From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555
Received for publication, July 31, 2002, and in revised form, September 9, 2002
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ABSTRACT |
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Acrolein (Fig. 1), the simplest
-Hydroxy-1,N2-propano-2'deoxyguanosine
(
-HOPdG) is a major deoxyguanosine adduct derived from
acrolein, a known mutagen. In vitro, this adduct has
previously been shown to pose a severe block to translesion synthesis
by a number of polymerases (pol). Here we show that both yeast and
human pol
can incorporate a C opposite
-HOPdG at ~190- and
~100-fold lower efficiency relative to the control deoxyguanosine and
extend from a C paired with the adduct at ~8- and ~19-fold lower
efficiency. Although DNA synthesis past
-HOPdG by yeast pol
was
relatively accurate, the human enzyme misincorporated nucleotides
opposite the lesion with frequencies of ~10
1 to
10
2. Because
-HOPdG can adopt both ring closed and
ring opened conformations, comparative replicative bypass studies were
also performed with two model adducts, propanodeoxyguanosine and
reduced
-HOPdG. For both yeast and human pol
, the ring open
reduced
-HOPdG adduct was less blocking than
-HOPdG, whereas the
ring closed propanodeoxyguanosine adduct was a very strong block.
Replication of DNAs containing
-HOPdG in wild type and xeroderma
pigmentosum variant cells revealed a somewhat decreased mutation
frequency in xeroderma pigmentosum variant cells. Collectively, the
data suggest that pol
might potentially contribute to both
error-free and mutagenic bypass of
-HOPdG.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated aldehyde, is an environmental contaminant and a
product of inborn metabolism. In organisms, acrolein is generated via a
number of pathways, such as the oxidation of polyamines and lipid
peroxidation (1, 2). Like many other bifunctional aldehydes, acrolein
reacts with DNA bases to form several DNA adducts, among which the
-hydroxy-1,N2-propano-2'deoxyguanosine
(
-HOPdG)1 was identified
as a major deoxyguanosine (dG) derivative (3, 4). Importantly,
-HOPdG has been detected in DNA from mammalian tissues (5-7),
suggesting that this adduct is generated in vivo. The
-HOPdG adduct is formed by conjugate addition of acrolein to
N2 of dG to produce
N2-(3-oxopropyl)dG. Ring closure at N1 leads to
the formation of the cyclic adduct (Fig. 1). In the nucleoside and
presumably in single-stranded DNA,
-HOPdG predominantly exists in
the cyclic form, such that at physiological pH, the ring open species
cannot be detected spectrophotometrically (8). However, in the presence of a reducing agent, the acyclic form can be trapped as the
N2-(3-hydroxypropyl) adduct (Fig. 1).
View larger version (22K):
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Fig. 1.
Structures of the acrolein and related
deoxyguanosine adducts.
Another dG derivative,
1,N2-propanodeoxyguanosine (PdG) (Fig. 1), whose
structure is similar to that of the ring closed -HOPdG, has been
extensively exploited as a model compound for the
-HOPdG and other
exocyclic dG adducts in both structural and biological studies. NMR
spectroscopy of the PdG-adducted oligodeoxynucleotides has revealed
that when placed opposite dC, PdG adopts a syn orientation within the duplex and introduces a localized structural perturbation that is pH- and sequence-dependent (9, 10). The inability of PdG to form normal Watson-Crick hydrogen bonds severely blocks DNA
synthesis both in vitro (11, 12) and in vivo
(13-16), and the replication that does occur results in mutations
(13-16). Specifically, PdG-induced base substitutions occurred at an
overall frequency of 7.8 × 10
2 and 7.5 × 10
2/translesion synthesis in the COS-7 (14) and in the
nucleotide excision repair-deficient human cells (16), respectively. In both strains, G to T transversions predominated.
Recently, the structure of the -HOPdG-containing
oligodeoxynucleotide was solved by NMR spectroscopy (17). These data
have indicated that within the duplex,
-HOPdG exists primarily in the ring open form. In such a conformation, the modified base participates in standard Watson-Crick base pairing by adopting a
regular anti orientation around the glycosidic torsion
angle, with the N2-propyl chain in the minor
groove pointing toward the solvent (17). The structural differences
between PdG and
-HOPdG within the duplex have led to the hypothesis
that the latter lesion would be less blocking for replication and less
mutagenic than the former.
Biological studies aimed to test the cytotoxic and mutagenic effects of
acrolein-modified DNAs and of site-specific -HOPdG adduct have
generated conflicting results. It is known that acrolein itself causes
mutations in both bacterial (18) and mammalian (19) systems and has
tumor-initiating activity (20). When a DNA vector was treated with
acrolein and propagated in human cells, the majority of mutations were
single, tandem, and multiple base substitutions that predominantly
occurred in G:C base pairs (21). However in bacteria,
-HOPdG, the
major acrolein-derived dG adduct, is not a strong block for DNA
synthesis nor a miscoding lesion (22-24). Analyses of mutations caused
by
-HOPdG in wild type Escherichia coli and in
polB, dinB, and umuDC deficient
strains revealed that in the absence of these "SOS" polymerases,
the efficiency and accuracy of the translesion synthesis were not
significantly affected (22). In contrast to the prokaryotic data,
-HOPdG caused mutations at an overall frequency of 7.4 × 10
2/translesion synthesis when a single-stranded,
site-specifically modified vector was propagated in COS-7 cells (24).
Interestingly, both the frequencies and types of mutations were
remarkably similar to those reported for the PdG adduct (14, 16).
However,
-HOPdG was shown to be only marginally miscoding (
1%
base substitution) when double-stranded vector was utilized (16). In
this investigation, a number of cell lines including HeLa, a nucleotide
excision repair-deficient xeroderma pigmentosum group A, and polymerase
-deficient xeroderma pigmentosum variant were examined.
Although replication across -HOPdG in vivo was
predominantly error-free (from 93 to 100% of the translesional
events), the adduct was shown to be a severe block and a miscoding
lesion during in vitro DNA synthesis by a number of
polymerases. Particularly, replication across
-HOPdG by the Klenow
exo
fragment of E. coli polymerase I was
significantly inhibited and extremely error-prone (22, 23).
-HOPdG
also strongly blocked DNA synthesis by two major eukaryotic
polymerases, pol
and pol
(24). In the presence of proliferating
cell nuclear antigen, little bypass of the adduct by pol
was
achieved, and it appeared to be highly mutagenic (24). We hypothesized
therefore that in mammalian cells, specialized, translesion DNA
synthesis polymerases (25, 26) are involved in promoting replication across
-HOPdG.
Among DNA polymerases proficient in translesion synthesis, yeast
polymerase (a product of the RAD30 gene) (27) and its human counterpart (a product of the RAD30A (XPV,
POLH) gene) (28, 29) both possess a unique ability to replicate
efficiently and accurately past a cis-syn cyclobutane
pyrimidine dimer (30, 31), the predominant DNA lesion caused by
ultraviolet irradiation. In the yeast Saccharomyces
cerevisiae, deletion of RAD30 confers moderate
sensitivity to UV irradiation and leads to increased UV-induced
mutagenesis (32). Mutations in the human RAD30A gene cause
the variant form of xeroderma pigmentosum (XPV), suggesting that
predisposition of XPV individuals to sunlight-induced skin cancer is
due to the lack of accurate translesion DNA synthesis across UV-induced
DNA lesions (28, 29, 33). Yeast and human pol
also efficiently
bypass a product of oxidative DNA damage, the 7,8-dihydro-8-oxoguanine,
and do so in a predominantly error-free manner (34). In addition,
several other DNA lesions were reported to be substrates for human
(35-39) and yeast (35, 40) pol
.
In the present study, the ability of yeast and human pol to perform
translesion DNA synthesis across
-HOPdG has been examined, and the
efficiency and fidelity of synthesis have been tested using
steady-state kinetic analyses. To further explore the bypass mechanism,
comparative studies were also performed with two model DNA adducts:
PdG, which mimics the cyclic form of
-HOPdG, and N2-(3-hydroxypropyl)dG, which is similar to
-HOPdG in its ring open form. In addition, the mutagenic potential
of
-HOPdG was tested in vivo in both human fibroblasts
and pol
-deficient XPV cells utilizing a site-specifically modified
single-stranded pMS2 vector.
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EXPERIMENTAL PROCEDURES |
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Materials--
T4 DNA ligase, T4 polynucleotide kinase, and
EcoRV were obtained from New England BioLabs (Beverly, MA).
S1 nuclease and proteinase K were purchased from Invitrogen.
[-32P]ATP was purchased from PerkinElmer Life
Sciences. Bio-Spin columns were purchased from Bio-Rad. Centricon 100 concentrators were obtained from Amicon Inc. (Beverly, CA).
Slide-A-Lyzer Dialysis Cassettes were obtained from Pierce.
Strains and Vectors-- Single-stranded pMS2 DNA was a generous gift from Dr. M. Moriya (State University of New York, Stony Brook, NY). SV40-transformed cTAG derived from XP4BE cells and SV80 normal human fibroblasts were obtained from Dr. Marila Cordeiro-Stone (University of North Carolina, Chapel Hill, NC). The E. coli DH10B cells used for amplification of transformed DNA isolated from mammalian cells were purchased from Invitrogen.
Oligodeoxynucleotides--
12-mer oligodeoxynucleotide modified
with -HOPdG, 5'-GCTAGC(
-HOPdG)AGTCC-3', was kindly provided
by Dr. T. M. Harris and Dr. C. M. Harris (Vanderbilt
University, Nashville, TN), and it was prepared by a previously
described procedure (8). The 24-mer oligodeoxynucleotide,
5'-GCAGTATCGCGC(PdG)CGGCATGAGCT-3', adducted with PdG was
synthesized as described (41) and was a generous gift from Dr. L. J. Marnett (Vanderbilt University, Nashville, TN). Nondamaged 12- and
24-mer with a dG in place of
-HOPdG or PdG, respectively, were
purchased from Midland Certified Reagent Co. (Midland, TX). All of the
other oligodeoxynucleotides were synthesized by the Molecular Biology
Core Laboratory of the National Institute of Environmental Health
Sciences Toxicology Center at the University of Texas Medical Branch
(Galveston, TX) and purified by electrophoresis through a 15%
denaturing PAGE (in the presence of 7 M urea).
Construction of site-specifically modified linear templates for
in vitro replication assays was done according to the
previously described procedure (24). Sequences of the resulting
oligodeoxynucleotides were identical:
5'-GCTAGCGAGTCCGCGCCAAGCTTGGGCTGCAGCAGGTC-3', where
the underlined G is either -HOPdG or nonadducted dG and 5'-GCAGTATCGCGCGCGGCATGAGCTGCGCCAAGCTTGGGCTGCAGCAGGTC-3',
where the underlined G is either PdG or nonadducted dG. To obtain the N2-(3-hydroxypropyl)dG-containing DNA substrate,
10 µl of 1 M NaBH4 dissolved in 1 M Hepes buffer (pH 7.4) were added twice to 200 µl of the
-HOPdG-adducted 38-mer oligodeoxynucleotide (1-2 µM). Each addition of the reducing agent was followed by incubation at room
temperature for 4 h. DNA was then dialyzed against 10 mM Tris-HCl (pH 7.0), 1 mM EDTA overnight using
Slide-A-Lyzer Dialysis Cassette (3,500 molecular weight cut
off). To confirm the completeness of reduction, the polypeptide
trapping technique was utilized (42) modified by A. J. Kurtz for
-HOPdG-containing DNAs. Briefly, probes of both
-HOPdG- and
reduced
-HOPdG-adducted oligodeoxynucleotides (50 nM)
were incubated with 50 mM lysine-tryptophan-lysine-lysine in the presence of 25 mM NaCNBH3 and 100 mM Hepes (pH 7.4) for 5 h. The reactions were
terminated by the addition of an equal volume of 95% (v/v) formamide,
20 mM EDTA, 0.02% (w/v) xylene cyanol, and 0.02% (w/v)
bromphenol blue and heating at 90 °C for 2 min. Next, DNAs were
resolved through a 15% denaturing PAGE and visualized with
PhosphorImager Screen. Under these conditions, no trapping was detected
in reactions with
-HOPdG-containing oligodeoxynucleotide, whereas
the
-HOPdG-containing DNA was completely complexed with the polypeptide.
Pol Purification--
Purifications of yeast pol
and
human pol
were done as described in Refs. 27 and 31, respectively.
DNA Polymerase Reaction--
The 21-mer oligodeoxynucleotides
were used as primers for in vitro polymerase reactions.
Their sequences were: 5'-CCTGCTGCAGCCCAAGCTTGG-3', which is
complementary to the 38-mer -HOPdG-containing template DNAs from
positions
9 to
29 relative to the site of lesion (
9 primer) as
well as complementary to the PdG-adducted 50-mer from positions
15 to
35 (
15 primer); 5'-AGCCCAAGCTTGGCGCGGACT-3' and
5'-AGCTTGGCGCAGCTCATGCCG-3', which are complementary from the position
1 to
21 to the
-HOPdG-containing template and the PdG-containing
template, respectively (
1 primers); and 5'-GCCCAAGCTTGGCGCGGACTC-3' and 5'-GCTTGGCGCAGCTCATGCCGC-3', which overlap the lesion site in
modified templates (0 primers). Primer oligodeoxynucleotides were
phosphorylated with T4 polynucleotide kinase using
[
-32P]ATP and purified using P-6 Bio-Spin columns
supplied with 10 mM Tris-HCl buffer (pH 7.4). The
-32P-labeled primers were mixed with the
oligodeoxynucleotide substrates at a molar ratio of 1:2 in the presence
of 25 mM Tris-HCl buffer (pH 7.6), 50 mM NaCl,
heated at 90 °C for 2 min, and cooled to room temperature overnight.
Primer extension and single-nucleotide incorporation experiments with
yeast pol were carried out as described (27) and with human pol
as in Ref. 31. Briefly, the reaction mixture (10 µl) contained 5 nM primer annealed to a template, 25 mM
Tris-HCl buffer (pH 7.5), 10 mM NaCl, 5 mM
MgCl2, 10% glycerol, 100 µg/ml of bovine serum albumin,
5 mM dithiothreitol, 100 µM of each of the
four dNTPs (primer extension experiments), or 10 µM
individually (single-nucleotide incorporation experiments), and yeast
or human pol
at the concentrations as indicated in the figure
legends. The reactions were incubated at 22 °C and terminated by the
addition of 4× excess of stop solution consisting of 95% (v/v)
formamide, 20 mM EDTA, 0.02% (w/v) xylene cyanol, and
0.02% (w/v) bromphenol blue. The reaction products were resolved
through a 20% denaturing PAGE and visualized by a PhosphorImager screen.
Steady-state Kinetic Analysis--
Steady-state kinetic assays
were carried out under the same conditions as the DNA polymerase assays
except that 1 nM yeast or human pol and 20 nM DNA substrates were used with various concentrations of
one of the four nucleotides. The reactions were quenched after 5 min.
Quantitative analyses were performed using a PhosphorImager screen and
Image-Quant 5.0 software (Molecular Dynamics, Sunnyvale, CA).
Calculations of rates of nucleotide incorporation were done as
described in Ref. 43. The rates of nucleotide incorporation were
graphed as a function of nucleotide concentration, and the
kcat and Km parameters were
obtained from the best fit of the data to the Michaelis-Menten equation.
Construction of Circular Single-stranded pMS2 DNA Modified with
-HOPdG--
The 12-mer oligodeoxynucleotides containing either
-HOPdG or a nondamaged dG were phosphorylated at the 5' end with ATP
and inserted into single-stranded pMS2 shuttle vector as described earlier (24). The two ligated samples were designated pMS2(dG) and
pMS2(
-HOPdG).
Mutagenesis Experiments--
Transfection of pMS2(dG) and
pMS2(-HOPdG) into cTAG and SV80 cells, isolation of DNA,
amplification in E. coli DH10B cells, and
differential hybridization analysis were done as previously described
(24). Hybridization with the progeny plasmid DNA was performed using
[
-32P]ATP-labeled 18-mer oligodeoxynucleotide probes
(5'-GATGCTAGCNAGTCCATC-3', where N refers to A, T, G, or C). Whatman
541 filters containing hybridized colonies were exposed to X-Omat AR
film overnight, and autoradiographs were developed to identify mutation
frequency and types of mutations. Representative colonies were
subjected to dideoxy sequencing (44) to confirm the presence of the
mutations. A 20-mer primer (5'-CCATCTTGTTCAATCATGCG-3') sequence around
100 nucleotides downstream of the adduct was used for sequencing the region containing the 12-mer oligodeoxynucleotide in progeny plasmid DNA.
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RESULTS |
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In Vitro Lesion Bypass with Yeast DNA Polymerase --
To
examine whether yeast pol
was able to replicate past a
-HOPdG
adduct, running start primer extension experiments were performed (Fig.
2A). A 21-mer primer was
annealed to the template DNA so that it allowed the addition of 9 nucleotides before encountering the adduct (
9 primer). On the
nondamaged DNA substrate, primers were efficiently extended by yeast
pol
(Fig. 2A, lanes 1-4). On the
-HOPdG-containing substrate (Fig. 2A, lanes
5-8), yeast pol
appeared to be capable of bypassing the
lesion and forming full-length products. However, DNA synthesis was
partially inhibited right before the DNA lesion and opposite from
it.
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To understand better the importance of ring opening during replication,
primer extension experiments were carried out using two model DNA
substrates: the PdG adduct, which is an analogue of the ring closed
form of the -HOPdG, and the reduced
-HOPdG, which is similar to
the ring open form of the natural adduct. In the case of the 50-mer
PdG-containing substrate, 21-mer primer was positioned on the template
so that the incorporation of 15 nucleotides was needed before reaching
the lesion (
15 primer). Because both efficiency and accuracy of the
DNA synthesis are known to be sequence-dependent (43, 45),
an additional nondamaged control 50-mer DNA template was utilized that
had the same sequence as the PdG-adducted template. These data revealed
that the PdG adduct was a much stronger block for replication by yeast
pol
than
-HOPdG. Under conditions that allowed an efficient
replication of the nondamaged DNA template (Fig. 2A,
lanes 13-16), DNA synthesis on the PdG-adducted template
was greatly inhibited one nucleotide before the lesion, and synthesis
was completely aborted after incorporating a nucleotide opposite the
lesion (Fig. 2A, lanes 17-20). However,
replication by yeast pol
beyond the PdG can be achieved but at much
higher concentrations of enzyme (data not shown). With the reduced
-HOPdG-adducted template (Fig. 2A, lanes
9-12), the bypass efficiency by yeast pol
seemed to be comparable with that on the
-HOPdG-adducted template.
The specificity of nucleotide incorporation by yeast pol opposite
and beyond the lesions was also tested. To identify the nucleotide that
is incorporated by this polymerase opposite the adducted base,
single-nucleotide incorporation experiments were carried out using
standing start DNA substrates in which 3' terminus of the primer was
located one nucleotide before the lesions (
1 primers) (Fig.
2B). On both nondamaged substrates, yeast pol
preferentially incorporated a C opposite G (Fig. 2B,
lanes 3 and 18). Incorporation of a T and to a
lesser extent an A and a G was also observed, especially on the 38-mer
template. Interestingly, incorporation of a correct nucleotide (C) was
predominant opposite each of the modified bases, namely the
-HOPdG
(Fig. 2B, lane 8), the reduced
-HOPdG (Fig. 2
B, lane 13), and the PdG (Fig. 2B, lane
23) adducts.
To test whether any misincorporation occurred past the lesion site, single-nucleotide incorporation experiments were carried out using DNA substrates in which the correct nucleotide (C) was primed with the adducted base (0 primers). No nucleotide misincorporation was observed on any of the adducted templates examined (data not shown).
Thus, yeast pol is capable of bypassing the
-HOPdG adduct,
and in contrast to all other polymerases tested so far (22-24), it
predominantly incorporates the correct nucleotide opposite and
downstream of the lesion. In addition, these data show that a cyclic
PdG is a much stronger block for replication by yeast pol
than an
acyclic reduced
-HOPdG, but neither of the model adducts seem to be
particularly miscoding for this polymerase.
In Vitro Lesion Bypass with Human DNA Polymerase
--
Primer extension reactions and single-nucleotide
incorporation experiments were carried out with human pol
(Fig.
3) using the same set of the
primer/templates as with the yeast enzyme. Similar to the yeast pol
, human polymerase was able to replicate past the
-HOPdG (Fig.
3A, lanes 5-8) and the reduced
-HOPdG lesions
(Fig. 3A, lanes 9-12). However, unlike yeast pol
, at higher enzyme concentrations human pol
appeared to bypass
the PdG adduct (Fig. 3A, lanes 17-20).
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Single-nucleotide incorporation experiments with human pol revealed
significant differences between the human and yeast enzymes in their
discrimination abilities during nucleotide insertion opposite the
-HOPdG adduct. Whereas yeast pol
preferentially incorporated the
correct nucleotide (C) opposite the lesion, human polymerase extended
the
1 primer almost equally well in the presence of A, C, and G (Fig.
3B, lanes 6-10). On the PdG-adducted template, the difference between these two polymerases was even more striking. In
contrast to the yeast pol
that incorporated a C opposite PdG, human
polymerase inserted A, G, and T better than the correct nucleotide
(Fig. 3B, lanes 21-25). Interestingly,
incorporation by human pol
is much more accurate opposite the
reduced
-HOPdG adduct (Fig. 3B, lanes 11-15)
than opposite the nonreduced adduct (Fig. 3B, lanes
6-10).
Single-nucleotide incorporation experiments were carried also out using
0 primers with the C primed with the adducted base. Yielding data
similar to that of the yeast pol , human polymerase preferentially
incorporated the correct nucleotide on all five substrates tested (data
not shown).
Efficiency of Nucleotide Incorporation and Extension--
To
compare the efficiency of translesion synthesis by yeast and human pol
, steady-state kinetic parameters kcat and
Km were first determined for the correct nucleotide
(C) incorporation opposite dG in two different sequence contexts and
also opposite
-HOPdG, reduced
-HOPdG, and PdG adducts. The
reactions were performed using the same 21-mer
1 primers as in the
single-nucleotide incorporation experiments. For yeast pol
, C is
incorporated opposite the ring closed PdG adduct with a
1600-fold lower efficiency (kcat/Km) than C is incorporated
opposite the unadducted dG (Table I). In
contrast, yeast pol
incorporates a C opposite the ring open reduced
-HOPdG with only a 12-fold lower efficiency than opposite the
unadducted dG. The efficiency of incorporation opposite the
-HOPdG
adduct is in between these two extremes with a 190-fold reduction
relative to the unadducted dG. The same trends were also observed with
human pol
(Table II).
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Next, the steady-state kinetic parameters were determined for the
extension from a C residue paired with the modified bases and were used
to determine the efficiency of extending from each adduct relative to
the extension from an unadducted dG (Tables I and II). For both yeast
and human pol , the efficiencies of extensions from the
-HOPdG
and the reduced
-HOPdG were reduced ~5-30-fold relative to the
unadducted dG. In contrast, the extension from the PdG was blocked to a
much greater extent, especially in the case of the yeast enzyme
(6800-fold; Table I).
Fidelity of Nucleotide Incorporation by Yeast and Human Pol Opposite
-HOPdG--
In the single-nucleotide incorporation
experiments, yeast and human pol
displayed different accuracies of
replication across the
-HOPdG adduct. To further evaluate the
accuracy of nucleotide incorporation opposite the lesion, kinetic
analyses were carried out using
1 primer, and the frequencies of
misincorporation were calculated as the ratio of
kcat/Km of the incorrect nucleotide to
the correct nucleotide (43). These data showed that yeast pol
synthesizes past
-HOPdG relatively accurately with efficiency of
incorporation of a C ~75 times higher than that of the next most
preferred nucleotide (G) (Table I). In contrast, human pol
discriminated poorly between the correct and wrong nucleotides incorporating opposite
-HOPdG. Particularly, high misincorporation frequencies were observed for A and G (Table II).
Mutagenicity of -HOPdG-modified Single-stranded pMS2 Vectors in
Normal Human Fibroblasts and XPV Cells--
Table
III shows the outcomes of in
vivo replication of pMS2 (dG) and pMS2 (
-HOPdG) in SV80 and XPV
cells. The data presented for XPV cells were obtained from five
independent experiments. All of the 1104 E. coli
transformants resulting from replication of modified pMS2 (
-HOPdG)
in XPV cells were picked and grown in 96-well plates. Hybridization
analysis revealed that 767 colonies hybridized with either one of the
four probes, whereas 337 colonies did not hybridize with any of the
four probes. Of those transformants that did not hybridize with any
sequence-specific probe, none of those hybridized to sequences
immediately upstream of the oligodeoxynucleotide ligation site,
suggesting that this deletion was not caused by the adduct. Although
96% of the hybridized transformants did not contain any targeted
mutations (Table III), 1.3% (10/767) were G to A transitions, 0.5%
(4/767) were G to C transversions, and 2.1% (16/767) were G to T
transversions. Sequencing of plasmid DNA prepared from these colonies
confirmed the presence of T, C, or A, respectively, opposite the site
of the adducted guanine. No mutations were observed when 192 colonies
were screened out of transformants obtained from nonadducted
pMS2(dG).
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When these experiments were repeated in SV80 normal human fibroblasts,
all of the 288 transformed colonies subsequently obtained from two
experiments were analyzed for mutations by differential hybridization
strategy. Although only 92 colonies hybridized with either one of
the four probes, 89% (82/92) contained the correct base opposite the
adducted guanine, whereas 8.6% (8/92) were G to T transversions, and
1.1% (1/92) were G to C and G to A mutations. None of the colonies
from the control pMS2(dG) transformants showed any mutation. Thus, XPV
cells appeared to have a lower mutation frequency (3.9%) when
compared with normal human fibroblast cells (11%).
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DISCUSSION |
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The -HOPdG adduct was not a significant block for replication
when site-specifically modified vectors were propagated in E. coli (22-24) or in mammalian cells (16, 24). In E. coli, the adduct appeared not to be miscoding (22-24). Depending
on the cell type and vector used, 93-100% of the translesion events
were nonmutagenic during in vivo replication in mammalian
cells (16, 24). Thus, in both prokaryotic and eukaryotic systems, DNA
polymerases exist that are able to synthesize past
-HOPdG
efficiently and in a predominantly error-free manner. On the other
hand, none of the polymerases examined in vitro so far,
namely, Klenow exo
fragment of E. coli pol I
(22, 23), calf thymus pol
(24), and human pol
(24), were able
to incorporate the correct nucleotide opposite this adduct. In the
present study, yeast pol
has been identified as the first
polymerase that possesses an ability to replicate across the
-HOPdG
adduct relatively accurately. Comparable efficiency of DNA syntheses
past
-HOPdG was also observed for human pol
, but this polymerase
displayed a much higher propensity for misincorporation.
Single-nucleotide experiments as well as steady-state analyses showed
that human pol
frequently incorporates an A or a G opposite
-HOPdG and therefore is likely to introduce G to T and G to C transversions.
We note that the observed kcat for C
incorporation opposite the undamaged G template (~5
min1; Table I) is slower than the rate of nucleotide
incorporation measured during processive synthesis (~80
min
1; Ref. 46) for yeast pol
. This suggests that
kcat reflects the rate of DNA release and thus
is an underestimate of the actual rate of nucleotide incorporation.
Nevertheless, because the observed Km is expected to
be decreased with the kcat in a compensatory manner, the efficiencies of nucleotide incorporation
(kcat/Km) determined under
steady-state conditions provide a measure of catalytic efficiencies of
the enzyme. More detailed kinetic studies are needed, however, to more
accurately define the mechanisms controlling the fidelity of pol
opposite these DNA adducts.
The nucleotide incorporation data for pol are in agreement with
results of the in vivo replication assays when
site-specifically modified single-stranded pMS2 vector was propagated
in XPV cells. Overall mutagenic frequency determined in the XPV cells
(3.9 × 10
2/translesion synthesis) was about two and
three times less than that in COS-7 (24) and normal human cells,
respectively. Importantly, lower frequencies of transversions
(particularly G to T) in XPV cells, but not G to A transitions,
accounted for the observed differential between two types of cells.
Thus, pol
might potentially contribute to the bypass of the
-HOPdG adduct in mammalian cells being responsible for both
error-free and error-prone replicative events.
Based on the NMR spectroscopy data, a model of error-free bypass of
-HOPdG has been proposed in which the incoming dCTP triggers a
structural rearrangement of the adduct from the ring closed to the ring
open form. This change allows the formation of the standard
Watson-Crick hydrogen bonds, stabilizes the structure, and facilitates
the subsequent extension reaction (17). To examine the role of ring
opening during replication by pol
, we compared the efficiency of
incorporation opposite
-HOPdG to the incorporation opposite the two
model adducts: PdG and reduced
-HOPdG. For both yeast and human pol
, cyclic PdG was a very strong block for the incorporation of a C
relative to the acyclic reduced
-HOPdG. For incorporation opposite
-HOPdG, both polymerases had an intermediate incorporation
efficiency. Ring opening was also important for the extension from a C
paired with the adduct. For both yeast and human pol
, relative
efficiencies of extension were similar when
-HOPdG- and reduced
-HOPdG-modified DNA substrates were used. By contrast, the cyclic
PdG adduct is a very strong block for extension by these polymerases,
especially for the yeast enzyme. Overall, these data are consistent
with the proposed model of de los Santos (17), such that ring opening
of
-HOPdG is essential not only for efficient incorporation opposite
the lesion by yeast and human pol
but also for efficient extension.
However, from these data it cannot be concluded whether the incoming
nucleotide causes the transformation of the adduct from the ring closed
to the ring open form or whether the equilibrium is shifted toward ring
open conformation by protein-DNA interactions in the polymerase active site.
The steady-state kinetic analyses and single-nucleotide incorporation
experiments have revealed significant differences between yeast and
human pol with respect to their accuracies of replication across
modified bases. For the human enzyme, frequencies of misincorporation opposite
-HOPdG were on average, 1 order of magnitude higher than
for the yeast enzyme. In addition, the incorporation by human pol
opposite PdG was extremely error-prone, whereas yeast pol
inserted
the correct nucleotide preferentially.
The proficient ability of yeast and human pol to replicate across
the ring open form of
-HOPdG strongly indicates that in spite of the
fact that it is located in the minor groove, the presence of this
adduct on the templating residue poses no significant hindrance to
these polymerases. This suggests the lack of any specific contact of
these enzymes with the minor groove of the templating residue, which
would permit pol
to replicate across DNA adducts, which protrude
into the minor groove.
Although DNA synthesis past -HOPdG by pol
is very efficient when
the adduct exists in its ring open form, in vivo replication data (16, this report) clearly show that pol
is not solely
responsible for bypass of this lesion in humans. Thus, another
polymerase is likely involved in translesion synthesis across
-HOPdG. The yet unidentified polymerase may be able to efficiently
bypass the ring closed form of
-HOPdG and perhaps other exocyclic dG adducts (1, 2), in which N1 modification prevents Watson-Crick pairing.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Dr. Masaaki Moriya (Department
of Pharmacological Sciences, State University of New York at Stony
Brook, NY) for the generous gift of pMS2 vector, Dr. Lawrence J. Marnett (Department of Biochemistry, Center in Molecular Toxicology,
Vanderbilt University at Nashville, TN) for the generous gift of
PdG-adducted oligodeoxynucleotide, and Dr. Marila Cordeiro-Stone
(Department of Pathology and Laboratory Medicine, University of
North Carolina, Chapel Hill, NC) for the generous gift of XPV cells. We
are grateful to Dr. Lubomir V. Nechev, Dr. Thomas M. Harris, and
Dr. Constance M. Harris (Department of Chemistry, Center in
Molecular Toxicology, Vanderbilt University, Nashville, TN) for
synthesis of -HOPdG-adducted oligodeoxynucleotide and for helpful
discussions. We also acknowledge the Molecular Biology Core
Laboratory at the National Institute of Environmental Health Sciences
Toxicology Center (University of Texas Medical Branch, Galveston, TX)
for oligodeoxynucleotide synthesis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institute of Health Grants ES06676 (to R. S. L.), ES00267 (to R. S. L.), and GM19261 (to L. P.).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.
Recipient of the Mary Gibbs Jones Distinguished Chair in
Environmental Toxicology from the Houston Endowment. To whom
correspondence should be addressed. Tel.: 409-772-2179; Fax:
409-772-1790; E-mail: rslloyd@utmb.edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M207774200
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ABBREVIATIONS |
---|
The abbreviations used are:
-HOPdG,
-hydroxy-1,N2-propano-2'deoxyguanosine;
dG, deoxyguanosine;
PdG, 1,N2-propanodeoxyguanosine;
pol, DNA
polymerase;
XPV, xeroderma pigmentosum variant.
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