by pol , we determined steady state kinetic
parameters for incorporating a single correct or incorrect nucleotide
onto a 3'-terminally matched primer-template. As expected for a
polymerase with a proofreading exonuclease, initial experiments with
exonuclease-proficient pol did not result in the accumulation of
improperly extended primers (data not shown). Subsequent experiments
utilized the exonuclease-deficient form of the enzyme. The p140 subunit
alone efficiently inserted dTMP opposite template A (Table
IV). The catalytic efficiencies of
inserting the three incorrect nucleotides were 25-60% as efficient as
insertion of dTMP, but significantly higher concentrations of the
incorrect dNTPs were required to achieve these efficiencies. The ratio
of the catalytic constants (kcat/Km) is an estimation of
the overall propensity of inserting a given nucleotide, and values for
incorrect nucleotides can be compared relative to the correct
nucleotide. As typified for a faithful DNA polymerase, p140
discriminated against insertion of the three incorrect nucleotides by
factors of 103-105 (Tables IV), and these data
are generally consistent with the error rates determined in the
gap-filling assays. Interestingly, inclusion of the p55 accessory
subunit had little effect on the kinetics of nucleotide insertion,
suggesting the effects of p55 on fidelity that were observed in the
gap-filling assays may stem from altered efficiencies of extending
mismatched or misaligned primer termini.
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Table IV
Single nucleotide insertion kinetics
Steady state kinetic values were measured for incorporation of a single
correct or incorrect dNMP residue onto a 3'-terminally matched
primer-template as described under "Experimental Procedures."
Reactions utilized 10-30 fmol of p140 Exo or p140
Exo · p55. All values are the averages of at least two
independent determinations.
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Mispair Extension--
The propensity of pol to insert a
single dTMP residue onto a variety of 3'-terminally mismatched
primer-templates was examined by steady state kinetic analysis. As
expected, mispaired 3' termini were extremely short lived when
incubated with wild-type p140 (mean kcat for
excision was >100/min, data not shown), so subsequent mispair
extension reactions were performed with exonuclease-deficient p140.
Whereas only nanomolar concentrations of dTTP were needed to permit
efficient extension of the matched G·C terminus by p140, Km(dTTP) values of 5-55 µM were
required for extension of A·C, T·C, G·G, and C·C
mispairs (Table V). The catalytic
efficiencies (kcat) of extending mispairs were
consistently less than 20% as efficient as extension of a paired
terminus at all dTTP concentrations. With values normalized to those
for a matched primer terminus, p55 enhanced the overall efficiency of
mismatch extension by factors ranging from about 2-fold for the A·C
mismatch to 70-fold for the C·C mismatch. The high concentration of
dTTP needed to extend a mismatched (C·C) primer-template and the
strong influence of p55 on efficiency of extension are illustrated in
Fig. 2. Nanomolar dTTP concentrations
were needed for efficient extension of a matched G·C terminus by
p140·p55. In contrast, micromolar dTTP concentrations were required
for extension of a 3'-terminal C·C mispair. Inclusion of p55 reduced
the concentration of dTTP needed for extension of the C·C mismatch
while it increased the kcat by 10-fold (Fig. 2
and Table V). Higher concentrations of dTTP also stimulated additional
misincorporation events.
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Table V
Kinetics of mispair extension
Steady state kinetic values were measured for incorporation of a single
dTMP residue onto the indicated 3'-terminally matched or mismatched
primer · templates, as described under "Experimental
Procedures." Reactions utilized 10-30 fmol of p140 Exo or
p140 Exo · p55. All values are the averages of at
least two independent determinations.
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Fig. 2.
Addition of p55 enhances extension of C:C
mismatches by exonuclease-deficient p140. Mispair extension
reactions ("Experimental Procedures") utilized oligonucleotide
substrates with a G·C pair or a C·C mispair at the 3' terminus of
the primer, as indicated. Reactions with G·C DNA included 10 fmol of
p140·p55 and dTTP at the indicated concentrations. Reactions with
C·C DNA included 0.5 pmol of p140 or p140·p55 and the indicated
concentrations of dTTP. The leftmost lane contained no
enzyme. Products were resolved by denaturing PAGE and visualized with a
PhosphorImager as described under "Experimental Procedures." The
position of the unextended oligonucleotide primer is indicated by the
arrow. The nucleotide sequence of the template strand is
indicated on the right side of the figure.
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DISCUSSION |
This study has several implications related to the accuracy of
human mitochondrial DNA transactions mediated by DNA polymerase .
Our results indicate that the base substitution fidelity of wild-type
human DNA polymerase is high, consistent with its central role in
mitochondrial DNA replication and repair. This high fidelity results
from high nucleotide selectivity, slow mismatch extension, and
efficient proofreading by the intrinsic 3'- to 5'-exonuclease. The data
with the recombinant proteins studied here are consistent with a number
of previous observations (30, 31, 62, 65) on the base substitution
fidelity of pol purified from natural sources. The data also reveal
that pol has high single-base addition and deletion fidelity for
errors at non-iterated and short repetitive sequences (Tables I and
III). Such events likely involve misalignments where the unpaired base
is at or near the primer terminus, at situations relevant to most of
the sequences in the 16,569-base pair human mitochondrial genome. The
data in Tables I and III and Fig. 1 further suggest that pol frameshift fidelity in most sequence contexts is enhanced by proofreading, as also indicated by previous studies of other DNA polymerases (66).
As predicted by the classical strand slippage hypothesis for synthesis
of repetitive sequences (57, 58), pol error rates increase with
increasing homopolymeric run length (Table III and Fig. 1). The
wild-type catalytic subunit and the p140·p55 complex both have low
frameshift fidelity in long runs despite the presence of an intrinsic
proofreading exonuclease that effectively proofreads most base
substitution intermediates. This suggests that proofreading efficiency
diminishes with increasing distance between the terminus and the
location of the extra nucleotide in the misaligned intermediate. This
effect of location can actually negate the contribution of proofreading
to the frameshift fidelity of pol in tracts of 6-8 nucleotides
(Table III). When combined with the increased error rate of pol as
a function of increasing run length (Fig. 1, results with
exonuclease-deficient p140), the minimal contribution of proofreading
implies that repetitive sequences in mtDNA are at relatively high risk
for insertion and deletion mutagenesis due to spontaneous strand
slippage during mitochondrial DNA replication. Consistent with this,
frameshift mutations within homopolymeric runs have been documented to
occur in vivo (67, 68). Interestingly, no homopolymeric runs
longer than eight nucleotides exist in human mtDNA, and only a single
homopolymeric run of eight nucleotides exists (1).
Strand slippage may also produce base substitution errors via a
transient misalignment (Table II). Such a dislocation mechanism has
been invoked previously to explain synthesis errors by two naturally
exonuclease-deficient DNA polymerases, pol and human immunodeficiency virus, type I-reverse transcriptase. This study of pol
provides the first evidence for substitutions by dislocation for
any family A DNA polymerase, and it also provides the first evidence
that dislocation errors can be suppressed by proofreading. Theoretically this could involve editing either the misaligned primer-template or the terminally mismatched intermediate. In the
absence of editing, the data in Table II indicate that p55 diminishes
the probability of extending misaligned termini (compare 64 × 106 to 18 × 106) while increasing the
probability of extending terminally mismatched primer termini (96 × 106 to 150 × 106). This shift in
mutational specificity is the first indication that a replication
accessory protein can modulate the rate of base substitutions arising
by dislocation. Although it suggests a model in which p55 promotes
realignment prior to continued DNA synthesis, addition of the accessory
subunit also seems to interfere with the ability of p140 to proofread
realigned but mispaired primer termini at dislocation loci. The two
competing effects, promoting realignment of primer-templates and
enhancing extension of mismatches, result in a clear base substitution
hot spot even with the wild-type p140·p55 enzyme complex (error rate
of 84 × 106).
Indeed, the results in Tables I, III, and V and Fig. 2 indicate that
p55 enhances the ability of the p140 pol catalytic subunit to
extend a variety of mismatched primer-templates but had no detectable
influence on the misinsertion rates of p140. This enhancement is
generally strongest for primer-templates wherein the mismatched or
unpaired bases can reside at or within a few base pairs of the primer
terminus, such as for base substitutions and frameshifts at
non-iterated or short repetitive sequences. In contrast, p55 has little
or no effect on frameshifts involving extra bases in runs of 5-8 base
pairs (Table III). Although the mechanistic explanation for this p55
enhancement must await further kinetic and structural analysis, the
error rate data suggest that p55 is somehow modulating the interactions
between p140 and the duplex primer-template that occur at and within
about 4-5 base pairs of the active site. The negligible p55 effect on
frameshift error rates in long homopolymeric tracts may also eventually
be instructive in light of its ability to enhance the processivity of
the catalytic subunit (47). Processivity factors, such as PCNA,
thioredoxin, and the 55-kDa accessory subunit of pol increase the
number of nucleotides incorporated each time a polymerase binds to DNA
by decreasing disassociation of the enzyme from DNA (47, 69, 70). Thus,
the enhanced binding afforded by these processivity factors reduces the
options available to the polymerase following a replication error.
Fidelity studies in vitro have shown that thioredoxin
increases base substitution errors for T7 polymerase copying
non-iterated DNA sequences (71), and gel-based oligonucleotide
extension assays have shown that PCNA enhances misincorporation by calf
thymus DNA polymerase 
by a factor of 27 (72). The reduction in base
substitution fidelity caused by the accessory subunit of pol is
consistent with this model; however, the analysis is somewhat more
complex for frameshift mutations. Thioredoxin increases the frequency
of 1 and 2 frameshift errors made by exonuclease-deficient T7 DNA
polymerase when copying both non-iterated sequences and short
homopolymeric runs (71). Similarly, p55 enhances 1 frameshifts for
random DNA sequences and shorter homonucleotide runs (Tables I and
III). Since frameshift mutations are caused by misalignment of the
primer and template strands during DNA synthesis, the fidelity effects
observed for these accessory factors could be explained by a reduced
opportunity for template realignment during
disassociation/reassociation events. In general homopolymeric runs are
particularly prone to frameshift mutations due to an increased
probability of slippage between the primer and template strands during
DNA synthesis. Misalignments in longer homonucleotide runs can be
stabilized by correctly paired nucleotides proximal to the primer
terminus, and such intermediates may not provoke transfer to the
exonuclease site. The three-dimensional structure of the T7
polymerase-thioredoxin complex shows that thioredoxin acts as a flap or
lid to the DNA-binding cleft of T7 DNA polymerase (73). Restricting the
geometry of the DNA-binding cleft may serve to suppress stabilization
of misaligned intermediates, resulting in an anti-mutator effect.
Indeed, +1 frameshift errors catalyzed by exonuclease-deficient T7 DNA
polymerase at a T5 run are reduced 46-fold by thioredoxin
(71).
We have shown that proofreading-deficient pol synthesizes DNA with
substantially reduced fidelity, thus predicting a mutator phenotype
in vivo. Consistent with this expectation, Zassenhaus and
co-workers(44) have clearly demonstrated that heart-specific overexpression of a transgene encoding exonuclease-deficient pol generated mutations in mtDNA and induced cardiomyopathy in their mice.
Interestingly, among 11 mtDNA mutations specifically attributed to the
transgene (as opposed to random errors or naturally occurring polymorphisms in the heart mtDNAs), 6-base substitution mutations occurred within homopolynucleotide runs (44). Similarly, stable episomal overexpression of exonuclease-deficient p140 in cultured human
cells resulted in a variety of point mutations, with C to T transitions
and G to A transitions dominating the mutant collection (43). The
subunit composition of pol in these two studies is uncertain.
Overexpression of the mutant catalytic subunit relative to natural
expression of the accessory subunit suggests pol may be functioning
as a monomer; however, the more processive heterodimer would likely
dominate synthesis of mtDNA in vivo. We and others (47, 49,
50, 63, 74, 75) have suggested the accessory subunit may help initiate
mtDNA synthesis. We have further suggested that efficient replication
of the mitochondrial chromosome requires the high processivity afforded
by the accessory subunit but that filling of short gaps during
mitochondrial base excision repair need not be processive and need not
require the accessory subunit (27, 45, 47). In Drosophila,
expression of the two pol genes is regulated by distinct mechanisms
(75), leaving open the possibility of unequal gene expression in human cells. Our results predict a scenario in which differential expression of the two subunits could modulate the fidelity of replication. Thus
age-related or tissue-specific changes in expression of the two
subunits might contribute to the accumulation of mutations associated
with mitochondrial disease and dysfunction. Additional articles
addressing mitochondrial DNA polymerase fidelity have been published
recently, indicating a calculated increase in fidelity upon the
addition of the accessory subunit for their in vitro reaction conditions and specific sequence context (76, 77). We also
observed enhanced fidelity at certain sequence contexts, but the
overall effect of the accessory subunit was to lower the fidelity of
DNA replication.
with and without
Exonucleolytic Proofreading and the p55 Accessory Subunit*
, and
TOP
ABSTRACT
INTRODUCTION
