From the Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7260
Received for publication, August 25, 2000, and in revised form, February 27, 2001
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ABSTRACT |
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DNA adducts formed by platinum-based anticancer
drugs interfere with DNA replication. The carrier ligand of the
platinum compound is likely to affect the conformation of the Pt-DNA
adducts. In addition, the conformation of the adduct can also change
upon binding of damaged DNA to the active site of DNA polymerase. From the crystal structures of pol Platinum-based chemotherapeutic drugs exert their cytotoxic effect
through binding to DNA.
cis-diaminedichloroplatinum(II)(cisplatin)1
(Fig. 1), which has been broadly used for cancer treatment since 1978, and
(trans-R,R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) (Fig. 1), which is currently in clinical trial in the
United States and has been approved in several European countries for
the treatment of tumors with intrinsic and acquired resistance to
cisplatin, both form the same types adducts on the DNA (1, 2). Although
the most prevalent lesions created by interaction of both these agents
with DNA are bifunctional intrastrand 1,2-d(GpG) cross-links, these
adducts differ in the carrier ligands they retain upon binding to DNA.
Pt-DNA adducts formed by oxaliplatin contain
(trans-R,R)-1,2-diaminocyclohexane
(dach) carrier ligands, whereas the adducts formed by cisplatin contain
cis-diammine carrier ligands. Structures of the DNA
containing cisplatin-GG adducts determined by NMR and x-ray
crystallography reveal significant distortion of base pairs in the
vicinity of the adduct (3-6). In addition, both the crystallographic
and NMR structures show that the cisplatin adduct causes a large
positive roll in the helix resulting in compression of the major groove
and widening of the minor groove. Although the conformation of
oxaliplatin-GG adducts has not yet been determined experimentally, a
recent molecular modeling study suggests that the overall conformation
of cisplatin- and oxaliplatin-GG adducts are likely to be very similar.
However, the bulky dach ring of the oxaliplatin adduct fills much of
the DNA major groove, causing it to be narrower and less polar in the
area of the adduct. These subtle differences in overall DNA conformation appear to influence the biological effects of the Pt-DNA
adducts. Thus, both the mismatch repair complex (7) and high mobility
group proteins (8) bind more strongly to cisplatin-GG adducts than to
oxaliplatin-GG adducts. In addition, eukaryotic DNA polymerases The conformation of Pt-DNA adducts in the active site of DNA
polymerases is likely to be different from that of adducted DNA in
solution. The fact that DNA templates are often bent upon binding to
the active site of DNA polymerases (11) supports this hypothesis. Unfortunately, whereas the conformation of mutagenic DNA adducts has
been determined on a variety of duplex DNAs in solution, relatively little is known about how binding of the DNA templates to a polymerase active site affects the conformation of the adduct and/or the nascent
base pair. The conformation of Pt-DNA adducts is clearly not identical
in all DNA polymerase active sites. Whereas pol In contrast to the situation with 6 nucleotide-gapped templates, the
conformations of both primed single-stranded and single nucleotide-gapped templates bound to the active site of pol Construction of Platinum Adduct-containing
Templates--
Primer-templates were constructed from synthetic
oligonucleotides as described previously (8). Briefly, platination
reactions were carried out with aquated derivatives of cisplatin
(Sigma) and the active biotransformation product of oxaliplatin,
Pt(dach)Cl2 (provided by Dr. S. D. Wyrick, UNC).
Platination of a 12-mer oligonucleotide containing a single GG sequence
within a StuI restriction site (TCTAGGCCTTCT)
was performed for 2 h at 37 °C in the dark with a 2:1
drug/oligonucleotide ratio. The oligonucleotides containing single
platinum adducts were separated from unplatinated oligonucleotides by
20% polyacrylamide gel electrophoresis.
The templates used for primer extension and steady-state kinetic
experiments are listed in Fig. 1. DNA templates (44-mer), with or
without platinum adduct, were constructed as described previously (8).
Briefly, platinated 12-mer or undamaged 12-mer (used for assembling
control DNA templates) were ligated with the 14-mer on the 5'-end and
an 18-mer on the 3'-end of the template using a 35-mer scaffold. After
16 h of ligation at 16 °C by T4 DNA ligase, templates were
restricted by StuI to ensure the absence of any unplatinated
oligonucleotides and purified on 12% denaturing polyacrylamide gels.
Control experiments were performed to ensure the purity of platinated
templates as described previously (8).
The 24- or 25-mer primers were 32P 5'-end-labeled. The
downstream oligonucleotides (19- and 18-mer) used to form single
nucleotide-gapped templates were 5'-end phosphorylated with unlabeled
ATP. DNA substrates were prepared by annealing undamaged or platinated
44-mer templates with primers and downstream oligonucleotides at a
1.2:1:2 molar ratio. Annealing efficiencies were >99%, as evidenced
by the different mobility of the 32P-labeled primers before
and after hybridization to the template on non-denaturing
polyacrylamide gels (data not shown).
Steady-State Polymerization Kinetics--
Recombinant human pol
Primer Extension Assays Using Individual Nucleotides--
To
compare the pattern of misincorporation opposite Pt-GG adducts on
primed single-stranded and gapped DNA templates, primer extension
assays were performed using each dNTP individually as described
previously (9). DNA templates (200 fmol expressed as primer termini)
were incubated with 5 fmol of pol Kinetic Misincorporation Assays--
To measure nucleotide
misinsertion opposite Pt-GG adducts, 150 fmol of single
nucleotide-gapped DNA substrates were incubated with 5 fmol of pol In a previous study (9) we performed a kinetic analysis of
nucleotide incorporation opposite Pt-GG adducts in a 5'-AGGC-3' sequence by pol ternary complexes it is
evident that undamaged gapped and primed single-stranded (non-gapped) DNA templates exist in very different conformations when bound to pol
. Therefore, one might expect that the constraints imposed on the
damaged templates by binding to the polymerase active site should also
affect the conformation of the Pt-DNA adducts and their ability to
inhibit DNA replication. In support of this hypothesis we have found
that the efficiency, carrier ligand specificity, site of discrimination
(3'-G versus 5'-G of the Pt-GG adducts), and fidelity of
translesion synthesis past Pt-DNA adducts by pol
are strongly
affected by the structure of the DNA template. Previous studies have
suggested that the conformation of Pt-DNA adducts may be affected by
the sequence context of the adduct. In support of this
hypothesis, our data show that sequence context affects the efficiency,
fidelity, and pattern of misincorporation by pol
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
,
, and
bypass oxaliplatin-GG adducts more readily than
cisplatin-GG adducts (8-10). Finally, the misincorporation frequency
of DNA polymerase
(pol
) is slightly greater with
oxaliplatin-GG adducts than with cisplatin-GG adducts on primed
single-stranded DNA (9). These data suggest that differences in the
conformation of oxaliplatin- and cisplatin-GG adducts can affect both
the efficiency and fidelity of translesion synthesis by DNA
polymerases, which could at least partially account for differences in
the cytotoxicity and mutagenicity of these Pt-DNA adducts.
,
,
, and
all bypass oxaliplatin adducts better than cisplatin adducts, HIV-1
reverse transcriptase does not discriminate between cisplatin and
oxaliplatin adducts (8-10). In addition, pol
,
,
, and
differ in the site of replication termination, the site of
discrimination between cisplatin- and oxaliplatin-GG adducts (the 3'-G
or the 5'-G) and the pattern of misincorporation opposite the adduct
(8-10). The conformation of the 6 nucleotide-gapped template bound to
pol
has not been directly determined but is likely to be different
from the conformation of primed single-stranded DNA bound to pol
(12). Studies on the efficiency, specificity (cisplatin
versus oxaliplatin), and fidelity of translesion synthesis by pol
on primed single-stranded and 6 nucleotide-gapped templates also suggest that the manner in which the adduct binds to the active
site of the DNA polymerase could have significant biological consequences. For example, pol
catalyzes 2.5-fold more translesion synthesis past oxaliplatin adducts than past cisplatin adducts in
experiments using single-stranded DNA, but does not appear to
differentiate between cisplatin and oxaliplatin adducts during 6 nucleotide gap filling DNA synthesis (9). In addition, the type of the
DNA template used also influences the effect of platinum adducts on the
nucleotide insertion fidelity of pol
(9).
are
known and are quite different. For the primed single-stranded template,
the double-stranded region is bound to the enzyme, whereas the
single-stranded region does not interact with the polymerase and thus,
has considerable flexibility (13). With a single nucleotide-gapped template both ends of the gap are bound to enzyme, resulting in a 90°
bend in the template (12, 14). Steady-state and presteady-state kinetic
analyses (15, 16) have shown that the catalytic efficiency and
nucleotide insertion fidelity of pol
are strongly influenced by the
type of DNA substrate (gapped versus non-gapped). Even though only the structure of primed single-stranded template is likely
to be relevant to translesion synthesis by pol
in vivo, usage of conformationally different DNA templates should serve as a
good model system to study influence of template structure on
translesion synthesis. Therefore, the present study was designed to
evaluate how the use of single nucleotide-gapped DNA templates would
affect the efficiency, carrier ligand specificity, and nucleotide insertion fidelity of DNA synthesis past Pt-DNA adducts by pol
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was generously provided by Dr. S. Wilson (NIEHS, National
Institutes of Health). The steady-state kinetic parameters
Km and Vmax for dCTP
incorporation were measured as a function of deoxynucleotide
concentration (17, 18). Single nucleotide-gapped DNA templates (150 fmol) were incubated at 37 °C in 10 µl-reaction mixtures
containing 50 mM Tris-HCl, pH 8.0, 10 mM
MgCl2, 2 mM dithiothreitol, 20 mM
NaCl, 200 µg/ml bovine serum albumin, 2.5% glycerol, 5 fmol of pol
, and variable concentrations of dCTP. To determine the efficiency of nucleotide incorporation opposite the 3'-G, dCTP concentrations ranged from 1 to 8 µM for the undamaged template and from
16 to 256 µM for the platinated templates. To determine
efficiency of nucleotide incorporation opposite the 5'-G, dCTP
concentrations ranged from 0.03 to 0.5 µM for the
undamaged template, from 16 to 256 µM for the
cisplatin-containing templates and from 0.25 to 4 µM for
the oxaliplatin-containing template. Reactions were terminated by
mixing with 0.7 volumes of formamide-loading dye containing 500 mM EDTA, 0.1% bromphenol blue in 90% formamide and were
then immediately transferred onto ice for 5 min. Products were resolved
by denaturing polyacrylamide gel electrophoresis (8 M urea,
16% acrylamide, 4 h at 2000 V) and then visualized and quantified
using a Molecular Dynamics PhosphorImager and ImageQuant software.
Initial time-course studies indicated that under standard conditions
product formation was linear (i.e. conforms to steady-state kinetics) for up to 4 min. Therefore, reactions were performed for 1 min for control DNA templates and for 2 min for platinated DNA
templates. Less than 20% of the primers were extended under these
conditions. The velocity of dCTP incorporation opposite the template
3'-G and 5'-G sites was determined as described previously (17, 18).
Vmax (the maximum reaction velocity) and
Km (dCTP concentration at which the reaction
velocity is half-maximal) were determined from a Hanes-Woolf plot by
linear least squares fit (18). Values for the apparent
kcat were calculated based on the assumption
that pol
was fully active. The efficiency of nucleotide insertion
by pol
was calculated as
kcat/Km. To facilitate
comparison of values for different platinum adducts, the relative
insertion efficiency frel was calculated as
frel = (kcat/Km for
Pt)/(kcat/Km for control).
Because the steady-state kinetic analysis utilized in these experiments produces an apparent rather than a true Vmax
(17, 18), Vmax and kcat
values in the literature are reported in a variety of units. The
kcat values we report were obtained by dividing
Vmax (in nM primer extended per min)
by the enzyme concentration (0.5 nM). To compare these data
with the Vmax and
Vmax/Km values reported in our previous kinetic analysis of translesion synthesis by pol
with different DNA templates (9), these kcat and
kcat/Km values must be divided by
30. To compare these data with Vmax values
reported as nM min
1, these
kcat values must be multiplied by 5.
at 37 °C for 30 min in 10-µl
reactions containing 5 mM of each dNTP individually.
and variable concentrations of dTTP or dATP. Reactions were performed
for 5 min for undamaged DNA templates and 10 min for platinated DNA
templates. dTTP concentrations ranged from 5 to 100 µM
for undamaged and from 250 to 5000 µM for platinated templates. dATP concentrations ranged from 25 to 400 µM
for undamaged and from 200 to 3600 µM for platinated
templates. The kinetics of nucleotide misincorporation opposite
platinated and undamaged GG sites were determined as described above.
The misinsertion frequency (fmis) was determined
as the ratio of insertion efficiencies (kcat/Km) for incorrect and
correct nucleotides. The relative misinsertion frequency was determined
as fmis.rel = (fmis-Pt)/(fmis-control).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
using primed single-stranded DNA templates and 6 or
5 nucleotide-gapped DNA templates. In those studies, cisplatin-GG adducts inhibited nucleotide incorporation to a greater extent than
oxaliplatin-GG adducts when primed single-stranded templates were used,
but pol
did not discriminate between cisplatin and oxaliplatin
adducts during short gap filling. In the present study we have examined
the catalytic efficiency and fidelity of pol
on DNA templates with
a single nucleotide gap opposite the 3'- or 5'-G of the 5'-AGGC-3'
sequence in both undamaged templates and templates containing a single
cisplatin- or oxaliplatin-GG adduct (Fig.
1). These experiments are of interest
because the conformation of this type of DNA template bound to pol
is very different from the templates with platinum adducts that have
been analyzed previously (14).
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Fig. 1.
Chemical structure of the platinum complexes
(A), configuration (B), and sequence
(C) of single nucleotide-gapped DNA templates used in
primer-extension and kinetic studies. The site-specifically
modified DNA templates were constructed using a combination of
synthetic oligonucleotides as described under "Experimental
Procedures." B shows a schematic representation of the DNA
template used to determine nucleotide incorporation opposite the 3'-
and 5'-G. The top panel of C shows the sequences
of the template, upstream primer, and downstream acceptor used to
measure nucleotide incorporation opposite the 3'-G. It also shows the
variations in the sequence of the template and primer that were used to
assess the effect of changes in the base 3' to the target site on the
efficiency of correct nucleotide incorporation and the pattern of
misinsertion. The bottom panel of C shows the
sequences of the template, upstream primer, and downstream acceptor
used to measure nucleotide incorporation opposite the 5'-G, along with
the changes in sequence of the template and acceptor used to assess the
effect of the base 5' to the target base. The GG sites of
platinum adduct binding are underlined. In addition to the
templates shown here, primed single-stranded templates (in which
downstream oligonucleotides were omitted) and templates with 5 or 6 nucleotide gaps (in which downstream oligonucleotide was a 14-mer) were
used to compare pattern of misincorporation on structurally different
DNA templates. These templates have been described previously
(9).
Effect of DNA Template Structure on Efficiency of dCTP Incorporation-- We performed steady-state kinetic assays for dCTP insertion opposite the template 3'- and 5'-G on undamaged templates and templates with cisplatin and oxaliplatin adducts by measuring nucleotide insertion at the first template site adjacent to the primer terminus (18). All experiments were carried out in the presence of a 30-fold excess of template over enzyme. The results are summarized in Table I. One striking observation was the 10-fold lower dCTP incorporation efficiency (kcat/Km) opposite the 3'-G relative to the 5'-G on undamaged single nucleotide-gapped DNA. This appears to be primarily an effect of sequence context on dCTP incorporation efficiency (see below). However, the influence of sequence context on the efficiency of dCTP incorporation opposite the guanines also depends on template structure, because no significant differences were observed in the kcat/Km for dCTP incorporation opposite the 3'- and 5'-G in the same sequence context with longer gapped or primed single-stranded DNA templates (9).
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The enzyme concentration, template concentration, and template sequence
used in these experiments are identical to those used in our previous
study of pol (9). Thus, the kinetic parameters obtained in this
study can be directly compared with the kinetic parameters reported in
our previous study (9). This allows a comparison of the efficiency and
fidelity of translesion synthesis past platinum-DNA adducts by pol
with single nucleotide-gapped templates (this study), 5 or 6 nucleotide-gapped templates (9), and primed single-stranded templates
(9). Such a comparison indicates that the use of single
nucleotide-gapped DNA results in an increase in the efficiency of
nucleotide insertion
(kcat/Km) by pol
on
undamaged templates. Thus, dCTP incorporation opposite the 3'- and 5'-G
was 2.5-30 and 10-120 times more efficient on single
nucleotide-gapped DNA than on the DNA with longer gaps and non-gapped
substrates, respectively. This is similar to the results reported
previously (15, 16).
Pt-GG adducts had different effects on the efficiency
(kcat/Km) of dCTP insertion
opposite the 3'-G and the 5'-G (Table I). There was a similar decrease
in dCTP insertion efficiency across from the 3'-G of both platinum
adducts (Table I). In contrast, the efficiencies of dCTP incorporation
opposite the 5'-G were very different depending on carrier ligand of
platinum adduct. There was a 170-fold decrease in insertion efficiency
for dCTP incorporation opposite the 5'-G of cisplatin adduct. This is
the most significant inhibition of pol catalytic activity by a
cisplatin adduct among all templates (gapped and non-gapped) tested
(compare Table I and results in Vaisman and Chaney, Ref. 9). On the other hand, there was relatively little inhibition of pol
catalytic efficiency opposite the 5'-G of the oxaliplatin adduct (Table I and
Ref. 9). Thus, our data show a very interesting variation in the
carrier ligand specificity for dCTP incorporation as the DNA template
changes from primed single-stranded to short-gapped (5 or 6 nucleotides) and to single nucleotide-gapped. With the primed
single-stranded template, the efficiency of dCTP incorporation was
about 2.5-fold greater for the oxaliplatin adduct opposite the 3'-G but
was about the same for the two adducts opposite the 5'-G (9). With the
short-gapped DNA (5 and 6 nucleotides), no significant differences were
evident between templates with cisplatin and oxaliplatin adducts in the
insertion efficiency of dCTP opposite either the 3'-G or the 5'-G (9).
With the single nucleotide-gapped template the efficiency of dCTP
incorporation was about 20-fold greater for the oxaliplatin adduct
opposite the 5'-G but was about the same for both adducts opposite the 3'-G (this study).
These differences could be related to the differences in the
conformation of the non-gapped and gapped DNA templates bound to pol
. The x-ray crystal structure of pol
with a non-gapped primed
template shows that the double-stranded region of the template is
aligned in the palm subdomain of the 31-kDa domain of polymerase (13,
19). The thumb region (C-terminal domain) closes around the DNA
template to form a binding pocket for the incoming dNTP (13, 19).
Whereas the double-stranded region and the template base for the
incoming dNTP are tightly constrained by the thumb and fingers in this
structure, the single-stranded region appears to have considerable
flexibility (13, 19). In contrast, with single nucleotide-gapped DNA
the amino-terminal 8-kDa domain of pol
binds to the downstream
5'-phosphate. This anchors both ends of the gap to the polymerase
active site and results in a 90° bend in the template DNA (12).
Although a crystal structure of pol
with a 6 nucleotide-gapped
template is currently unavailable because of the difficulty in
obtaining an appropriate crystal (12), it is likely that the
conformation of the templates with longer gaps are different from the
conformation of single nucleotide-gapped DNA. For example, with the one
nucleotide-gapped DNA, the 5'-phosphate in the DNA gap is hydrogen
bonded to Lys-68 (14), whereas for the DNA with a gap greater than one
nucleotide, the 5'-phosphate is hydrogen bonded to Lys-35, Lys-68,
Lys-72, and Lys-84 (20).
It is possible that the intrinsic conformation of the Pt-DNA adducts in
primed single-stranded and single nucleotide-gapped DNA may also be
different in solution. For example, dramatic conformational differences
were observed in NMR solution structures of the
()-trans-anti-[BP]dG adduct in single nucleotide-gapped
and primed single-stranded DNA templates (21, 22). However, because of
the severe constraints imposed on the gapped DNA when it binds to the
active site of pol
, we feel that the greatest conformational
differences between Pt-DNA adducts on the templates we have used are
likely to be those imposed by the enzyme. These conformational
differences probably play a critical role in determining the
specificity, site and level of inhibition of nucleotide incorporation
by the different Pt-DNA templates.
Nucleotide Misincorporation Opposite GGs on Undamaged and
Platinated Templates--
The conformation of the adduct at the active
site of a DNA polymerase could also affect its mutagenicity and the
spectrum of mutations that are produced. To obtain qualitative
information about misincorporation across from the platinum adducts,
extension studies by pol were performed in the presence of 5 mM of either dCTP, dGTP, dTTP, or dATP. Fig.
2 shows the results of these experiments when both single-stranded and gapped templates were utilized. The
presence of a platinum adduct did not influence the apparent pattern of
nucleotide misincorporation. (Because the pattern of misincorporation
was identical for templates with cisplatin and oxaliplatin adducts,
only gels for templates with oxaliplatin adducts are shown). However,
as could be predicted based on the different structures of pol
complexes with the non-gapped and gapped DNA templates, both the
location (3'-G versus 5'-G) and spectrum (dNTP inserted) of
misincorporation by pol
varied significantly depending on the
structure of the DNA template (Fig. 2). dTTP misincorporation was
detected for most DNA templates, except for incorporation opposite the
5'-G on single-stranded templates. None of the other nucleotides were
inserted opposite the 3'-G or 5'-G by pol
with primed
single-stranded DNA templates and templates with 5 or 6 nucleotide
gaps. However, the pattern of misincorporation opposite the template
3'-G and 5'-G with single nucleotide-gapped DNA templates was
different. Pol
inserted dTTP much more readily than dATP opposite
the 3'-G. Little or no misincorporation of dGTP opposite the 3'-G could
be detected, even following a prolonged reaction time and at increased
enzyme concentrations (data not shown). However, misincorporation of all three nucleotides was observed across from the 5'-G (Fig. 2).
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To characterize the misincorporation efficiency on undamaged and
platinated single nucleotide-gapped templates more quantitatively and
to compare these data to previously reported kinetic parameters with
non-gapped templates and templates with 5 or 6 nucleotide gaps (9), a
steady-state kinetic analysis of dTTP and dATP incorporation was
performed (Table II). Chagovetz et
al. (16) have reported previously that the nucleotide insertion
fidelity of pol was 10-100 times higher on single
nucleotide-gapped DNA compared with 6 nucleotide-gapped DNA and
primer-template with no gaps. In contrast to this report, we observed
an increased frequency (fmis) of dTTP
misincorporation opposite the undamaged 3'- and 5'-G residues for
single nucleotide-gapped DNA compared with 5 or 6 nucleotide-gapped DNA
and single-stranded DNA. The most likely explanation for the
differences between our results and previous data (16) is the influence
of the neighboring sequence context. The influence of sequence context
on the fidelity of DNA polymerases has been reported previously (23,
24). For example, Mendelman et al. (24) reported that the
misincorporation frequency for DNA polymerase
and AMV RT varied by
10-fold for the same mispair in different locations of the DNA
template.
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Cisplatin- and oxaliplatin-GG adducts had a similar effect on the efficiency (kcat/Km) of dTTP misincorporation with single nucleotide-gapped templates (Table II). Both adducts decreased the efficiency of dTTP incorporation by 30 to 40-fold opposite the 3'-G and 75 to 100-fold opposite the 5'-G. However, because cisplatin- and oxaliplatin-GG adducts had different effects on the efficiency of correct (dCTP) nucleotide incorporation (9), they also had very different effects on the frequency of misincorporation (fmis = (kcat/Km)dTTP/(kcat/Km)dCTP; Table II). The frequency of dTTP misincorporation opposite the 3'-G was 1.6-fold less for oxaliplatin adducts than for cisplatin adducts. This was because of both a slight decrease in the efficiency of dTTP incorporation (Table II) and a slight increase in the efficiency of dCTP incorporation (Table I) for oxaliplatin adducts compared with cisplatin adducts. Opposite the 5'-G the frequency of dTTP misincorporation was 16-fold less for oxaliplatin adducts than for cisplatin adducts, primarily because of the relatively high efficiency of dCTP incorporation opposite the 5'-G of oxaliplatin adducts (Table I). This pattern of dTTP misincorporation was significantly different from that observed in the same sequence context with templates of different structure. The frequency of dTTP misincorporation opposite the 3'-G was 1.6 to 1.8-fold greater for oxaliplatin adducts than for cisplatin adducts with single-stranded and 6 nucleotide-gapped DNA templates (9). The pattern of dTTP misincorporation opposite the 5'-G could not be quantitatively compared with those templates because of the low frequency of dTTP misincorporation opposite the 5'-G with single-stranded and 5 nucleotide-gapped DNA templates.
In the qualitative experiments to determine the pattern of
misincorporation with single nucleotide-gapped DNA, a significant misinsertion of dATP was observed opposite the 5'-G (Fig. 2). Therefore, we also determined kinetic parameters for dATP incorporation at this site (Table II). The qualitative assessment of misincorporation (Fig. 2) suggested that dTTP and dATP were misincorporated with similar
efficiencies opposite the 5'-G on undamaged templates. However,
quantitative measurements (Table II) showed that pol favored
misincorporation of dTTP by 6.5-fold compared with dATP on undamaged
templates. This apparent discrepancy is explained by the fact that
difference in the efficiency of dATP and dTTP incorporation was
determined primarily by the differences in the Km
values. Because qualitative determination of the pattern of
misincorporation was performed at saturating dNTP concentrations, the
Km-governed discrimination could not be detected in
those experiments. This finding indicates the importance of quantitative analysis of primer extension on damaged and undamaged templates. In contrast to nucleotide incorporation on the undamaged template, pol
catalyzed dTTP and dATP misincorporation with similar
efficiency on platinated templates (Table II). Thus, it is clear that
both cisplatin- and oxaliplatin-GG adducts increase the frequency of
dATP misincorporation relative to dTTP misincorporation. Finally, in
comparing the oxaliplatin- and cisplatin-GG adducts, the frequency of
dATP misincorporation opposite the 5'-G was 17-fold greater for the
cisplatin adducts than for the oxaliplatin adducts.
The Effect of Sequence Context on the Efficiency and Fidelity of
pol --
As described above, the efficiency of dCTP incorporation
was 10-fold greater opposite the 5'-G than opposite the 3'-G of the 5'-AGGC-3' sequence on an undamaged single nucleotide-gapped DNA template (Table I). In addition, the qualitative pattern of
misincorporation on the single nucleotide-gapped template was
dTTP~dATP
dGTP for the 5'-G and dTTP
dATP for the 3'-G (Fig.
2). With a primed single-stranded DNA template, dTTP misincorporation
was seen opposite the 3'-G and no misincorporation could be observed
opposite the 5'-G (Fig. 2). These data suggest that sequence context,
as well as template structure, strongly influences both the efficiency
and fidelity of pol
. To test this possibility, we systematically
altered the bases 5' and 3' to the GG sequence with both single
nucleotide-gapped and single-stranded templates. For the kinetic
analysis of the dCTP incorporation, the measurements were made with the
single nucleotide-gapped DNA template only (Fig. 1C),
because no kinetic differences had been observed previously for the 3'-
and 5'-G in primed single-stranded and longer gapped DNA templates (9). As can be seen in Table III, alteration
of the base 5' to the GG sequence had only a small effect on the
efficiency of dCTP incorporation opposite the 5'-G. In contrast, the
efficiency of dCTP incorporation was strongly influenced by the base 3'
to the adduct. The efficiency of dCTP incorporation opposite the 3'-G
was low with a pyrimidine 3' to the GG sequence. However, when a purine
was 3' to the GG sequence, the efficiency of dCTP incorporation
opposite the 3'-G was increased significantly to a value that is
similar to that seen opposite the 5'-G in the 5'-AGGC-3' sequence.
These data can best be explained by a model in which a purine 3' to the
templating G stabilizes it in the active site of pol
through
stacking interactions. These data also suggest that the greater
efficiency of dCTP incorporation opposite the 5'-G in the original
5'-AGGC-3' sequence was primarily because of the presence of the
adjacent 3'-G.
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To test how local DNA sequence context influences nucleotide
misinsertion, we carried out primer extension reactions with individual
nucleotides on templates with various nearest neighbors adjacent to the
target site (Fig. 3). On single
nucleotide-gapped DNA, with a purine 3' to the target site, dTTP
misincorporation was about 2 times higher than dATP misincorporation
(Fig. 3B). With a pyrimidine 3' to the target site, pol inserted dTTP 50-85-fold more efficiently than dATP (Fig.
3A). In addition, the type of pyrimidine 3' to the target
site strongly influenced dNTP misincorporation. Levels of both dTTP and
dATP misincorporation were 10-60-fold higher with C 3' to the target
site than with T 3' to the target site. In contrast, the template base
5' to the target site did not produce a significant impact on the
relative levels of dTTP and dATP misincorporation. On primed
single-stranded DNA templates, dTTP misincorporation could be detected
only when C was 3' to the target site (Fig. 3, C and
D). These data need to be interpreted with caution, because
they have not been confirmed by detailed kinetic analysis of
misincorporation in different sequence contexts. However, the data do
suggest that the previously observed differences in the pattern of
misincorporation opposite the 3'- and 5'-G in the 5'-AGGC-3' sequence
are largely because of effects of the sequence context on the
specificity of misincorporation. These data are also in agreement with
previous studies that showed that the base 3'to the target site has a
significant effect on the misincorporation efficiency by pol
(23,
24). We have not examined the effect of sequence context on the
efficiency and fidelity of translesion synthesis past cisplatin- and
oxaliplatin-GG adducts because of the need to locate the platinum
adduct in a restriction site for purposes of purification of the
platinated 44-mer and because of the difficulty obtaining a unique
platinum adduct in a GG sequence with G either 3' or 5' to the adduct. However, the sequence context of Pt-GG adducts is known to affect their
mutagenicity (25, 26) and Pilch et al. (27) have recently shown that sequence context affects the conformation of Pt-GG adducts.
Thus, it is likely that sequence context will also affect both the
efficiency and fidelity of translesion synthesis past Pt-GG adducts by
pol
and other DNA polymerases.
|
Summary--
In our previous reports (9) we have shown that
eukaryotic DNA polymerases pol ,
,
, and
all bypass
oxaliplatin-GG adducts to a greater extent than cisplatin-GG adducts.
However, the efficiency and accuracy of this bypass was different for
the each polymerase. This suggested that translesion replication past Pt-DNA adducts was determined by both the structure of the adduct and
the DNA polymerase involved in translesion synthesis. In the present
study we show that the replication blocking potential and mutagenicity
of Pt-DNA adduct is likely to be strongly influenced by constraints
imposed on the template when it binds to the active site of the
polymerase. In support of this hypothesis we have shown that for
translesion synthesis past Pt-DNA adducts by pol
the specificity
(cisplatin versus oxaliplatin), the site of discrimination
(3'-G versus 5'-G), and the pattern of misincorporation is
strongly dependent on the DNA template structure. These differences are
most likely because of changes in the conformation of Pt-DNA adducts
when structurally different DNA templates are bound to the active site
of pol
. Thus, whereas the conformation of DNA adducts is most often
studied in solution in the absence of any protein, these data suggest
that conformational studies of DNA adducts in the active site of the
polymerases responsible for translesion synthesis will be important for
an understanding of mutagenesis mechanisms. Our data also show that
sequence context affects the efficiency of correct nucleotide
incorporation and the pattern of misincorporation by pol
. Whereas
these data were obtained with undamaged DNA, sequence context is also
likely to affect the efficiency and fidelity of translesion synthesis
past Pt-DNA adducts as well. Elucidation of structural features of Pt-DNA adducts in the active site of pol
will be required to define
the mechanism of these effects.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Wilson (NIEHS, National
Institutes of Health) for providing pol and Dr. S. D.
Wyrick (University of North Carolina) for providing us with the
Pt(dach)Cl2. We are indebted to Dr. J. T. Reardon and Dr.
P. E. Juniewicz for critical reading of the manuscript, and to Dr.
W. A. Beard for helpful discussions of the data.
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FOOTNOTES |
---|
* This research was supported by a research contract from Sanofi-Synthelabo Pharmaceuticals.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.
Present address: Section on DNA Replication, Repair, and
Mutagenesis, NICHD, National Institutes of Health, Bethesda, MD
20892-2725.
§ To whom correspondence should be addressed. Tel.: 919-966-3286; Fax: 919-966-2852; E-mail: Stephen_Chaney@med.unc.edu.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M007805200
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ABBREVIATIONS |
---|
The abbreviations used are: cisplatin, cis-diaminedichloroplatinum(II); oxaliplatin, (trans-R,R)-1,2-diaminocyclohexaneoxalatoplatinum(II); Pt(dach)Cl2, (trans-R,R)-1,2-diaminocyclohexanedichloroplatinum(II); dach, (trans-R,R)-1,2-diaminocyclohexane; pol, polymerase; HIV-1 RT, human immunodeficiency virus 1 reverse transcriptase; HMG, high mobility group.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Eastman, A. (1987) Pharmacol. Ther. 34, 155-166[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Woynarowski, J. M.,
Chapman, W. G.,
Napier, C.,
Herzig, M. C. S.,
and Juniewicz, P.
(1998)
Mol. Pharmacol.
54,
770-777 |
3. | Gelasco, A., and Lippard, S. J. (1998) Biochemistry 37, 9230-9239[CrossRef][Medline] [Order article via Infotrieve] |
4. | Herman, F., Kozelka, J., Stoven, V., Guittet, E., Girault, J. P., Huynh-Dinh, T., Igolen, J., Lallemand, J. Y., and Chottard, J. C. (1990) Eur. J. Biochem. 194, 119-133[Abstract] |
5. | Takahara, P. M., Rosenzweig, A. C., Frederick, C. A., and Lippard, S. J. (1995) Nature 377, 649-652[CrossRef][Medline] [Order article via Infotrieve] |
6. | Yang, D., van Bloom, S. S. G. E., Reedijk, J., van Bloom, J. H., and Wang, A. H. J. (1995) Biochemistry 34, 12912-12920[Medline] [Order article via Infotrieve] |
7. | Fink, D., Nebel, S., Aebi, S., Zheng, H., Cenni, B., Nehme, A., Christen, R. D., and Howell, S. B. (1996) Cancer Res. 56, 4881-4886[Abstract] |
8. | Vaisman, A., Lim, S. E., Patrick, S. M., Copeland, W. C., Hinkle, D. C., Turchi, J. J., and Chaney, S. G. (1999) Biochemistry 38, 11026-11039[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Vaisman, A.,
and Chaney, S. G.
(2000)
J. Biol. Chem.
275,
13017-13025 |
10. | Vaisman, A., Masutani, C., Hanaoka, F., and Chaney, S. G. (2000) Biochemistry 39, 4575-4580[CrossRef][Medline] [Order article via Infotrieve] |
11. | Kunkel, T. A., and Wilson, S. H. (1998) Nat. Struct. Biology. 5, 95-99[Medline] [Order article via Infotrieve] |
12. | Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., and Kraut, J. (1996) Biochemistry 35, 12742-12761[CrossRef][Medline] [Order article via Infotrieve] |
13. | Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903[Medline] [Order article via Infotrieve] |
14. | Sawaya, M. R., Prasad, R., Wilson, S. H., Kraut, J., and Pelletier, H. (1997) Biochemistry 36, 11205-11215[CrossRef][Medline] [Order article via Infotrieve] |
15. | Ahn, J., Kraynov, V. S., Zhong, X., Werneburg, B. G., and Tsai, M. D. (1998) Biochem. J. 331, 79-87[Medline] [Order article via Infotrieve] |
16. |
Chagovetz, A. M.,
Sweasy, J. B.,
and Preston, B. P.
(1997)
J. Biol. Chem.
272,
27501-27504 |
17. |
Boosalis, M. S.,
Petruska, J.,
and Goodman, M. F.
(1987)
J. Biol. Chem.
262,
14689-14696 |
18. | Creighton, S., Bloom, L. B., and Goodman, M. F. (1995) Methods Enzymol. 262, 232-256[Medline] [Order article via Infotrieve] |
19. | Beard, W. A., and Wilson, S. H. (1998) Chem. Biol. 5, R7-R13[Medline] [Order article via Infotrieve] |
20. | Beard, W. A., and Wilson, S. H. (2000) Mutat. Res. 460, 231-244[Medline] [Order article via Infotrieve] |
21. | Cosman, M., Hingerty, B. E., Geacintov, N. E., Broyde, S., and Pate, D. J. (1995) Biochemistry 34, 15334-15350[Medline] [Order article via Infotrieve] |
22. | Feng, B., Gorin, A., Kolbanovskiy, A., Hingerty, B. E., Geacintov, N. E., Broyde, S., and Patel, D. J. (1997) Biochemistry 36, 13780-13790[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Efrati, E.,
Tocco, G.,
Eritja, R.,
Wilson, S. H.,
and Goodman, M. F.
(1997)
J. Biol. Chem.
272,
2559-2569 |
24. |
Mendelman, L. V.,
Boosalis, M. S.,
Petruska, J.,
and Goodman, M. F.
(1989)
J. Biol. Chem.
264,
14415-14423 |
25. | Bubley, G. J., Ashburner, B. P., and Teicher, B. A. (1991) Mol. Carcin. 4, 397-406[Medline] [Order article via Infotrieve] |
26. | de Boer, J. G., and Glickman, B. W. (1989) Carcinogenesis 10, 1363-1367[Abstract] |
27. | Pilch, D. S., Dunham, S. U., Jamieson, E. R., Lippard, S. J., and Breslauer, K. J. (2000) J. Mol. Biol. 296, 803-812[CrossRef][Medline] [Order article via Infotrieve] |