From the Laboratory of Molecular Genetics and
Laboratory of Structural Biology, NIEHS, National Institutes
of Health, Research Triangle Park, North Carolina 27709, the
§ Institute for Molecular and Cellular Biology, Osaka
University and CREST Japan Science and Technology Corporation, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan, and the ¶ Institute of
Physical and Chemical Research (RIKEN), Wako-shi, Saitama
351-0198, Japan
Received for publication, September 29, 2000, and in revised form, November 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human DNA polymerase The UmuC/DinB superfamily of DNA polymerases includes human DNA
polymerase Current models suggest that pol Materials--
Materials for the SV40 replication fidelity assay
were from previously described sources (17-20), and
exonuclease-deficient human pol SV40 Replication Fidelity Measurements--
SV40
origin-dependent replication reactions were performed using
extracts of human TK6 and HeLa cells as described previously (18, 20). When analyzed by agarose gel electrophoresis, the products
generated in the reactions listed in Table I were similar to those seen
in earlier studies (16-18, 21). Analysis of the lacZ mutant
frequency of the replication products by introduction into an E. coli lacZ Processivity Analysis--
Reactions (25 µl) contained 40 mM Tris-HCl (pH 8.0), 10 mM
dithiothreitol, 6.5 µg of BSA, 60 mM KCl, 2.5%
glycerol, 10 mM MgCl2, 1 mM dNTPs,
5 nM 30-mer template primed at a 1.2 to 1 molar ratio with
a 5'-32P-labeled 20-mer oligonucleotide and 0.005 nM pol Kinetic Analysis of Mismatch Extension--
Reactions (25 µl)
were as above except that they contained 200 nM template
primed at 1.2 to 1 molar ratio with a 5'-32P-labeled
primer, 2 nM pol Previous studies indicated that the fidelity of replication of
undamaged M13mp2 DNA by extracts of human TK6 or HeLa cells is high
(17-19, 21). In this study, inclusion of human pol , the product of the skin
cancer susceptibility gene XPV, bypasses UV photoproducts
in template DNA that block synthesis by other DNA polymerases. Pol
lacks an intrinsic proofreading exonuclease and copies DNA with low
fidelity, such that pol
errors could contribute to mutagenesis
unless they are corrected. Here we provide evidence that pol
can
compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that
pol
has low processivity and extends mismatched primer termini less
efficiently than matched termini. These properties could provide an
opportunity for extrinsic exonuclease(s) to proofread pol
-induced
replication errors. When we tested this hypothesis during replication
in human cell extracts, pol
-induced replication infidelity was
found to be modulated by changing the dNTP concentration and to be
enhanced by adding dGMP to a replication reaction. Both effects are
classical hallmarks of exonucleolytic proofreading. Thus, pol
is
ideally suited for its role in reducing UV-induced mutagenesis and skin
cancer risk, in that its relaxed base selectivity may facilitate
efficient bypass of UV photoproducts, while subsequent proofreading by
extrinsic exonuclease(s) may reduce its mutagenic potential.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(pol
),1
the product of the XPV (Rad30A) skin cancer
susceptibility gene (1, 2). Human pol
has the ability to
efficiently copy cis-syn thymine-thymine dimers in template
DNA (1). Mutations in XPV that inactivate pol
(1, 2)
render cells hypermutable by UV radiation (3-7) and defective in
replicating DNA containing UV photoproducts (Ref. 8 and references
therein). These facts demonstrate an important role for pol
in
modulating UV-induced mutagenesis and in reducing the risk of human
skin cancer. Kinetic analysis reveals that human pol
inserts
incorrect nucleotides opposite undamaged (9, 10) and damaged (10)
template bases more efficiently than most other DNA polymerases.
Moreover, human pol
lacks an intrinsic proofreading exonuclease
activity (9), and its base substitution error rates when copying
undamaged DNA are much higher than are those of most other eukaryotic
polymerases, whether they have proofreading activity or not (9). We (9) and others (10) have suggested that this generally relaxed
discrimination ability during DNA synthesis may be critical to the
ability of human pol
to bypass certain DNA lesions that impede
synthesis by other DNA polymerases (1, 8, 11, 12).
competes with other replicative
polymerases for 3'-OH termini at a replication fork (reviewed in Refs.
13-15). Given the intrinsically low fidelity of pol
, mechanisms
may exist to prevent errors by pol
from reducing the accuracy of
chromosomal replication. We previously suggested (9) two obvious error
correction mechanisms, exonucleolytic proofreading of pol
mistakes
by a separate exonuclease(s) and post-replication DNA repair of
mismatches generated by pol
. To test the proofreading hypothesis,
here we examine the effects of pol
on the accuracy of replication
of double-stranded DNA catalyzed by the replication apparatus in
extracts of human cells. For this we used the SV40 replication system,
whose protein requirements are similar to those of human chromosomal
replication (16). We first establish replication conditions under which
pol
reduces replication fidelity, suggesting that pol
can
indeed compete with other polymerases during semiconservative DNA
replication. We then demonstrate that pol
has two intrinsic
biochemical properties, low processivity and slow mismatch extension,
that could allow a separate exonuclease to compete for mismatched
primer termini at the replication fork. Finally, we demonstrate that
pol
-induced replication infidelity depends on the dNTP
concentration and is increased in the presence of a deoxynucleoside
monophosphate, both classical hallmarks of exonucleolytic proofreading.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was described earlier (1, 9).
-complementation host strain and plating to
score wild-type (blue) and mutant (colorless and light blue) plaques
was performed as described previously (20).
. Ten-µl aliquots were removed after 5, 15, or
30 min at 37 °C and analyzed by electrophoresis in a 16%
polyacrylamide gel in parallel with products of sequencing reactions on
the same template. Product bands were quantified by phosphorimagery.
, and either dATP or dGTP. The
template-primers are shown in the legend to Table III. Aliquots were
removed at 2, 4, 6, and 8 min and products separated by electrophoresis
in 16% polyacrylamide gels and quantified by phosphorimagery.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
in a
replication reaction reduced fidelity, as indicated by the concentration-dependent increase in the frequency of
lacZ mutants among the M13mp2 products of semiconservative
replication (Table I, Experiment 1). No
increase in mutant frequency was observed when excess human pol
was
included or when pol
was incubated with the extract in the absence
of the SV40 large T-antigen that is required for replication from the
SV40 origin. In fact the mutant frequencies under both reaction
conditions were within the range of frequencies (3-7 × 10
4) for control DNA that had not been
replicated in the extract.
Replication infidelity induced by pol
4 to
7 × 10
4.
DNA sequence analysis of 28 independent lacZ mutants from
the reaction containing 100 nM pol revealed the
presence of 17 single-base substitutions, 11 of which were consistent
with incorporation of dGTP opposite T. One clone had a tandem
double-base substitution and seven contained a single nucleotide
deletion. This error specificity is remarkably similar to that of pol
during gap-filling synthesis (9). A 14-nucleotide insertion was
also recovered and three of the lacZ mutants contained two
widely separated sequence changes, circumstances not encountered in
previous studies of replication fidelity. Single base substitution and
frameshift error rates calculated from these data are higher than for
replication in the absence of added pol
(Table
II, Experiment 1). Overall, these data
suggest that pol
is capable of competing with other DNA polymerases
at the replication fork and generates both base substitution and
frameshift mutations.
|
We previously suggested that the mutagenic potential of pol in
human cells might be reduced if pol
-dependent errors
were proofread (9). Several experiments were conducted to test this hypothesis. First, primer extension reactions were performed using the
lacZ template and a 1000-fold molar excess of
template-primer over enzyme, a condition that results in a single cycle
of processive synthesis. Analysis of reaction products (Fig.
1) demonstrated that pol
polymerizes
one to ten nucleotides per cycle of enzyme binding dissociation and
that the probability of termination of processive synthesis after each
incorporation varies between about 40 and 70%. This result
quantitatively confirms the earlier observation (8) that human pol
has low processivity, a property that could provide an exonuclease
access to a template-primer containing a mismatch.
|
All DNA polymerases studied to date extend mismatched template-primers
less efficiently than matched termini. To determine whether this is
also the case with human pol , we performed extension reactions to
obtain steady-state kinetic constants from which extension efficiencies
for matched and mismatched termini were calculated. The results (Table
III) indicate that pol
extended mismatched termini less efficiently than matched termini by
factors of from 3-fold (T
G mismatch) to more than 100-fold (G
A
mismatch). These data are consistent with the recent qualitative
demonstration that human pol
extends mismatched termini less
efficiently than matched termini with undamaged DNA and at sites of DNA
damage (8) and with a recent report that yeast pol
also extends mismatched termini less efficiently than matched termini (22).
|
For polymerases having intrinsic proofreading exonucleases, slow
polymerization increases the opportunity for movement of the primer
terminus to an exonuclease active site for excision of a misinserted
nucleotide (recently reviewed in Ref. 23). While pol lacks an
intrinsic proofreading activity (9), it is possible that editing could
be performed by a separate exonuclease that is physically associated
with the replication machinery, as is the case in E. coli.
To determine whether replication errors induced by
exonuclease-deficient pol
could be edited by an extrinsic exonuclease, replication fidelity was examined in a human extract under
conditions known to modulate proofreading activity. One hallmark of
proofreading is the "next nucleotide effect" (reviewed in Ref. 24).
Since the probability of polymerization from a mismatch (or a
misalignment) depends on the concentration of the next correct
nucleotides to be incorporated, at high dNTP concentrations polymerization is favored over editing, and fidelity is reduced. This
approach has already been used successfully to detect proofreading of
base substitution (18, 19) and frameshift errors (19, 25) produced by
the human replication complex. However, in reactions containing
equimolar dNTPs, a next nucleotide effect is difficult to detect with
the M13mp2 forward mutation assay, because replication fidelity is very
high (17-21, 25). Thus, replication in an extract to which excess pol
was not added generated products with lacZ mutant
frequencies that were similar to unreplicated DNA control values (Table
I, Experiment 2). In contrast, when replication reactions were
performed in the presence of pol
, fidelity decreased as the dNTP
concentration was increased from 10 to 1000 µM (Table I,
Experiment 2). The fact that the fidelity was higher in reactions lacking exogenous pol
indicates that the majority of errors being
proofread were dependent on pol
.
A second hallmark of proofreading activity is reduced replication
fidelity in the presence of a high concentration of dNMP (18-20), the
product of proofreading exonucleases. In the present study, inclusion
of 2 mM dGMP in a replication reaction containing 100 µM dNTPs and to which pol had been added (Table I,
Experiment 3) also reduced replication fidelity (lacZ mutant
frequency of 45 × 10
4 compared with
20 × 10
4 for the equivalent reaction
without dGMP). Again, the lower fidelity was dependent on pol
. Both
results imply that pol
-induced replication errors can be proofread.
We also examined whether pol competes for primer termini on
the leading strand, the lagging strand, or both. Here we employed a
strategy used previously (19), in which excess dGTP is included in a
replication reaction to monitor misincorporation of dGTP during lagging
strand replication of the M13mp2 viral (+) strand or during leading
strand replication of the complementary (
) strand. A reaction
containing 50 nM pol
and a 50-fold molar excess of dGTP
generated replication products whose lacZ mutant frequency
was strongly elevated relative to control values (Table I, Experiment
4). Sequence analysis of 29 independent lacZ mutants recovered from this reaction revealed 33 single base substitutions, 31 of which reflected misincorporation of dGMP. Thirty were due to
misincorporation opposite template T, the most frequent error generated
by pol
during gap-filling DNA synthesis (9). Of these, nine were
consistent with pol
misincorporation during replication of the
leading strand and 21 reflected pol
misincorporation during
replication of the lagging strand (Table II). The error rates
calculated from these data are much higher than those observed for
replication with excess dGTP in the absence of excess pol
(Table
II). Notably, seven independent lacZ mutants contained the
same T to C substitution at template nucleotide 121. At this site,
misinsertion of dGTP opposite this T is followed by correct incorporation of dGTP, which was present at 1000 µM.
Thus, this hot spot may reflect misinsertion by pol
followed by
suppression of proofreading due to the high dGTP concentration.
Overall, these data imply that pol
competes with the enzymatic
machinery that replicates both the leading and lagging strands.
At first glance, the idea that pol is intrinsically inaccurate
seems paradoxical in light of its role in suppressing UV-induced mutagenesis (3-7) and lowering the incidence of skin cancer (1, 2). In
fact, we believe that the properties of pol
are ideally suited for
this critical role. We have suggested that the intrinsically low
fidelity of pol
is a reflection of relaxed selectivity that facilitates highly efficient bypass of UV photoproducts that block other replicative polymerases. This implies that the low fidelity observed in vitro is not simply due to the lack of an
accessory protein that might enhance pol
selectivity in
vivo. We further suggest that a high probability of termination of
processive synthesis (Fig. 1) and slow extension of mismatches (Table
III) facilitates proofreading (Table I). Proofreading could be
performed by exonucleases intrinsic to replicative DNA polymerases
and
or by a separate exonuclease (26, 27). In the former case, the
exonuclease active site of pol
or pol
could directly bind the
mismatch for immediate removal, as seen with the replicative T7 DNA
polymerase (28). Even if pol
extends a mismatch for a few
nucleotides (note that discrimination for extension of a T
G mismatch
in Table III is only 3-fold), pol
or
may still be able to edit
a mismatch. This is suggested by studies (reviewed in Ref. 23) showing
that some polymerases contact the DNA for five base pairs upstream of
the polymerase active site, such that the presence of an embedded mismatch could still promote excision over polymerization. It is also
possible that errors that escape proofreading could be corrected by
mismatch repair (29, 30), thereby further contributing to the fidelity
of the overall bypass process. Finally, our data indicating that pol
can compete for 3'-OH termini during replication by leading and
lagging strand replication proteins suggest that genome instability
could result from conditions that promote this competition, such as a
change in the ratio of pol
relative to other polymerases. This
could result from overexpression of pol
, from a reduction in
polymerization efficiency due to mutations, or from reduced expression
of other polymerases. As one example of the latter, decreased cellular
levels of pol
have recently been shown (31) to promote genomic
instability by an unknown mechanism.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank William A. Beard and Youri Pavlov for critical evaluation 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@niehs.nih.gov.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.C000690200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
pol , DNA
polymerase
;
pol
, DNA polymerase
;
pol
, DNA polymerase
;
pol
, DNA polymerase
.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K., and Hanaoka, F. (1999) Nature 399, 700-704[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Johnson, R. E.,
Kondratick, C. M.,
Prakash, S.,
and Prakash, L.
(1999)
Science
285,
263-265 |
3. | Maher, V. M., Ouellette, L. M., Curren, R. D., and McCormick, J. J. (1976) Nature 261, 593-595[Medline] [Order article via Infotrieve] |
4. | Myhr, B. C., Turnbull, D., and DiPaolo, J. A. (1979) Mutat. Res. 62, 341-353[Medline] [Order article via Infotrieve] |
5. | Wang, Y. C., Maher, V. M., and McCormick, J. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7810-7814[Abstract] |
6. | Wang, Y. C., Maher, V. M., Mitchell, D. L., and McCormick, J. J. (1993) Mol. Cell. Biol. 13, 4276-4283[Abstract] |
7. | Waters, H. L., Seetharam, S., Seidman, M. M., and Kraemer, K. H. (1993) J. Invest. Dermatol. 101, 744-748[Abstract] |
8. |
Masutani, C.,
Kusumoto, R.,
Iwai, S.,
and Hanaoka, F.
(2000)
EMBO J.
19,
1-10 |
9. | Matsuda, T., Bebenek, K., Masutani, C., Hanaoka, F., and Kunkel, T. A. (2000) Nature 404, 1011-1013[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Johnson, R. E.,
Washington, M. T.,
Prakash, S.,
and Prakash, L.
(2000)
J. Biol. Chem.
275,
7447-7450 |
11. |
Masutani, C.,
Araki, M.,
Yamada, A.,
Kusumoto, R.,
Nogimori, T.,
Maekawa, T.,
Iwai, S.,
and Hanaoka, F.
(1999)
EMBO J.
18,
3491-3501 |
12. |
Johnson, R. E.,
Prakash, S.,
and Prakash, L.
(1999)
Science
283,
1001-1003 |
13. |
Johnson, R. E.,
Washington, M. T.,
Prakash, S.,
and Prakash, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12224-12226 |
14. |
Woodgate, R.
(1999)
Genes Dev.
13,
2191-2195 |
15. |
Friedberg, E. C.,
Feaver, W. J.,
and Gerlach, V. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5681-5683 |
16. | Waga, S., and Stillman, B. (1994) Nature 369, 207-212[CrossRef][Medline] [Order article via Infotrieve] |
17. | Roberts, J. D., and Kunkel, T. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7064-7068[Abstract] |
18. | Roberts, J. D., Thomas, D. C., and Kunkel, T. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3465-3469[Abstract] |
19. |
Izuta, S.,
Roberts, J. D.,
and Kunkel, T. A.
(1995)
J. Biol. Chem.
270,
2595-2600 |
20. | Roberts, J. D., and Kunkel, T. A. (1993) Methods Mol. Genet. 2, 295-313 |
21. | Thomas, D. C., Roberts, J. D., Sabatino, R. D., Myers, T. W., Tan, C. K., Downey, K. M., So, A. G., Bambara, R. A., and Kunkel, T. A. (1991) Biochemistry 30, 11751-11759[Medline] [Order article via Infotrieve] |
22. | Haracska, L., Yu, S.-L., Johnson, R. E., Prakash, L., and Prakash, S. (2000) Nat. Genet. 25, 458-461[CrossRef][Medline] [Order article via Infotrieve] |
23. | Kunkel, T. A., and Bebenek, K. (2000) Annu. Rev. Biochem. 69, 497-529[CrossRef][Medline] [Order article via Infotrieve] |
24. | Kunkel, T. A. (1988) Cell 53, 837-840[Medline] [Order article via Infotrieve] |
25. | Roberts, J. D., Nguyen, D., and Kunkel, T. A. (1993) Biochemistry 32, 4083-4089[Medline] [Order article via Infotrieve] |
26. |
Höss, M.,
Robins, P.,
Naven, T. J.,
Pappin, D. J.,
Sgouros, J.,
and Lindahl, T.
(1999)
EMBO J.
18,
3868-3875 |
27. |
Mazur, D. J.,
and Perrino, F. W.
(1999)
J. Biol. Chem.
274,
19655-19660 |
28. | Johnson, K. A. (1993) Annu. Rev. Biochem. 62, 685-713[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Wang, H.,
Lawrence, C. W.,
Li, G. M.,
and Hays, J. B.
(1999)
J. Biol. Chem.
274,
16894-16900 |
30. |
Liu, H.,
Hewitt, S. R.,
and Hays, J. B.
(2000)
Genetics
154,
503-512 |
31. |
Kokoska, R. J.,
Stefanovic, L.,
DeMai, J.,
and Petes, T. D.
(2000)
Mol. Cell. Biol.
20,
7490-7504 |
32. |
Mendelman, L. V.,
Petruska, J.,
and Goodman, M. F.
(1990)
J. Biol. Chem.
265,
2338-2346 |