From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061
Received for publication, October 3, 2000
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ABSTRACT |
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DNA polymerase DNA polymerase The high fidelity of replicative DNA polymerases arises, in part,
because their active sites are intolerant of the distorted geometry
resulting from mispairs between the template residue and the incoming
nucleotide (7). Steady state kinetic studies of yeast and human Pol The accuracy of synthesis by DNA polymerases depends on the frequency
of incorporation of incorrect nucleotides into DNA and on the frequency
of extension of the mismatched primer termini. Extension of mismatched
primers is a critical step in mutation fixation, because in the absence
of efficient extension, the mismatched nucleotide can be excised by a
proofreading exonuclease, or if the mismatch is not excised, cell death
may ensue as a result of incomplete DNA synthesis. Thus, for a mutation
to be expressed, extension from the misincorporated nucleotide must
occur. To better understand how Pol DNA Substrates--
DNA substrates containing all possible
correct base pairs or mispairs at the 3' primer terminus were generated
using four different oligodeoxynucleotide primers and four
oligodeoxynucleotide templates. The four 45-nucleotide primers have the
following sequence: 5'-GTTTT CCCAG TCACG ACGAT GCTCC GGTAC TCCAG TGTAG
GCATN, where N is G, A, T, or C. The four 52-nucleotide templates have
the following sequence: 5'-TTCGT ATNAT GCCTA CACTG GAGTA CCGGA GCATC GTCGT GACTG GGAAA AC, where N is G, A, T, or C. The various
combinations of primers and templates were annealed by mixing 1 µM 32P-end labeled primer with 1.5 µM template in 50 mM Tris-HCl, pH 7.5, and
100 mM NaCl and heating to 90 °C for 2 min before slowly cooling to room temperature over several hours.
Steady state Kinetics Assays--
Yeast and human Pol We examined the steady state kinetics of nucleotide incorporation
by Pol (Pol
) functions in
error-free replication of UV-damaged DNA, and in vitro it
efficiently bypasses a cis-syn T-T dimer by incorporating
two adenines opposite the lesion. Steady state kinetic studies have
shown that both yeast and human Pol
are low-fidelity enzymes, and
they misincorporate nucleotides with a frequency of
10
2-10
3 on both
undamaged and T-T dimer-containing DNA templates. To better understand
the role of Pol
in error-free translesion DNA synthesis, here we
examine the ability of Pol
to extend from base mismatches. We find
that both yeast and human Pol
extend from mismatched base pairs with
a frequency of ~10
3 relative to matched
base pairs. In the absence of efficient extension of mismatched primer
termini, the ensuing dissociation of Pol
from DNA may favor the
excision of mismatched nucleotides by a proofreading exonuclease. Thus,
we expect DNA synthesis by Pol
to be more accurate than that
predicted from the fidelity of nucleotide incorporation alone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(Pol
)1 functions in
error-free replication of UV-damaged DNA, and mutations in the gene
encoding this enzyme result in increased UV mutability in both yeast
and humans (1). In humans, inactivation of Pol
causes the variant
form of the cancer prone syndrome xeroderma pigmentosum (2, 3). Pol
is unique among eukaryotic DNA polymerases in its ability to
efficiently replicate DNA containing a cis-syn T-T dimer,
and it does so by incorporating two adenines across from the two
thymines of the dimer (3-6).
have indicated that it is a low-fidelity enzyme, misincorporating
nucleotides with a frequency of
10
2-10
3 on
undamaged DNA (5, 8). Remarkably, however, Pol
synthesizes DNA
opposite a T-T dimer with the same efficiency and accuracy as opposite
undamaged T residues (5, 6). The low fidelity of Pol
may reflect an
unusual tolerance of its active site for deviant geometry arising from
distorting template lesions such as a T-T dimer.
, with a low nucleotide insertion
fidelity, can function in an error-free pathway of translesion DNA
synthesis in vivo, here we examine the ability of Pol
to
extend from base mispairs. We find that yeast and human Pol
extend
from mismatched primer-templates with a frequency of
~10
3 relative to matched primer-templates.
This implies that Pol
, which has a low processivity, will have a
greater likelihood of dissociating from the DNA template after the
incorporation of an incorrect nucleotide than a correct one. That would
lower the error rate of DNA synthesis in vivo, because the
mismatched primer terminus could then be subjected to the proofreading
3'
5' exonuclease activity of other protein factors.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
were
expressed in and purified from yeast strain BJ5464 as described (4, 5).
The steady state kinetics of single nucleotide incorporation were
measured by incubating 1 nM yeast or human Pol
with 20 nM DNA substrate in 25 mM sodium phosphate, pH
7.0, buffer containing 5 mM magnesium chloride, 5 mM dithiothreitol, 10 µg/ml bovine serum albumin, and
10% glycerol for 10 min at 25 °C. For nucleotide incorporation
following a correctly base paired or mispaired primer terminus, the
concentration of dATP was varied from 0 to 5 µM or from 0 to 2000 µM, respectively. Reactions were quenched after
10 min by adding 10 volume of loading buffer (95% formamide, 0.03%
bromphenol blue, and 0.3% cyanol blue). Samples were then run on 10%
polyacrylamide sequencing gels to separate the unextended and extended
DNA primers. Gel band intensities were quantified using a
PhosphorImager and ImageQuant software (Molecular Dynamics). The
observed rate of nucleotide incorporation was calculated by dividing
the amount of reaction product formed by the 10-min incubation time.
The observed rate of nucleotide incorporation was then plotted as a
function of nucleotide concentration, and the apparent
Km and Vmax parameters were
obtained from the best fit to the Michaelis-Menten equation using
nonlinear regression (Sigma Plot 4.0). The intrinsic efficiency of
mismatch extension, fexto, which is a
constant that represents the efficiency of extending mismatched termini
in competition with matched termini at equal DNA concentrations, was
calculated as described (7, 9, 10) using the following equation:
fexto = (Vmax/Km)mismatch/(Vmax/Km)matched.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
following the correctly base paired and mispaired termini in
primer-template substrates (7, 9, 10). For example, the rate of
incorporation of an A residue by yeast Pol
opposite a template T
residue following a G·C base pair or an A·C, T·C, or C·C
mispair was measured over a broad range of dATP concentrations (Fig.
1A). Gel band intensities were
evaluated, and the rate of nucleotide incorporation was plotted as a
function of nucleotide concentration. As shown in Fig. 1B,
these plots yield curves typical of Michaelis-Menten kinetics. The
apparent values of Vmax and
Km for extension of each primer terminus were
obtained from the best fit to the Michaelis-Menten equation using
nonlinear regression. The frequency of mispair extension (fexto), which is the ratio of the apparent
Vmax/Km of extension from the
mispair to the apparent
Vmax/Km of extension from a
correct base pair, was then calculated (7, 9, 10).
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Fig. 1.
Mismatch extension by yeast Pol
. A, dAMP incorporation by yeast
Pol
opposite a template T following a G·C base pair or
A·C, T·C, and C·C mispairs.
Yeast Pol
(1 nM) was incubated with DNA substrate (20 nM) and varying concentrations of dATP at 25 °C for 10 min. B, observed rate of nucleotide incorporation by yeast
Pol
following a G·C base pair or A·C,
T·C, and C·C mispairs graphed as a function
of dATP concentration. The obtained Vmax and
Km parameters are listed in Table I.
As shown in Table I, for the
incorporation of an A residue following a G·C base pair, the apparent
Km for yeast Pol is 0.20 µM, and
the Vmax is 0.28 nM/min, whereas for
the incorporation of an A following an A·C mispair, the apparent
Km is 21 µM, and the
Vmax is 0.25 nM/min, respectively.
Thus, for the A·C mispair, fexto is
8.5 × 10
3, and similarly, the
fexto values for the T·C and C·C
mispairs are 1.5 × 10
3 and 1.1 × 10
3, respectively. The
fexto values were determined for all the
possible mispairs, and overall, yeast Pol
extends from mispairs with
an average frequency of 3.1 × 10
3
(Table I).
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For most DNA polymerases, the frequency of extension from a given
mispair (fexto) is approximately the same as
the frequency of incorporating that same mispair
(finc; Refs. 7, 10). Fig.
2A compares the fexto values with the previously reported
finc values (8) for yeast Pol for each
possible mispair. Points lying above the dashed line
represent mispairs with a higher efficiency of extension than
insertion, whereas those below the line indicate mispairs with a lower
efficiency of extension than insertion. Because most of the points lie
near or below the dashed line, yeast Pol
is somewhat less
efficient at extending from mispairs than at forming mispairs.
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We also examined human Pol for its ability to extend from base
mispairs. As was observed for yeast Pol
, the
fexto values for human Pol
range from
10
2 to 10
3, with an
average of 2.5 × 10
3 (Table
II), and a comparison of
fexto values with the previously published
finc values (5) indicates that human Pol
is
also somewhat less efficient at extending from mispairs than at
inserting mispaired bases (Fig. 2B).
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Pol replicates through a cis-syn T-T dimer with the same
efficiency and fidelity as through undamaged T nucleotides (5, 6).
Furthermore, our steady state kinetic analyses of base mispair
extension across from the T-T dimer indicate that these mispairs are
also inefficiently extended and with the same frequency as mispairs in
undamaged DNA.2
When compared with other DNA polymerases, the mispair extension ability
of Pol is greater than that of the high-fidelity DNA polymerase
,
the fexto of which ranges from
10
3 to 10
6 (10).
However, its mispair extension ability is considerably lower than that
of the most promiscuous extender of mispairs known, yeast Pol
, which
extends from mispaired template primer termini with a frequency of
10
1 to 10
2 (11).
Pol
plays an essential role in mutagenic bypass of DNA lesions, and
it specifically functions in damage bypass by extending from
nucleotides placed opposite DNA lesions by another DNA polymerase (11).
Pol has low processivity (5, 8), and thus it has a modest
probability (0.2-0.3) of dissociating from the DNA template after each
nucleotide incorporation. Our observation that both yeast and human
Pol
extend from mismatched primer termini with a frequency of
~10
3 relative to a matched primer terminus
implies that Pol
has a substantially higher probability of
dissociating from the primer terminus after the incorporation of an
incorrect nucleotide than a correct nucleotide. Dissociation of Pol
would prevent mutation fixation, because any mispairs left in DNA would
then be subject to removal by the proofreading exonucleolytic activity
of Pol
or other proofreading exonucleases. Thus, DNA synthesis by
Pol
would be more accurate than is indicated from the fidelity of nucleotide incorporation (finc) values. Because
Pol
extends from mismatched bases opposite a T-T dimer with the same
efficiency as from undamaged DNA, we predict that the error frequency
during T-T dimer bypass will also be lower than that suggested from the finc values for the incorporation of wrong
nucleotides opposite the two T nucleotides of the T-T dimer (5, 6).
We expect the activity of Pol to be restricted to DNA synthesis
during damage bypass. The Rad6-Rad18 complex, which is essential for
damage bypass and which contains ubiquitin conjugating and DNA binding
activities (12), may be crucial for modulating the specific targeting
of Pol
to sites where replication has stalled at a DNA lesion and
for ensuring the dissociation of Pol
from DNA once the lesion has
been bypassed. Furthermore, association with other protein factors may
increase the fidelity of nucleotide incorporation by Pol
. Thus,
in vivo, damage bypass by Pol
would be much more accurate
than 10
2-10
3, the
frequency of nucleotide misincorporation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM19261.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: Sealy Center for
Molecular Science, University of Texas Medical Branch, 6.104 Medical
Research Bldg., 11th and Mechanic Sts., Galveston, TX 77555-1061. Tel.:
409-747-8601; Fax: 409-747-8608; E-mail: lprakash@scms.utmb.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M009049200
2 M. T. Washington, R. E. Johnson, S. Prakash, and L. Prakash, unpublished observations.
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ABBREVIATIONS |
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The abbreviation used is:
Pol, polymerase
.
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REFERENCES |
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