From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
Received for publication, July 27, 2000, and in revised form, October 25, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nucleotide insertion opposite
8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase
High fidelity DNA replication is critical to the preservation of
genomic stability and the avoidance of mutations that can disrupt the
regulation of complex biological systems. Cells contain several DNA
polymerases and complex DNA repair systems to preserve genomic
integrity (1, 2). Accurate replication is disrupted by the presence of
covalent DNA-chemical adducts, which can be misread and lead to
mutations and cancer (3). Understanding the miscoding events induced by
modified DNA is important in understanding risks of environmental
chemicals, as well as aspects of chemotherapeutic treatment.
Misincorporation is primarily a kinetic phenomenon and not simply
thermodynamic. Work with several DNA adducts and artificial DNA bases
clearly indicates that both the identity of incorporated bases and
their frequency of substitution are functions of which polymerase is
used as a catalyst (4-10). Our own work on how polymerases influence
misincorporation has been focused on
8-oxoG1 (9, 11-14). 8-OxoG
is a relatively simple adduct in that the only chemical attached to the
DNA is one atom of oxygen, and it was selected as a model because of
its relatively high mutagenicity and lack of polymerase blockage. This
lesion is generally regarded as being the most abundant of those
induced by oxidative damage (15-17).
Polymerases derived from prokaryotic systems have been used extensively
as models for mechanistic studies because of their availability, the
general lack of need for complex accessory proteins, and the
availability of structural and mechanistic information (18). The
question arises as to how relevant findings made with these enzymes are
to mammalian and other eukaryotic polymerases, particularly those
polymerases that will copy past sites of DNA damage. Recent studies
indicate the presence of low fidelity, distributive polymerases in both
prokaryotes and eukaryotes that are involved in translesion DNA
synthesis (6, 19, 20). However, the extent to which the mammalian forms
are able to copy past chemical lesions other than photodamaged
pyrimidines is largely unexplored, and the question of how lesions
interact with replicative DNA polymerases is important. Recently we
used purified calf thymus DNA pol Some work has been done on the replication of mammalian pol We studied the incorporation of dCTP and dATP opposite 8-oxoG in an
oligonucleotide primer-template complex with purified calf thymus pol
Materials--
Primer and template oligonucleotides were
purchased from Midland Certified Reagent Co. (Midland, TX) and purified
as described previously (9, 21) or were purchased (gel-purified) from Operon Technologies (Alameda, CA). UltraPure Grade dNTPs were obtained
from Amersham Pharmacia Biotech (Uppsala, Sweden). The thio-substituted
dNTPs, (Sp)- Enzymes--
Human PCNA was expressed in Escherichia
coli and purified to electrophoretic homogeneity as described (25)
with modifications (21). The concentration of PCNA was estimated by the
calculated
pol End Labeling of Primer and Primer/Template
Annealing--
Oligonucleotide primers (Table I) were 5' end-labeled
with [ Steady-state Reactions--
pol
A similar procedure was used for incorporation of 8-oxo-dGTP opposite A
and C. A complementary oligonucleotide to the 24/36G-mer was used with
a C or A in the position of the incoming dNTP (9). The concentrations
of pol Pre-steady-state Kinetics--
Pre-steady-state rapid-quench
experiments were performed utilizing a KinTek Quench Flow Apparatus
(Model RQF-3, KinTek, Austin, TX). Reactions were initiated by rapid
mixing of dNTP in Buffer A with a primer/template/pol Kd Determination of dCTP or dATP Binding to pol
Determination of KdDNA for pol
Steady-state Kinetics of dCTP or dATP Incorporation Opposite
8-OxoG--
The steady-steady kinetics of elongation of the 5'
32P-labeled 24/36 8-oxoG-mer duplex DNA (Table
I) to 25/36 8-oxoG-mer product were
measured as a function of dCTP or dATP concentration dependence using a
large excess of 24/36 8-oxoG-mer (100 nM) relative to pol
PCNA has been reported to increase the processivity of pol Steady-state Kinetics of 8-Oxo-dGTP Incorporation Opposite Template
C or A--
Previous studies with other polymerases had shown
asymmetry in the pairing of 8-oxoG with C and A, depending on whether
the 8-oxoG was in the DNA template or the incoming nucleotide (9). These studies were repeated with pol Pre-steady-state Kinetics of Nucleotide Incorporation Opposite
8-OxoG--
Pre-steady-state kinetic analysis of dCTP or dATP
incorporation opposite 8-oxoG by pol
When PCNA was excluded from the pre-steady-state reactions, no apparent
burst of dCTP or dATP incorporation opposite 8-oxoG was observed, even
at pol Determination of Phosphorothioate Elemental Effect--
The rate
of polymerization was examined for a phosphorothioate elemental effect
to determine if the rate of a single nucleotide incorporation event is
influenced by phosphodiester bond formation (chemistry step).
Replacement of the Next Correct Nucleotide Insertion Beyond C:8-OxoG or A:8-OxoG Base
Pairs in the Presence or Absence of PCNA--
Pre-steady-state kinetic
analysis of the next correct base (dGTP) insertion beyond C:8-oxoG or
A:8-oxoG pairs was examined to determine whether pol Determination of KddNTP for Binding
of Nucleotides to pol Determination of KdDNA for pol
The major features of the catalytic mechanism of correct
nucleotide insertion into unmodified DNA by pol (pol
) was examined by steady-state and pre-steady-state rapid
quench kinetic analyses. In steady-state reactions with the accessory
protein proliferating cell nuclear antigen (PCNA), pol
preferred to
incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an
order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased Km). Pre-steady-state kinetic
analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and
oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol
preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol
replication fidelity at
8-oxoG depends upon contributions from Km,
KddNTP, and rates of phosphodiester
bond formation, and PCNA is an important accessory protein for
incorporation and extension at 8-oxoG adducts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, considered to be the main
leading strand DNA polymerase (1, 2), in a series of steady-state and
pre-steady-state kinetic experiments and concluded that the major
features of the catalytic mechanism were very similar to those
established in the prokaryotic models (21). However, questions about
the processing of chemical-DNA adducts remain.
past
DNA adducts and misincorporation. O'Day et al. (22)
reported that the presence of the accessory protein PCNA enabled pol
bypass of cyclobutane thymine dimers, as judged by qualitative polyacrylamide gel electrophoresis experiments. Using similar approaches, Mozzherin et al. (23) demonstrated that PCNA
stimulated pol
incorporation and bypass at abasic sites and 8-oxoG
and C8-(2-aminofluorenyl)guanine modifications
(but not C8-(2-acetamidofluorenyl)guanine). The
increase in the amount of extended products was 2.5-fold for 8-oxoG and
C8-(2-aminofluorenyl)guanine but more for abasic
sites. The mechanism was concluded to involve extension of the new
primer terminus (resulting from incorporation), facilitated by a
decreased pol
:DNA off-rate (koff) by PCNA.
However, other mechanisms could not be excluded. Other work by
Mozzherin et al. (24) indicated that the presence of PCNA
led to more errors in the incorporation of nucleotides in unmodified
oligonucleotides, a result also rationalized in terms of a decreased
DNA koff for pol
due to PCNA (24).
and recombinant human PCNA, using steady-state and pre-steady-state
kinetic approaches. The results show a strong dependence on the
presence of PCNA for the polymerization rate constant
(kpol) and extension beyond the lesion, and the
effects of phosphorothioate substitution of dNTPs suggest that the
rate-limiting step may be phosphodiester bond formation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S-dNTPs, were purchased from
U. S. Biochemical Corp. (Cleveland, OH), and
[
-32P]dATP and [3H]dTTP were
obtained from PerkinElmer Life Sciences (Boston, MA). 8-Oxo-dGTP was
prepared and rigorously characterized as described elsewhere (9).
First trimester fetal calf thymus was purchased from
Pel-freez (Rogers, AR) and the sources of reagents used for the purification of pol
are described elsewhere (21). The bacterial expression vector pT7/PCNA (expressing human PCNA) was a generous gift of Dr. B. Stillman (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) obtained from Prof. E. Fanning (Vanderbilt University).
280 value of 17.7 mM
1 cm
1.
was purified from first trimester fetal calf thymus as
described by Podust et al. (26) with modifications (21). The
amount of pol
was determined by quantitative amino acid analysis of
the individual polypeptides of pol
in the Vanderbilt facility
following separation by SDS-polyacrylamide gel electrophoresis and
transfer to a polyvinylidene fluoride membrane (21). The preparations
obtained using the five-step procedure contained three polypeptides,
molecular mass 50, 116, and 125 kDa (21). The 125-kDa protein is
the catalytic subunit of pol
(27), the 50-kDa protein is an
accessory protein reported to be required to bind PCNA (28), and the
116-kDa protein is a proteolytic product of the p125 subunit. p116
lacks ~80 amino acids of the N terminus of p125 and has been found in
many thymus pol
preparations (21, 27, 29). In a recent report,
Schumacher et al. (27) found that the 116-kDa proteolytic
product retained polymerase activity to the same extent as the
full-length p125 for replication factor C-independent
incorporation of nucleotides into primer/template DNA, in support of
the work of Wu et al. (29). The concentrations of pol
in
the reactions in this report were determined by the limiting amount of
subunit (p50 or p116 + p125).
-32P]dATP (3000 Ci mmol
1) and T4
polynucleotide kinase and annealed to the template in a ratio of 1:1.5
(primer:template) (9).
(containing 0.031 pmol of
the limiting subunit of pol
) was added to a mixture (5 µl volume)
containing annealed 5' 32P-labeled 24/36G-mer or 24/36
8-oxoG-mer primer:template (200 nM), 72 nM
PCNA, 0.4 mg of bovine serum albumin ml
1, 50 mM bis-Tris-HCl (pH 6.5), and 2 mM
dithiothreitol. The reactions were initiated with the addition of an
equal volume of Buffer A (50 mM bis-Tris-HCl (pH 6.5) and
12.5 mM MgCl2) and dNTP (2 × final
concentration) at varying concentrations. Final concentrations were 50 mM bis-Tris-HCl (pH 6.5), 0.2 mg of bovine serum albumin ml
1, 100 nM 24/36G-mer or 24/36 8-oxoG-mer,
3.1 nM pol
, 36 nM PCNA, 6 mM
MgCl2, and 1 mM dithiothreitol in 10 µl. The
reactions were run at 37 °C in triplicate and quenched with 20 µl
of 20 mM EDTA (pH 7.4) after 5 min. The products were
separated by denaturing polyacrylamide gel electrophoresis (16%
acrylamide (w/v), 1.5% bisacrylamide (w/v), 8.0 M urea)
and the amount of primer extended was quantitated utilizing a Molecular
Dynamics Model 400E PhosphorImager (Molecular Dynamics, Sunnyvale, CA)
and Image Software version 3.3. kcat and
Km values were determined by nonlinear regression
using a k·cat computer program (Biometallics, Princeton, NJ). In
steady-state reactions performed in the absence of PCNA, the pol
concentration was increased to 27 nM and reaction time to
10 min.
and PCNA were 3.1 and 72 nM, respectively, in
the buffer described above. Reactions were done in the presence of
varying concentrations of 8-oxo-dGTP for 5 min (opposite C) or 10 min
(opposite A), and the results were analyzed as for other steady-state experiments.
solution with
or without PCNA at 37 °C. The final concentrations of the reactants
were 10-95 nM pol
(based on the limiting subunit of
pol
), 50 mM bis-Tris-HCl (pH 6.5), 6 mM
MgCl2, 1 mM dithiothreitol, 180 or 400 nM PCNA, 0.2 mg bovine serum albumin ml
1, and
100 nM primer/template. The reactions were quenched with equal volumes of 0.6 M EDTA at times varying from 5 ms to
10 s. The products were analyzed as described for steady-state
assays. kpol (maximum rate of nucleotide
incorporation) was determined by a fit of the data to the burst
equation: y = A(1
e
kpt) + ksst, where A = burst amplitude,
kp = first-order rate constant, t = time, and kss = steady-state rate of nucleotide incorporation.
·PCNA·DNA--
The kinetic Kd value for dCTP
or dATP binding to pol
·PCNA·24/36 8-oxoG-mer complexes was
estimated by pre-steady-state rapid-quench analysis. The dNTP
concentration dependence of the pre-steady-state burst rates was
examined by varying the concentration of dNTP and measuring the
pre-steady-state burst rates of dNTP incorporation into the 24/36
8-oxoG-mer duplex DNA. The values of kpol were
determined as described above. The pre-steady-state rates were plotted
against [dNTP] and the data was fit to the hyperbola
kobs = [kpol[dNTP]/([dNTP] + Kd)] to determine Kd (30).
·24/36 8-OxoG-mer Complexes--
The Kd values
for productive pol
binding to 24/36 8-oxoG-mer or 24/36G-mer
complexes in the presence of PCNA were determined by pre-steady-state
rapid quench analysis. The DNA concentration was varied and the changes
in the burst amplitude were measured to determine enzyme active site
concentration and the Kd for binding DNA. The
Kd was determined by plotting the burst amplitudes
against the concentration of DNA and then fitting the data to the
quadratic equation [E·D] = 0.5(Kd + Et + Dt)
[0.25(Kd + Et + Dt)2
Et Dt]1/2, where
Et = [total enzyme], Dt = [total DNA], and Kd = dissociation constant for
the reaction E + D
E·D.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(3.1 nM). The steady-state kcat
values for dCTP or dATP incorporation opposite 8-oxoG in the presence
of PCNA were 0.0055 s
1, similar to the
kcat for dCTP incorporation into unmodified DNA (0.0035 s
1) (21). Km values for dCTP
and dATP incorporation opposite 8-oxoG were determined to be 1.3 and
4.1 µM, respectively, an order of magnitude higher
than the Km for
correct nucleotide incorporation into unmodified DNA, 0.067 µM (Table
II).2
Primer/template complexes
Steady-state kinetic parameters for pol
replication and the bypass of abasic sites and DNA adducts, such as
8-oxoG, C8-(2-aminofluorenyl)guanine, and
cyclobutane thymine dimers (22, 23). When PCNA was omitted, the
kcat for dCTP and dATP incorporation opposite
8-oxoG was either similar or 16-fold lower (0.0040 and 0.00035 s
1, respectively) than in reactions containing PCNA
(Table II). The Km values were 400 and 5-fold higher
(510 and 19 µM) for dCTP and dATP incorporation, respectively.
, in the presence of PCNA. The
steady-state kcat values estimated for
incorporation of 8-oxo-dGTP opposite C and A were 0.0030 (± 0.0003)
and 0.0019 (± 0.0004) s
1, respectively. As in the case
of the prokaryotic polymerases (9), the Km values
for incorporation of 8-oxo-dGTP were much higher than for incorporation
of dCTP or dATP opposite template 8-oxoG, 0.65 (±0.16) mM
for 8-oxo-dGTP incorporation opposite C and 13 (±5) mM
opposite A. Thus, the efficiency
(kcat/Km) of 8-oxo-dGTP
incorporation is 3 orders of magnitude lower than for dCTP or dATP
incorporation opposite template
8-oxoG.3
was performed to discern
contributions of specific steps in the catalytic cycle important for
fidelity of incorporation opposite 8-oxoG. The rate of polymerization
(kpol) can be measured by rapid quench kinetics
and includes rates of dNTP binding, any associated conformational
changes, and phosphodiester bond formation. The incorporation of dCTP
or dATP into the 24/36 8-oxoG-mer by pol
in the presence of PCNA
showed biphasic kinetics, fitted to an initial burst followed by a
linear phase (Fig. 1). The burst phase
represents the rate of single nucleotide incorporation in the first
turnover of the enzyme. kpol was determined to
be 22 s
1 for dCTP incorporation and 21 s
1
for dATP incorporation. These values were similar to the rate of dCTP
incorporation opposite unmodified G (28 s
1). The
amplitude of the burst corresponds to the concentration of "active"
enzyme present in the reactions. The amplitude was ~4 nM
for dCTP incorporation and ~2 nM for dATP incorporation, indicating that 8 and 4% of the pol
is in an active conformation in these reactions, respectively. These values were calculated based
upon the 11 nM burst product formed in reactions containing 51 nM pol
for dCTP incorporation opposite unmodified G,
or 36%, from this preparation of pol
(results not shown).
View larger version (17K):
[in a new window]
Fig. 1.
Pre-steady-state kinetics of nucleotide
insertion opposite 8-oxoG by pol in the
presence of PCNA. A, dCTP incorporation opposite
8-oxoG. pol
(51 nM) was incubated with 100 nM 24/36 8-oxoG-mer and PCNA (180 nM), and
reactions were initiated by mixing with a solution of 200 µM dCTP (
). The reactions were quenched with EDTA at
various times and the rate of polymerization
(kpol) was determined. The solid line
is a fit to the burst equation ("Experimental Procedures").
For analysis of the phosphorothioate elemental effect
(
S-dCTP), dATP was replaced with
S-dCTP
(
). B, dATP incorporation opposite 8-oxoG. Reactions were
done as described above except in the presence of dATP (
) or
S-dATP (
).
concentrations of 95 nM (Fig.
2). The apparent rates of polymerization
in the absence of PCNA were 0.032 and 0.018 s
1 for dCTP
and dATP incorporation, respectively, 3 orders of magnitude less than
with PCNA and indicating that PCNA is essential for formation of an
active pol
complex capable of nucleotide incorporation opposite
8-oxoG.
View larger version (13K):
[in a new window]
Fig. 2.
Pre-steady-state rapid quench kinetics of
nucleotide insertion opposite 8-oxoG by pol in the absence of PCNA. pol
(95 nM) was
incubated with 100 nM 24/36 8-oxoG-mer in the absence of
PCNA and mixed with a solution of 1 mM dCTP (
) or dATP
(
). The reactions were quenched with EDTA at various times and the
rate of polymerization (kpol) was determined by
linear regression (solid line).
-phosphate group of a dNTP with a
phosphorothioate can reduce the rate of kpol by
~102-fold if the rate of polymerization is a direct
function of the chemistry step. The rates of dCTP and dATP
incorporation by pol
in the presence of PCNA were reduced by a
factor of 900-1000 (Fig. 1). The results are interpreted to mean that
phosphodiester bond formation may be the rate-limiting step for both
dCTP and dATP incorporation opposite 8-oxoG and that the rate of
polymerization (kpol) is a direct reflection of
this chemical step.
has a
preference for extension of either base pair. In the presence of PCNA
(Fig. 3A), pol
efficiently extended the A:8-oxoG base pair (kpol = 27 s
1) an order of magnitude faster than C:8-oxoG base pairs
(kpol = 1 s
1). When PCNA was
omitted (Fig. 3B), rates of next correct base pair insertion
decreased 70-fold for A:8-oxoG base pairs (0.38 s
1) and
4-fold for C:8-oxoG base pairs (0.24 s
1) and no bursts of
nucleotide incorporation occurred.
View larger version (15K):
[in a new window]
Fig. 3.
Pre-steady-state rapid quench kinetics of the
next correct base insertion (dGTP) beyond C:8-oxoG or A:8-oxoG base
pairs in the presence or absence of PCNA. A, pol (10.6 nM), PCNA (400 nM), and 100 nM 25C/36 8-oxoG-mer or 25A/36 8-oxoG-mer were preincubated
and mixed with a solution of 200 µM dGTP. The reactions
were quenched at various times with EDTA and the pre-steady-state burst
rates of product formation were determined. The plot is a fit to the
burst equation ("Experimental Procedures"). B, pol
(10.6 or 15.9 nM) was preincubated with 100 nM
25C/36 8-oxoG-mer or 25A/36 8-oxoG-mer, respectively, mixed with 200 µM dGTP for various times. Rates of the reactions were
determined by linear regression (solid line).
·PCNA·24/36 8-OxoG-mer Complexes--
The
pre-steady-state rate of nucleotide incorporation into the 24/36
8-oxoG-mer primer/template was determined as a function of the
concentration of dCTP (results not shown) or dATP (Fig. 4). The
KddCTP for DNA modified with 8-oxoG was 8 µM and the KddATP was 42 µM. These values are considerably higher than the
KddNTP for dCTP binding to unmodified DNA
(0.93 µM) (21).
View larger version (21K):
[in a new window]
Fig. 4.
dATP concentration dependence of the
pre-steady-state burst rate for incorporation opposite 8-oxoG.
A, pol (17 nM), PCNA (400 nM),
and 100 nM 24/36 8-oxoG-mer were preincubated and then
mixed with varying concentrations of dATP (100 µM,
;
50 µM,
; 25 µM,
; 12.5 µM,
; 6.3 µM,
). The plot is a fit to
the burst equation ("Experimental Procedures"). B, burst
rates measured in A were plotted against [dATP] and the
Kd was determined by a fit to a hyperbola
(solid line).
·24/36 8-OxoG-mer--
KdDNA, which
describes the reaction E·PCNA + DNA
E·DNA·PCNA, was determined by examining the DNA
concentration dependence on the burst amplitude of dCTP incorporation
by pol
. A fixed amount of pol
was incubated with increasing
concentrations of 24/36G-mer or 24/36 8-oxoG-mer and the reactions were
initiated with the addition of saturating dCTP for 100 ms, at which
maximal burst amplitudes were observed with negligible effect from
multiple turnovers of the enzyme (Fig.
5). The KdDNA
for the reaction pol
·PCNA + 24/36G-mer
pol
·PCNA·24/36G-mer was determined to be 64 ± 10 nM (21) and the KdDNA was
2-fold higher, 124 (± 24) nM, with the
8-oxoG-modified DNA. When PCNA was omitted, no appreciable burst of
nucleotide incorporation occurred even with 1 µM DNA
(results not shown).
View larger version (17K):
[in a new window]
Fig. 5.
Determination of
KdDNA. Burst amplitudes for
dCTP (200 µM) incorporation by pol (77 nM) in the presence of PCNA (750 nM) were
plotted against the concentration of 24/36G-mer (
) or 24/36
8-oxoG-mer (
) (3.9-250 nM). The solid line
is a fit to the quadratic equation to estimate
KdDNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
are similar to those of the prokaryotic DNA polymerases (21). A single nucleotide incorporation reaction is defined by several consecutive steps involving DNA binding, nucleotide binding, conformational changes, and
phosphodiester bond formation (Fig. 6).
The kinetic parameters of normal nucleotide incorporation by pol
are similar in magnitude to prokaryotic polymerases and the
rate-limiting step is most likely a conformational change preceding
phosphodiester bond formation (21, 30-32). The goals of this study
were to examine the kinetics of nucleotide incorporation opposite
8-oxoG by mammalian DNA pol
, to establish the steps in the
polymerase catalytic cycle important for fidelity, and to determine the
roles of the pol
accessory protein PCNA on replication at DNA
adducts. The results presented here indicate that replication by pol
at 8-oxoG adducts was most notably affected at the levels of
phosphodiester bond formation, nucleotide binding, and efficiency of
base pair extension beyond 8-oxoG, as found previously for prokaryotic
polymerases (11, 12). PCNA was also found to be an essential component
for efficient nucleotide incorporation opposite 8-oxoG and base pair
extension beyond the adduct.
View larger version (14K):
[in a new window]
Fig. 6.
Proposed polymerase mechanism. A
mechanism is shown with a possible nonproductive ternary complex
(E§) (14).
Steady-state Reactions--
In steady-state reactions with PCNA,
the efficiency of nucleotide incorporation by pol was decreased by
at least a factor of 12 for dATP and dCTP incorporation opposite 8-oxoG
compared with reactions with unmodified G. This decrease in nucleotide insertion efficiency at 8-oxoG is within the 1-3 orders of magnitude range of the decrease seen with prokaryotic polymerases (11, 12). The
decrease in insertion efficiency at 8-oxoG adducts is dominated by an
increased Km value for dCTP or dATP insertion (Table
II). A direct interpretation of this change is unknown because of the
complexity of the catalytic mechanisms of polymerases (18).
In the absence of PCNA, the efficiency of dCTP incorporation opposite
unmodified G or 8-oxoG was affected primarily at the level of
Km. The Km value of dCTP
incorporation opposite unmodified G and 8-oxoG increased by a factor of
18 and 300, respectively, whereas the kcat value
was not as affected. The absence of PCNA decreased the insertion
efficiency of pol by 30- and 500-fold for dCTP incorporation
opposite G and 8-oxoG, respectively. These results indicate that PCNA
is an important factor for efficient incorporation of the correct
nucleotide not only opposite unmodified bases but even more importantly
for insertion opposite DNA adducts, such as 8-oxoG.
The steady-state kcat value is dominated by the
rate of enzyme dissociation (koff) from the DNA
or some other step following product formation, as clearly demonstrated
by the burst kinetics in the biphasic pre-steady-state plots of the
reaction course (Figs. 1 and 3). It is interesting to note that in the
absence or presence of PCNA, the kcat value is
similar to that for correct nucleotide incorporation opposite G or
8-oxoG (Table II). This result suggests that the rate of dissociation
(koff), if described by
kcat, is probably not affected by PCNA in
correct nucleotide incorporation reactions. However, in the case of the
misincorporation of dATP opposite 8-oxoG, the
kcat is decreased 16-fold when PCNA is omitted,
indicating that the enzyme is either dissociating at a slower rate or
that other steps in the cycle have been perturbed and become
rate-limiting. These results suggest that PCNA may be more important
for the activity of pol in misincorporation than normal
incorporation reactions (23). However, the results do not support a
view that PCNA acts primarily by lowering the Kd for
the complex between pol
and DNA (33-35), where a lower
kcat would be expected in the presence of PCNA.
The lack of a burst of nucleotide incorporation without PCNA for both
dCTP and dATP incorporation reactions opposite 8-oxoG (Fig. 2)
indicates that the rate-limiting step is at or prior to phosphodiester
bond formation. The lower kcat for dATP
incorporation compared with dCTP suggests that there may be differences
in KddNTP (step 2, Fig. 6) and/or
conformational changes (steps 3 or 8, Fig. 6) for the misincorporation reaction.
Pre-steady-state Reactions--
A burst of nucleotide
incorporation opposite 8-oxoG occurred for incorporation of either dCTP
or dATP (kpol = 22 and 21 s1,
respectively) with rates similar to dCTP incorporation opposite unmodified G (28 s
1). The difference in the efficiency of
nucleotide insertion opposite 8-oxoG compared with unmodified G is due
to an increase in KddNTP (reduction in dNTP
binding affinity compared with dCTP insertion opposite G, 7- and
45-fold for dCTP and dATP incorporation opposite 8-oxoG, respectively)
and less to changes in KdDNA values
(2-fold) (Table III). pol
incorporated dCTP 6-fold more efficiently
(kpol/KddNTP) than
dATP. The misinsertion frequency [f = (kpol/KddATP)A/(kpol/KddCTP)C]
was determined to be 0.172, which corresponds to 14%
misincorporation of dATP opposite 8-oxoG (Table III).
|
PCNA increased the affinity of the pol for unmodified DNA
interaction by only 5-fold in our earlier work (21). Due to the lack of
a burst of nucleotide incorporation opposite 8-oxoG in the absence of
PCNA (Fig. 2), the affinity of the pol
interaction with DNA
(KdDNA) could only be estimated to be
>1000 nM (>3-fold greater than the
KdDNA value for pol
-unmodified DNA
interaction). However, the presence of 8-oxoG in the template reduced
the KdDNA only minimally (2-fold) in the
presence of PCNA (Table III), as in the case of the prokaryotic
polymerases (11, 12). The possibility does exists that PCNA may have a
major role in the interaction of pol
with modified DNA (see below),
although further experiments are needed to discern this.
To examine whether the chemistry step is rate-limiting in a single
turnover reaction by pol (at an 8-oxoG site), the
-phosphorus of
dCTP or dATP was replaced with a sulfur group and the rate of
nucleotide insertion was measured by pre-steady-state kinetics. The
rate of incorporation of these
S-dNTPs (kpol)
was 103-fold less than for dATP or dCTP incorporation,
suggesting that phosphodiester bond formation becomes rate-limiting
during replication reactions at 8-oxoG. Similar results have also been
obtained with the prokaryotic pol T7 and E. coli pol
I
and II
(11, 12). (Except in the case of
E. coli pol II exo
(11), no thio effect is
observed during normal incorporation.)
PCNA has been shown to be important for pol extension beyond DNA
adducts, including thymine dimers,
C8-(2-aminofluorenyl)guanine, and 8-oxoG (22,
23). The results presented here confirm that PCNA is critical for
nucleotide insertion past both 8-oxoG:C and 8-oxoG:A base pairs (Fig.
3). There is a preference for extension of 8-oxoG:A mispairs (27-fold),
as for pol T7 exo
, E. coli pol I
exo
and II exo
, and HIV-1 RT (11, 12). The
rate of the next correct base incorporation beyond 8-oxoG:A base pairs
(27 s
1) was similar to rates of normal base pair
insertion (28 s
1). Although C is the preferred base
inserted opposite 8-oxoG, the ability of pol
to extend the mispair
can contribute to the mutagenicity of 8-oxoG.
Effects of Accessory Proteins--
Some of the effects of PCNA on
incorporation have already been described. A considerable body of
literature has accrued to demonstrate physical interactions of PCNA
with both the p50 (28) and p125 (36) subunits of pol . The PCNA
trimer appears to form a circular "clamp" around DNA (37). The
stimulatory effect of PCNA is seen with short primer-template complexes
(Figs. 1-3) (21) as well as longer nucleic acids (for which "loading
factors," e.g. replication protein A, replication protein C (38), are required). Exactly how PCNA functions is still unresolved.
PCNA has been demonstrated to promote pol -catalyzed DNA replication
past thymidine dimers (22) and, more recently, past abasic sites,
C8-(2-aminofluorenyl)guanine, and 8-oxoG but not
C8-(2-acetamidofluorenyl) guanine (23).
Misincorporation in normal DNA (with the four normal dNTPs) is
increased in in vitro pol
experiments by the presence of
PCNA (24). This effect could be due to promotion of extension beyond
incorrect base pairs or to increased catalysis of misincorporation
per se. This study clearly indicates that the extension
beyond the A:8-oxoG base pair predominates (over C:8-oxoG pairs) and
that PCNA stimulates this reaction 70-fold. Mozzherin et al.
(24) and McConnell et al. (28) proposed that the
principal effect of PCNA stimulation of pol
is an increased
affinity of pol
for DNA (2000-fold). Electrophoretic gel shift
mobility results were presented as evidence, but exactly how these
assays relate to events in catalysis is yet unknown. Interestingly, in
steady-state assays the KmdNTP value was
mainly affected in correct nucleotide incorporation (Table I) (21). Is
there a direct effect of PCNA on processivity, as proposed by others
(23, 24)? In assays in which single base incorporation is measured
(e.g. this work), kcat is probably approximated by k7 (Fig. 6), the
koff rate (see below). However, in steady-state
kinetic experiments in which a single dNTP is incorporated, there is
little effect of PCNA on kcat (Table II). McConnell et al. (28) estimated the t1/2 of the pol
-oligonucleotide complex to be 2.65 h, in the
presence of PCNA, using a filter binding assay. In that work the
authors did not express "Vmax" in
quantitative terms, so no direct interpretation can be made. However,
this t1/2 corresponds to
k7 < 0.3 h
1, which is 2 orders of magnitude slower than the steady-state kcat in our own experiments (Table II).
Qualitative evidence also indicates that the presence of dNTP
significantly destabilizes the pol
·PCNA·DNA complex (33).
We did, as in Ref. 23, find that PCNA stimulated polymerization beyond
8-oxoG lesions. The extent of stimulation by PCNA was estimated by
Mozzherin et al. (23) to be 2.5-fold. In our pre-steady-state experiments, the stimulation of dNTP incorporation beyond either 8-oxoG:A or 8-oxoG:C pairs by PCNA was much greater (Fig.
3). The extent of stimulation by PCNA might have been underestimated in
the previous steady-state work (23) because of the contribution of the
inherent exonuclease activity of pol (21), which can obscure
steady-state experiments. Another possibility for the difference seen
is that Mozzherin et al. (23) used a mixture of dNTPs in
their experiments, the effect of preferential extension beyond an
8-oxoG:A pair in the presence of PCNA (Fig. 3A) might have
been diminished because of the lower efficiency of incorporation of
dATP opposite 8-oxoG (Table II).
The role of PCNA is probably more complex than simply increasing the
affinity of pol with oligonucleotides. A possible explanation (Fig.
6) is that PCNA alters the rates of conversion of the pol
·DNA
complex to and from an inactive complex. Further analysis will be
required to address the hypothesis. Another issue is that of other
accessory proteins. Additional components of yeast pol
have been
identified (39), and Lee and associates (35) have presented evidence
that an additional 12- and two 25-kDa subunits may be associated with
mammalian pol
, as well as the 125- and 50-kDa proteins and PCNA
(40, 41). The work to date has involved only association studies (40)
and no function has been associated with these gene products yet. These
additional subunits may have contributions to the activity and fidelity
of pol
, particularly when blocking lesions are a consideration.
However, we do feel that the 3-protein pol
system we are using here
(125- and 50-kDa proteins and PCNA) is a useful one in that up to 90%
burst amplitudes with some pol
preparations are observed in the
pre-steady-state experiments (Fig. 3A), and appreciable
bursts are seen even at incorporation opposite 8-oxoG (Fig. 1).
Incorporation of 8-Oxo-dGTP by pol --
There is potential for
8-oxo-dGTP to be incorporated by DNA polymerases (42, 43) and cause A
C transversions (43). Efficiencies of 8-oxo-dGTP incorporation
opposite A or C by prokaryotic polymerases are 4 orders of magnitude
lower than normal dGTP incorporation opposite C (9). The ability of pol
to incorporate 8-oxo-dGTP opposite A or C was also examined using
steady-state kinetics, and a similar low nucleotide insertion
efficiency of 8-oxo-dGTP was found for pol
. As in the case of the
prokaryotic polymerases (9), the very poor kinetic efficiency for
incorporation opposite C or A is the result of an unfavorable
Km. We would presume that the fraction of the dGTP
pool accounted for by 8-oxo-dGTP is similar to the situation in DNA,
suggesting that ~1/106 of the dNTP pool is 8-oxo-dGTP
(15-17, 44). The actual 8-oxo-dGTP pool size has not been reported but
preliminary estimates are very
low.4 With a catalytic
efficiency for 8-oxo-dGTP incorporation ~10
3 that of dGTP
by pol
(this work), the probability of inserting 8-oxo-dGTP should
be ~10
9. The significance of the MutT "repair"
enzyme in mammalian systems (45) is an issue in that the tendency to
insert 8-oxo-dGTP appears to be low. We raise the possibility that the
critical substrate for this enzyme may not be 8-oxo-dGTP but instead a
yet unknown nucleotide. However, an alternative explanation we cannot
rule out is that other mammalian polymerases insert 8-oxo-dGTP with high efficiency.
Conclusions--
We have studied the replication events at 8-oxoG
catalyzed by mammalian pol because there is rather general
agreement that this is the main polymerase involved in leading strand
DNA replication (1, 2). Also, conservation of sequence identity among
all of the components of the system is extensive. However, the
3-protein system we used here is a simplification of the cellular
situation, in which coupling with pol
and possibly other proteins
exist. Nevertheless, the system we use does provide a means of studying the interactions of pol
with DNA and carcinogen-modified DNA. Another point is that a number of relatively nonprocessive polymerases have been identified and probably make important contributions to the
outcome of the processing of modified DNA (6, 46). At this time it is
not clear exactly which of these become involved in replication past
8-oxoG lesions in mammalian cells.
In pre-steady-state experiments, pol incorporated both dCTP and
dATP opposite 8-oxoG with similar rates as for correct nucleotide incorporation into unmodified DNA. The efficiency of incorporation opposite 8-oxoG was dominated by a reduced
KddNTP and there was a preference for
incorporation of dCTP opposite 8-oxoG. Although pol
displayed a
preference for correct incorporation, extension beyond A:8-oxoG pairs
predominated. An issue central to many experiments presented in this
paper is the role of PCNA. The presence of PCNA was more essential to
the efficiency of nucleotide incorporation opposite 8-oxoG than
reactions with unmodified DNA (21). PCNA was also found to be essential
to the extension beyond A:8-oxoG or C:8-oxoG base pairs. The
mechanistic role of PCNA in these reactions could involve alterations
of koff (k7 of Fig. 6),
dNTP binding, or conformational effects of the enzyme. Our work can be
interpreted in terms of a role for altering rates of conversion of pol
·DNA complexes to unproductive forms and vice versa. Evidence for
the existence and importance of such unproductive complexes with
prokaryotic replicative polymerases has been presented from this (14)
and other laboratories (47-50). We propose that such inactive
complexes are also an issue with mammalian polymerases such as pol
.
With all of these postulated unproductive complexes, there is no
structural information available. Another current deficit is the lack
of ability to monitor changes in real time, although some possibilities
exist (13, 32) and are the subject of future efforts.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the contributions of K. M. Downey, A. G. So, and M. Carastro (University of Miami School
of Medicine) and V. N. Podust, E. Fanning, J. B. Wheeler, and
A. N. Mican (Vanderbilt University) for their assistance in the
purification of pol . We also thank E. Howard (Protein Chemistry
Core Facility (Vanderbilt University) for quantitative amino acid
analysis of pol
, B. Stillman (Cold Spring Harbor Laboratory) for
the human PCNA bacterial expression vector, and L. L. Furge
(Kalamazoo College) and A. N. Mican for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by U. S. Public Health Service Grants R35 CA44353 and P30 ES00267.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.
Supported in part by U. S. Public Health Service Grant F32
CA75731. Current address: Dept. of Drug Metabolism and
Pharmacokinetics, Novartis Pharmaceuticals Corp., Bldg. 405, Room 462A,
59 Rte. 10, East Hanover, NJ 07936-1080. Tel.: 973-781-3119; Fax:
973-781-5023; E-mail: heidi.einolf@pharma.novartis.com.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Medical Research Building I, 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: guengerich@toxicology.mc.vanderbilt.edu.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M006696200
1
The abreviations used are: 8-oxoG,
8-oxo-7,8-dihydrodeoxyguanosine; pol, polymerase; PCNA, human
proliferating cell nuclear antigen; G, guanine; A, adenosine; dNTP,
deoxynucleotide triphosphate; S-dNTP,
-thio-substituted dNTPs;
bis-Tris-HCl, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane-HCl; HIV-1 RT, human immunodeficiency virus-1 reverse transcriptase.
2 Using the kcat and Km values, a "misincorporation frequency" (f) of 0.33 can be estimated, with caveats of the limitations of these single nucleotide incorporation experiments, defined as f = [(kcat/Km)A/(kcat/Km)c], estimated % misincorporation = (f/[1 + f]) × 100. The preference for incorporation of dCTP opposite 8-oxoG is reflected in a misinsertion frequency (f) of 0.33, which corresponds to 24% misincorporation (for dATP).
3
A misincorporation frequency (f) of 0.03 can be calculated, again with the caveats of limitations of single
nucleotide incorporation experiments (see Footnote 2), corresponding to
3% misincorporation (of 8-oxo-dGTP opposite A) (Table II). This value
is an order of magnitude lower than for dATP incorporation opposite
template 8-oxoG, emphasizing the asymmetric nature of polymerases.
Also, the 3% misincorporation frequency is much lower than observed for any other polymerases examined, except for E. coli pol
II exo (9).
4 C. K. Mathews, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kornberg, A., and Baker, T. A. (1992) DNA Replication , W. H. Freeman, New York |
2. | Friedberg, E., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis , American Society of Microbiology, Washington, D. C. |
3. | Searle, C. E. (1984) Chemical Carcinogens , Vol. 1 and 2 , American Chemical Society, Washington, D. C. |
4. | Morales, J. C., and Kool, E. T. (2000) J. Am. Chem. Soc. 122, 1001-1007[CrossRef] |
5. | 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] |
6. |
Johnson, R. E.,
Prakash, S.,
and Prakash, L.
(1999)
Science
283,
1001-1004 |
7. | Langouët, S., Müller, M., and Guengerich, F. P. (1997) Biochemistry 36, 6069-6079[CrossRef][Medline] [Order article via Infotrieve] |
8. | Langouët, S., Mican, A. N., Müller, M., Fink, S. P., Marnett, L. J., Muhle, S. A., and Guengerich, F. P. (1998) Biochemistry 37, 5184-5193[CrossRef][Medline] [Order article via Infotrieve] |
9. | Einolf, H. J., Schnetz-Boutaud, N., and Guengerich, F. P. (1998) Biochemistry 37, 13300-13312[CrossRef][Medline] [Order article via Infotrieve] |
10. | Kim, M-S., and Guengerich, F. P. (1998) Chem. Res. Toxicol. 11, 311-316[CrossRef][Medline] [Order article via Infotrieve] |
11. | Lowe, L. G., and Guengerich, F. P. (1996) Biochemistry 35, 9840-9849[CrossRef][Medline] [Order article via Infotrieve] |
12. | Furge, L. L., and Guengerich, F. P. (1997) Biochemistry 36, 6475-6487[CrossRef][Medline] [Order article via Infotrieve] |
13. | Furge, L. L., and Guengerich, F. P. (1998) Biochemistry 37, 3567-3574[CrossRef][Medline] [Order article via Infotrieve] |
14. | Furge, L. L., and Guengerich, F. P. (1999) Biochemistry 38, 4818-4825[CrossRef][Medline] [Order article via Infotrieve] |
15. | Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S., Miura, K., Ohtsuka, E., and Nishimura, S. (1987) Nature 327, 77-79[CrossRef][Medline] [Order article via Infotrieve] |
16. | Ames, B. N., and Gold, L. S. (1991) Mutation Res. 250, 3-16[Medline] [Order article via Infotrieve] |
17. | Kasai, H., Crain, P. F., Kuchino, Y., Nishimura, S., Ootsuyama, A., and Tanooka, H. (1986) Carcinogenesis 7, 1849-1851[Abstract] |
18. | Johnson, K. A. (1993) Annu. Rev. Biochem. 62, 685-713[CrossRef][Medline] [Order article via Infotrieve] |
19. | Goodman, M. F. (1998) Nature 395, 221-223[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Tang, M.,
Bruck, I.,
Eritja, R.,
Turner, J.,
Frank, E. G.,
Woodgate, R.,
O'Donnell, M.,
and Goodman, M. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9755-9760 |
21. |
Einolf, H. J.,
and Guengerich, F. P.
(2000)
J. Biol. Chem.
275,
16316-16322 |
22. | O'Day, C. L., Burgers, P. M. J., and Taylor, J. S. (1992) Nucleic Acids Res. 20, 5403-5406[Abstract] |
23. |
Mozzherin, D. J.,
Shibutani, S.,
Tan, C. K.,
and Downey, K. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6126-6131 |
24. |
Mozzherin, D. J.,
McConnell, M.,
Jasko, M. V.,
Krayezsky, A. A.,
Tan, C. K.,
Downey, K. M.,
and Fisher, P. A.
(1996)
J. Biol. Chem.
271,
31711-31717 |
25. | Fien, K., and Stillman, B. (1992) Mol. Cell. Biol. 12, 155-163[Abstract] |
26. | Podust, V. N., Georgaki, A., Strack, B., and Hübscher, U. (1992) Nucleic Acids Res. 20, 4159-4165[Abstract] |
27. |
Schumacher, S. B.,
Stucki, M.,
and Hübscher, U.
(2000)
Nucleic Acids Res.
28,
620-625 |
28. | McConnell, M., Miller, H., Mozzherin, D. J., Quamina, A., Tan, C. K., Downey, K. M., and Fisher, P. A. (1996) Biochemistry 35, 8268-8274[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Wu, S. M.,
Zhang, P.,
Zeng, X. R.,
Zhang, S. J.,
Mo, J.,
Li, B. Q.,
and Lee, M. Y.
(1998)
J. Biol. Chem.
273,
9561-9569 |
30. |
Kati, W. M.,
Johnson, K. A.,
Jerva, L. F.,
and Anderson, K. S.
(1992)
J. Biol. Chem.
267,
25988-25997 |
31. | Patel, S. S., Wong, I., and Johnson, K. A. (1991) Biochemistry 30, 511-525[Medline] [Order article via Infotrieve] |
32. | Frey, M. W., Sowers, L. C., Millar, D. P., and Benkovic, S. J. (1995) Biochemistry 34, 9185-9192[Medline] [Order article via Infotrieve] |
33. |
Ng, L.,
McConnell, M.,
Tan, C-K.,
Downey, K. M.,
and Fisher, P. A.
(1993)
J. Biol. Chem.
268,
13571-13576 |
34. | Xiao, W., Derfler, B., Chen, J., and Samson, L. (1991) EMBO J. 10, 2179-2186[Abstract] |
35. | Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913[Medline] [Order article via Infotrieve] |
36. |
Zhang, P.,
Mo, J-Y.,
Perez, A.,
Leon, A.,
Liu, L.,
Mazloum, N.,
Xu, H.,
and Lee, M. Y. W. T.
(1999)
J. Biol. Chem.
274,
26647-26653 |
37. |
Mozzherin, D. J.,
Tan, C-K.,
Downey, K. M.,
and Fisher, P. A.
(1999)
J. Biol. Chem.
274,
19862-19867 |
38. | Hindges, R., and Hübscher, U. (1997) Biol. Chem. 378, 345-362[CrossRef] |
39. |
Burgers, P. M. J.,
and Gerik, K. J.
(1998)
J. Biol. Chem.
273,
19756-19762 |
40. |
Liu, L.,
Mo, J.,
Rodriguez-Belmonte, E. M.,
and Lee, M. Y. W. T.
(2000)
J. Biol. Chem.
275,
18739-18744 |
41. | Mo, J., Liu, L., Leon, A., Mazloum, N., and Lee, M. Y. W. T. (2000) Biochemistry 39, 7245-7254[CrossRef][Medline] [Order article via Infotrieve] |
42. | Maki, H., and Sekiguchi, M. (1992) Nature 355, 273-275[CrossRef][Medline] [Order article via Infotrieve] |
43. | Pavlov, Y. I., Minnick, D. T., Izuta, S., and Kunkel, T. A. (1994) Biochemistry 33, 4695-4701[Medline] [Order article via Infotrieve] |
44. | Cadet, J., Berger, M., Douki, T., and Ravanat, J. (1998) in Physiology, Biochemistry, and Pharmacology (Blaustein, M. P. , Greger, R. , Grunicke, H. , John, I. R. , Mendell, L. M. , Pette, D. , Schultz, G. , and Schweiger, M., eds) , pp. 1-86, Springer-Verlag, Berlin |
45. |
Sakumi, K.,
Furuichi, M.,
Tsuzuki, T.,
Kakuma, T.,
Kawabata, S.-I.,
Maki, H.,
and Sekiguchi, M.
(1993)
J. Biol. Chem.
268,
23524-23530 |
46. |
Friedberg, E. C.,
Feaver, W. J.,
and Gerlach, V. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5681-5683 |
47. | Suo, Z., Lippard, S. J., and Johnson, K. A. (1999) Biochemistry 38, 715-726[CrossRef][Medline] [Order article via Infotrieve] |
48. | Suo, Z., and Johnson, K. A. (1997) Biochemistry 36, 12459-12467[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Suo, Z.,
and Johnson, K. A.
(1998)
J. Biol. Chem.
273,
27259-27267 |
50. | Wöhrl, B. M., Krebs, R., Goody, R. S., and Restle, T. (1999) J. Mol. Biol. 292, 333-344[CrossRef][Medline] [Order article via Infotrieve] |