From the
Laboratory of Molecular Genetics and
¶Laboratory of Structural Biology, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709
Received for publication, April 22, 2003
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ABSTRACT |
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INTRODUCTION |
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Multiple proteins exist in mammalian cells that theoretically could
participate in each step in BER. By virtue of its DNA polymerase and dRP lyase
activities (2), DNA polymerase
(pol
) has a major role in single nucleotide BER of uracil in
mammalian cells (3,
4). Three other human DNA
polymerases also have dRP lyase activity, pol
(5), pol
(6), and pol
(7), and pol
and pol
can substitute for pol
in BER reactions in vitro that
repair uracil paired opposite adenine or guanine
(6,
7). Such enzymatic redundancy
is interesting because the base substitution fidelity of these four DNA
polymerases differs over a remarkable range when they work alone to fill gaps
in DNA. For misincorporation of dGMP opposite template thymine,
proofreading-proficient human mitochondrial pol
has an error rate of
105
(8), human pol
has an
error rate of >101
(6,
911),
and human pol
(1214)
and pol
(15) have
error rates between these two values. Moreover, while the latter three
polymerases lack intrinsic exonuclease activity, human APE is reported to have
3' to 5' exonuclease activity that can excise nucleotides
misinserted by pol
(16). Thus APE or a
exonuclease such as TREX 1
(17,
18) may proofread errors made
by exonuclease-deficient DNA polymerases during BER.
The number of DNA bases subject to BER due to modification by normal
cellular processes like depurination, deamination, oxidation, and alkylation
has been estimated to exceed 10,000 per day
(19). Exposure of cells to
physical and chemical agents in the environment can further increase the
number of modified bases. Thus errors arising during BER could give rise to
one or more mismatches per day that, if uncorrected, could result in base
substitutions. For example, uracil-initiated BER in mammalian cell-free
extracts involving replacing one to eight nucleotides generated substitution
error frequencies ranging from 5.2 to 7.2 x
104
(2022).
That more than one DNA polymerase can generate BER errors in cell-free
extracts was demonstrated by differences in BER error specificity in extracts
of pol (+/+) and (/) mouse cells
(22).
To investigate the enzymes and mechanisms responsible for determining the
fidelity of BER pathways, here we describe an assay to determine error rates
during BER reactions reconstituted with purified proteins. We measure base
substitution error rates for repair of uracil opposite adenine or guanine, in
single nucleotide BER reactions reconstituted with purified proteins, one of
which is human pol . The results establish the minimum fidelity of this
single nucleotide BER reaction and indicate that BER fidelity is at least
severalfold higher than is the fidelity of single nucleotide gap filling DNA
synthesis by pol
alone.
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EXPERIMENTAL PROCEDURES |
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Preparing Gapped DNADuplex DNA was formed by annealing two oligonucleotides (5'-AATTCCAGCTCGGTACCGGGCTAGCCTTTGGAGTCGACCTGCAGA-3') and (5'-AATTTCTGCAGGTCGACTCCAAAGGCTAGCCCGGTACCGAGCTGG-3'). This duplex was inserted into the EcoRI site of M13mp2. The resulting modified M13mp2 phage was used to prepare single-stranded DNA and double-stranded DNA as described previously (23). Gapped DNA was prepared by hybridizing circular plus strand phage DNA to the minus strand of the large duplex fragment produced by an EcoRI/PstI double digestion of the double-stranded DNA, as described previously (23). The gapped DNA was separated by agarose gel electrophoresis and purified from gel slices by electroelution using an Elutrap (Schleicher and Schuell).
Substrates for BERFor experiments with a G:U substrate, a duplex was formed by annealing a 45-mer (5'-CCAGCTCGGTACCGGGCTAGCCTTTGGAGTCGACCTGCAGAAATT-3') and a 45-mer (3'-GGTCGAGCCATGGCCCGATUGGAAACCTCAGCTGGACGTCTTTAA-5'). For experiments with the A: U substrate, a duplex was prepared by annealing a 45-mer (5'-CCAGCTCGGTACCGGGCTAGCCTTTGGAGTCGACCTGCAGAAATT-3') and a 45-mer (3'-GGTCGAGCCATGGCCCGAUCGGAAACCTCAGCTGGACGTCTTTAA-5').
In Vitro Base Excision Repair AssaysBER reactions were
performed according to a previously described procedure
(24) with modifications. The
reaction mixture contained 4 pmol of uracil containing substrate, 50
mM Hepes, pH 7.5, 2 mM dithiothreitol, 0.2 mM
EDTA, 100 µg/ml bovine serum albumin, and 10% glycerol. Reactions (12
µl) were initiated by adding 400 fmol of UDG and incubated at 37 °C for
5 min. The reaction (40 µl) was then supplemented with 5 mM
MgCl2, 4 mM ATP, 10 nM APE, 10 nM pol ,
100 nM DNA ligase I or T4 DNA ligase (20 units/µl), 10
µM each of dATP, dCTP, dGTP, dTTP, and either 0.3
µM [
-32P]dCTP (for G·U substrate) or
0.3 µM [
-32P]dTTP (for A·U substrate)
and incubated at 37 °C for 10 min. Reactions were stopped by adding 4
µl of 0.5 M EDTA. Molar ratios of pol
to substrate were
as follows: 1:1 for wild type pol
, 4:1 for R283K pol
, and 16:1
for R283A pol
.
Preparation of Repaired DNAThe repaired duplex DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Precipitated DNA was dissolved and the DNA was digested with PstI, followed by heat inactivation of PstI for 20 min at 80 °C. After adding an equal volume of gel loading buffer and incubating for 2 min at 95 °C, the DNA were separated by electrophoresis in a 12% polyacrylamide gel containing 7 M urea in 89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.8. The repaired 35-mer oligonucleotide was located by autoradiography and excised, the gel slice was crushed and soaked in water, and the single-stranded oligonucleotide was recovered by ethanol precipitation. The amount of repaired DNA recovered was calculated from the radioactivity recovered, with estimated yields of about 50%.
Hybridizing the Repaired Oligonucleotide to Gapped DNAThe repaired (or unrepaired control) oligonucleotide was incubated for 3 min at 75 °C and then mixed in 10-fold molar excess with the gapped DNA in a buffer containing 300 mM NaCl, 30 mM sodium citrate. This mixture was incubated at 48 °C for 3 min, 42 °C for 10 min and then slowly cooled to room temperature.
Measurement of Reversion FrequencyDNA samples were introduced into Escherichia coli MC1061 by electroporation, cells were plated, M13 plaque colors were scored, and reversion frequencies were calculated as described previously (23). DNA was isolated from collections of independent revertants to define the sequence change responsible for the dark blue plaque phenotype.
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RESULTS AND DISCUSSION |
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Measurements of Single Nucleotide BER FidelityA BER
reaction was performed with human UDG, APE, DNA pol , and DNA ligase I.
Reaction conditions were similar to those previously demonstrated to replace
uracil paired opposite A or G with a single correct nucleotide
(24). Control experiments (not
shown) demonstrated that UDG removed >95% of the uracil present in the
duplex substrate in the reaction. The DNA products of complete BER reactions
included the expected 45 base pair ligated DNA characteristic of complete BER
(Fig. 1C, lanes
2 and 3). Shorter chain length products were also observed that
result from uracil removal, APE incision of the DNA backbone, and
incorporation of the radiolabeled dNTP but without ligation. The 27-base pair
product (Fig. 1C,
lanes 1, 2, and 4) results from incorporation of one dNTP,
while the slightly longer products result from limited strand displacement.
Following digestion of the duplex products of BER with PstI, repaired
35-mer minus strand products (e.g.
Fig. 1C, lane
5) were isolated and hybridized in 10-fold molar excess to gapped M13
DNA, and the reversion frequency of these circular M13 genomes was determined.
Repair of uracil opposite template A in reactions containing correct dTTP but
no incorrect nucleotides resulted in products whose reversion frequency was
3.4 x 104
(Table I, Experiment 1). The
products of a BER reaction containing an equimolar concentration of all four
dNTPs, thus providing the opportunity to misincorporate nucleotides, yielded a
similar reversion frequency of 5.6 x 104.
Both values are similar to the reversion frequency of 3.9 x
104 observed when an unrepaired uracil containing
35-mer was hybridized to the gap (Table
I, line 2). Thus, pre-existing errors introduced during chemical
synthesis of oligonucleotides contribute significantly to reversion
frequencies in this range. Similar results were obtained for BER reactions
that replaced uracil with dCTP opposite template G
(Table I, Experiment 2).
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DNA from independent blue plaque revertants were then sequenced. Among 24
revertants from reactions containing A-U duplex and only dTTP
(Table II, Experiment 1), 19
contained a TGG codon consistent with misincorporation of dCTP opposite A.
Since no dCTP was present in the reaction, these data represent the background
noise in the assay. Among 24 revertants recovered from BER reactions
containing equal amounts of all four dNTPs, 17 contained a TGG codon
consistent with misincorporation of dCTP opposite A, 4 contained a TTG codon
consistent with misincorporation of dATP, and no revertant contained a TCG
codon reflecting dGTP misincorporation. This allows calculation of mismatch
specific reversion frequencies (second number in columns 48 in
Table II), as well as
"less than or equal to" error rates for the mismatches scored at
the A and G of the TAG codon (third number in columns 48). For wild
type pol -dependent single nucleotide BER performed with equimolar
concentrations of all four dNTPs, these rates range from
0.3 to
2.8
x 104. Error rates could be even lower,
with these data establishing the minimum base substitution fidelity of base
excision repair conducted by UDG, APE, DNA ligase 1, and wild type DNA
polymerase
.
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The Fidelity of Single Nucleotide BER Using Inaccurate Reaction ConditionsBER was then performed in reactions intended to generate base substitutions at higher rates. One approach employed reactions containing a 50-fold excess of incorrect dNTPs. With the A-U substrate, dCTP, dGTP, and dATP were present at 500 µM, and correct dTTP was present at 10 µM. This reaction generated products with a reversion frequency of 11 x 104 (Table I, Experiment 1). Sequence analysis revealed no increase in rate for the A-dATP or A-dGTP mismatches, but the error rate for A-dCTP was 12 x 104, well above the background value for this mismatch (Table II). A reaction with the G-U substrate and a 50-fold molar excess of dATP and dGTP over dCTP (to force the two transversions than can be scored) resulted in error rates for the G-dATP and G-dGTP mismatches of 4.5 x 104 and 5.3 x 104, respectively (Table II). Both values are higher than those observed for these mismatches in parallel reactions performed with equimolar dNTPs.
The second approach to increase error rates employed variants of DNA
polymerase containing lysine or alanine in place of arginine 283. In
comparison to wild type pol
, these polymerases were previously shown to
have reduced base substitution fidelity during gap-filling synthesis
(1214,
25), with the fidelity of the
R283A variant being lower than that of R283K. A BER reaction containing
equimolar dNTPs and the R283K variant repaired the A-U substrate with rates
for the A-dATP and A-dGTP mismatches that were not substantially different
from the values observed with wild type pol
for these same mismatches
(Table II, compare Experiments
1 and 3). This is consistent with the observation that the R283K replacement
did not strongly increase error rates for these mismatches during gap filling
(14). However, for the A-dCTP
mismatch, the BER error rate with R283K was higher (4.5 x
104) than that observed with wild type pol
(
2 x 104), consistent with the lower
fidelity of R283K for the A-dCTP mismatch during gap filling
(14). A BER reaction
containing equimolar dNTPs and the R283A variant repaired the A-U substrate
with error rates for the A-dCTP and A-dATP mismatches that were both more than
10-fold higher than observed with wild type pol
(Table II).
Comparison of Error Rates for BER and Gap Filling by pol
AloneWe previously determined error rates for wild type pol
and the R283K/A variants during synthesis to fill a gap containing a
single template adenine (13,
14). Those studies employed
equal concentrations of the four dNTPs and T4 DNA ligase to seal the nick
following gap filling. To compare those results to BER fidelity, BER fidelity
measurements were also performed with the R283K and R283A variants in
reactions containing T4 DNA ligase rather than DNA ligase 1. The results were
similar (Tables I and
II, Experiment 4), suggesting
that the ligase was not a critical determinant of fidelity under these
conditions. We also measured error rates for wild type pol
during
single base (adenine) gap-filling synthesis
(14) in the presence of a
50-fold excess of dCTP, dATP, and dGTP over dTTP. The data allow comparison of
the fidelity of pol
gap filling to the fidelity of pol
-dependent
BER, under conditions in which BER generates one or more of the three possible
substitution errors at rates above the background noise of the assay. The
results show that BER error rates (Fig.
2, gray bars) are severalfold lower than error rates for
gap filling by pol
alone (black bars). The differences suggest
that the nucleotide selectivity of pol
may be enhanced during BER by
the other proteins in the reaction, or perhaps by the presence of the 5'
dRP group, which is not excised until after polymerization has occurred. This
could result from increased efficiency of correct nucleotide incorporation to
enhance fidelity (26).
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Another possibility for the higher apparent fidelity of BER is proofreading
by the reported 3' to 5' exonuclease activity of APE
(16). The 38-fold
differences observed (Fig. 2) are consistent with removal of 6787% of misinsertions. If this is due
to APE proofreading, this efficiency is modest compared with the 90 to >99%
efficiency of editing by most DNA polymerases with intrinsic exonucleases. In
the latter cases, the contribution of proofreading to fidelity depends on the
rate of excision of the misinserted nucleotide relative to the rate of
extension of the mismatched primer terminus. For enzymes like pol and
pol
, the polymerase-exonuclease competition for the DNA substrate is
between two active sites present in the same protein. Excision is usually
strongly preferred over mismatch extension, whose rate is normally low and
highly sensitive to the concentration of the next correct dNTP to be
incorporated. However, proofreading of errors made by exonuclease-deficient
polymerases like pol
during single nucleotide BER could differ in at
least two ways. Since only a single nucleotide is incorporated, there is no
need for further extension, so proofreading efficiency during single
nucleotide BER may not depend on the concentration of the next correct
nucleotide. This could be advantageous for BER reactions that need to occur
throughout the cell cycle regardless of whether dNTP concentrations are as
high as in the S phase or following DNA damage
(27) or as low as in the
G1 phase. Also, once the mismatched DNA is generated, the
competition for this nicked, mismatched DNA substrate is between the ligase
active site and the exonuclease active site in two proteins separated in the
single nucleotide BER reaction pathway by pol
. Substrate partitioning
between proteins normally separated by two other enzymatic steps (polymerase
and dRP lyase) represents a novel extension of the general idea that the
product of one enzymatic step in BER is directly handed off to the next enzyme
in the pathway (28,
29). That idea was put forth
to explain how coordinating the BER step could avoid generating cytotoxic BER
intermediates, whereas the current refinement would avoid not the cytotoxic
but rather the mutagenic consequences of aberrant BER. Moreover, proofreading
by a separate exonuclease may allow regulation of BER fidelity. For example,
proofreading may suppress mutagenesis resulting from environmental stress, but
it may be counterproductive for putative uracil-initiated BER reactions that
could promote somatic hypermutation of immunoglobulin genes (reviewed in Ref.
30).
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FOOTNOTES |
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Present address: Inst. for Molecular and Cellular Biology, Osaka
University, 1-3, Yamada-oka, Suita, Osaka 565-0781, Japan.
|| To whom correspondence should be addressed: Laboratory of Molecular Genetics, NIEHS, NIH, DHHS, P. O. Box 12233, 111 T. W. Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-2644; Fax: 919-541-7613; E-mail: kunkel{at}niehs.nih.gov.
1 The abbreviations used are: BER, base excision repair; AP,
apurinic/apyrimidinic; APE, AP endonuclease; dRP,
5',2'-deoxyribose-5-phosphate; pol, polymerase; UDG, uracil DNA
glycosylase.
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REFERENCES |
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