Mammalian Translesion DNA Synthesis across an Acrolein-derived
Deoxyguanosine Adduct
PARTICIPATION OF DNA POLYMERASE
IN ERROR-PRONE SYNTHESIS IN
HUMAN CELLS*
In-Young
Yang
,
Holly
Miller
,
Zhigang
Wang§,
Ekaterina G.
Frank¶,
Haruo
Ohmori
,
Fumio
Hanaoka**, and
Masaaki
Moriya

From the
Laboratory of Chemical Biology, Department
of Pharmacological Sciences, State University of New York at Stony
Brook, Stony Brook, New York 11794-8651, the § Graduate
Center for Toxicology, University of Kentucky, Lexington, Kentucky
40536, the ¶ Section on DNA Replication, Repair and Mutagenesis,
NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725, the
Laboratory of Genetic Information Analysis, Department of
Genetics and Molecular Biology, Institute for Virus Research, Kyoto
University, Kyoto 606-8507, Japan, and the ** Graduate School
of Frontier Biosciences, Osaka University, Suita,
Osaka 565-0871, Japan
Received for publication, December 9, 2002, and in revised form, January 23, 2003
 |
ABSTRACT |
-OH-PdG, an acrolein-derived deoxyguanosine
adduct, inhibits DNA synthesis and miscodes significantly in human
cells. To probe the cellular mechanism underlying the error-free and
error-prone translesion DNA syntheses, in vitro primer
extension experiments using purified DNA polymerases and site-specific
-OH-PdG were conducted. The results suggest the involvement of pol
in the cellular error-prone translesion synthesis. Experiments with
xeroderma pigmentosum variant cells, which lack pol
, confirmed this
hypothesis. The in vitro results also suggested the
involvement of pol
and/or REV1 in inserting correct dCMP opposite
-OH-PdG during error-free synthesis. However, none of
translesion-specialized DNA polymerases catalyzed significant extension
from a dC terminus when paired opposite
-OH-PdG. Thus, our results
indicate the following. (i) Multiple DNA polymerases are involved in
the bypass of
-OH-PdG in human cells. (ii) The accurate and
inaccurate syntheses are catalyzed by different polymerases. (iii) A
modification of the current eukaryotic bypass model is necessary to
account for the accurate bypass synthesis in human cells.
 |
INTRODUCTION |
During the last several years, many new DNA polymerases
(pol)1 have been discovered
in prokaryotes and eukaryotes (1-3). Several of these
polymerases, such as eukaryotic pol
, pol
, pol
, pol
, and
REV1 and Escherichia coli pol IV and pol V, are thought to
be involved in translesion DNA synthesis. With the exception of pol
, which belongs to the B family, they share extensive sequence
homology and comprise a new polymerase family designated the Y family
(4). These polymerases are different from replicative polymerases in
several aspects, i.e. they replicate more efficiently across
altered bases and catalyze both accurate and inaccurate translesion DNA
syntheses, they have more flexible and larger catalytic pockets (5-7)
that give them the ability to tolerate damaged template bases, and they
show reduced fidelity when copying unmodified DNA (8-14). Their
ability to catalyze translesion synthesis has been studied extensively
in vitro using various DNA lesions as substrates, but
knowledge of their roles in translesion synthesis in mammalian cells is
still very fragmentary. Among these polymerases, pol
, which is
defective in cells of xeroderma pigmentosum variant (XPV) patients, was
shown to catalyze accurate and efficient translesion synthesis across
certain UV photoproducts (15), whereas human pol
(16) and REV1 (17)
are involved in inaccurate syntheses across UV photoproducts. One
recent study using pol
-defective mouse cells has shown that the
enzyme is involved in the error-free translesion synthesis across a
benzo[a]pyrene-dG adduct(s) (18). Two eukaryotic
translesion synthesis pathways have been proposed (19-23). In one
pathway, both insertion and extension steps are catalyzed by one DNA
polymerase. In the other pathway, extension is catalyzed by a DNA
polymerase, such as pol
or pol
, which is different from the one
inserting a nucleotide opposite a DNA lesion.
In this research, we conducted translesion synthesis studies in
vitro and in vivo to probe the cellular bypass
mechanism for an acrolein-derived dG adduct. Acrolein, the simplest
member of the
,
-unsaturated aldehyde family, is widely found in
the environment and is also produced endogenously. It initiates urinary
bladder carcinogenesis in rats (24) and is mutagenic in bacteria (25, 26) and cultured cells (27-29). Acrolein reacts with dG residues in
DNA to form two pairs of stereoisomeric exocyclic propano adducts (Fig.
1), namely the 8R and
8S isomers of
3H-8-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (
-OH-PdG) and the 6R and 6S isomers of
3H-6-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (
-OH-PdG).
-OH-PdG predominates over
-OH-PdG (30-32) and has been detected in DNA isolated from human and animal tissue (30, 33).
Lipid peroxidation is suspected to be the major endogenous source (30).
Comparative genotoxic studies with a site-specific adduct in human
cells have shown that
-OH-PdG is less blocking than is
-OH-PdG
(34) and is bypassed with high fidelity (34, 35).
-OH-PdG, on the
other hand, miscodes substantially in human cells with a frequency of
10-12% per bypass synthesis, with G
T being predominant (34). As
-OH-PdG strongly inhibits DNA synthesis (34), it is likely that the
translesion polymerases are involved in bypassing this adduct. This
leads to the following questions: (i) which translesion polymerase is
responsible for the correct and incorrect syntheses; and (ii) whether
these syntheses are catalyzed by one polymerase or by different
polymerases. Here, we show the following. (i) Multiple DNA polymerases
are involved in the bypass synthesis. (ii) Pol
participates in
incorrect synthesis. (iii) The current eukaryotic bypass model (19-23)
does not seem to account for the error-free bypass of this adduct.
 |
EXPERIMENTAL PROCEDURES |
Oligonucleotides--
The procedures for the synthesis,
purification, and characterization of oligonucleotides containing
-OH-PdG have been described (37). The 13-mer (5'-CTCCTCXATACCT-3')
and 28-mer (5'-CTGCTCCTCXATACCTACACGCTAGAAC-3'), in which X represents
-OH-PdG, were the same oligonucleotides as those used in our
previous study (34). The 13-mer and 28-mer were used in the translesion
synthesis studies in vivo (human cells) and in
vitro, respectively. The 16-mer (5'-GTTCTAGCGTGTAGGT-3'), 18-mer
(5'-GTTCTAGCGTGTAGGTAT) and 19-mer (5'-GTTCTAGCGTGTAGGTATN-3', in which
N stands for A, G, C, or T) were employed as primers in the experiments
of read-through nucleotide incorporation opposite
-OH-PdG and primer
extension from a terminus opposite
-OH-PdG, respectively. The 28-mer
template contained the entire sequence of the 13-mer. All unmodified as
well as modified oligonucleotides were purified by electrophoresis in
denaturing 20% polyacrylamide gel and formed a single band following purification.
DNA Polymerases and Proliferating Cell Nuclear Antigen
(PCNA)--
Human Pol
(38), pol
(39), pol
(13), REV1 (40),
calf thymus pol
(41), and Saccharomyces cerevisiae pol
(19) were purified as described. The 3'
5' exonuclease
(exo)-proficient Klenow enzyme was obtained from New England BioLabs
(Beverly, MA); human PCNA was a gift from Paul A. Fisher (State
University of New York, Stony Brook, NY).
Primer Extension Reaction--
The 28-mer template and a
5'-32P-end-labeled primer were mixed at a molar
ratio of 1:2, heated at 70 °C for 5 min, and annealed by slow
cooling. Reaction mixtures (10 µl) contained 40 mM
bis-Tris (pH6.8), 6 mM MgCl2, 10 mM
dithiothreitol (DTT), 40 µg/ml bovine serum albumin (BSA), and 14 ng/µl PCNA for pol
; 40 mM Tris-HCl (pH 8.0), 30 mM KCl, 5 mM MgCl2, 10 mM DTT, and 250 µg/ml BSA for pol
and pol
(42);
25 mM KH2PO4 (pH 7.0), 5 mM MgCl2, 5 mM DTT, 100 µg/ml BSA
and 10% glycerol for REV1 (43) and pol
(19); 40 mM
Tris-HCl (pH 8.0), 5 mM MgCl2, 10 mM
-mercaptoethanol (replacing DDT used in the original
buffer), 250 µg/ml BSA and 2.5% glycerol for pol
(44); and 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2,
and 7.5 mM DTT for the Klenow enzyme. The final
concentration of dNTP was 10 µM for incorporation
experiments and 100 µM each in extension and read-through
experiments. A primed template was added at a concentration of 40 nM. The amounts of polymerases added are indicated in the
legends to Figs. 3-7. Reactions with pol
were incubated at
30 °C for 30 min, and those with the other enzymes were at 37 °C
for 10 min. Following reaction, 7 µl of a formamide dye mixture (95%
formamide, 0.1% xylene cyanol, 0.1% bromphenol blue, and 20 mM EDTA) was added, and aliquots (4 µl) were subjected to
electrophoresis in denaturing (8 M urea) 20% polyacrylamide gel at 2300 V for 2.5 h. Radioactive bands were detected and, if necessary, quantified by a PhosphorImager and ImageQuant software (Amersham Biosciences).
Cell Lines--
The SV40-transformed human XPV cell lines CTag
(45) and XP30RO(sv) (46) were obtained from M. Cordeiro-Stone
(University of North Carolina, Chapel Hill, NC) and J. Cleaver
(University of California, San Francisco, CA), respectively. CTag and
XP30RO(sv) were established from XP4BE and XP30RO (GM3617),
respectively. XP4BE and XP30RO cells contain a four-nucleotide
(positions 289-292) and a 13-nucleotide (positions 343-355) deletion,
respectively, in the coding region of one allele of the XPV
gene and produce severely truncated proteins due to the new stop codons
generated (47, 48). The other allele is not transcribed in either cell line. Cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (100 µg/ml), and streptomycin (100 µg/ml) at 37 °C in 5% CO2. An
expression vector containing a mouse XPV cDNA
(mXPV) was constructed as follows. A NotI
fragment containing mXPV was isolated from
pGEM-mXPV (49) and cloned in the correct orientation into
the NotI site of pIRESneo2 (Clontech),
which has the G418 resistance gene. The construct, pIRES-mXPV, was introduced into CTag cells by the FuGENE6
method (Roche Molecular Biochemicals) according to a manufacturer's
protocol. Transfected cells were selected for G418 (Mediatech, Herndon, VA) resistance at 500 µg/ml medium. pIRESneo2 is designed to
translate a cloned gene and the G418 resistance gene from the same
transcript. As this transcript contains an internal ribosome entry site
between the cloned gene and the G418 resistance gene, the
mXPV gene and the G418 resistance gene are independently
translated. Furthermore, translation of the G418 resistance gene is
designed to be less efficient than that of the cloned gene. Therefore,
all G418-resistant cells are expected to express mXPV. To
further assure the collection of mXPV-expressing cells,
G418-resistant cells were irradiated with UV at 2J/m2 and
then cultured in the presence of 1 mM caffeine (49). Almost all cells transfected with the empty pIRESneo2 vector died after 4 days, whereas cells transfected with pIRES-mXPV survived.
Following two cycles of this phenotypic selection, surviving cells were used as the host for site-specific experiments. Finally, the
transcription of the mXPV gene was confirmed by RT-PCR
(reverse transcriptase-polymerase chain reaction) using RNeasy Mini Kit
(Qiagen) and SuperScript One-Step RT-PCR Kit (Invitrogen).
Translesion Synthesis Studies in Human Cells--
The shuttle
vector, pBTE, was described previously (35). This vector is stably
maintained in human cells and confers blasticidin S resistance to host
human and E. coli cells. Expression of the resistance gene
is driven by the SV40 early promoter in human cells and the EM7
bacterial promoter in E. coli. The construction of
double-stranded DNA plasmid containing site-specific
-OH-PdG has
been described (34) and is shown in Fig.
2 together with the experimental
strategy.
-OH-PdG was incorporated into the leading strand template.
An important feature of this construct is that the adduct was inserted
opposite a unique SnaBI site (5'-TACGTA-3') with mismatches
on both sides of the adduct (Fig. 2); thus, only the unmodified
complementary strand contains the SnaBI site. Progeny plasmids derived from the unmodified strand and excision repair events
are sensitive to SnaBI digestion, whereas those derived from
translesion synthesis are not. Hence, progeny derived from translesion
synthesis can be selectively collected for fidelity analysis by
digesting with SnaBI prior to E. coli
transformation.

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Fig. 2.
Outline of experimental procedure
(A), construction of modified plasmid
(B), and oligonucleotide probes used for sequence
analysis of progeny plasmid (C). In
panels B and C, note mismatches at the
sequence of 3'-AXC (highlighted). X represents -OH-PdG.
Probe S (overscored) hybridizes only to unmodified strand.
Probes L and R detect plasmid containing a 13-mer insert. Probes G, T,
A, C, and D determine targeted events. , single base deletion.
|
|
CTag/pIRES and CTag/pIRES-mXPV cells were seeded at 1 × 106 cells/25-cm2 flask, cultured overnight,
then transfected overnight with 1 µg of a DNA construct by the
FuGENE6 method. Where indicated, cells were treated with mitomycin C at
1 µg/ml medium for 50 min in an incubator, after which the medium was
replaced with a fresh medium, and transfection was begun immediately.
The next day, cells were detached by treating with trypsin-EDTA and
replated in a 75-cm2 flask. The following day, blasticidin
S (Invitrogen) was added to the culture medium at 5 µg/ml. Resistant
cells were collected after 5 or 6 days. The progeny plasmid was
purified by the method of Hirt (50) and treated with DpnI (2 units) for 1 h to remove residual input DNA.
To establish the apparent efficiency of translesion DNA synthesis,
DpnI-treated plasmid was used to transform E. coli. To determine coding events at the site of
-OH-PdG, the
DpnI-treated plasmid was digested with SnaBI
prior to transformation. One-tenth to one-fifth of the recovered
plasmid was electroporated into E. coli DH10B ElectroMAX (25 µl) (Invitrogen) by an E. coli Pulser (Bio-Rad), after
which 975 µl of YT (2×) medium (36) was added, and the bacteria were
cultured for 40 min at 37 °C. Portions of the transformation mixture
were plated onto YT (1×) plates containing blasticidin S (50 µg/ml)
and ampicillin (100 µg/ml). After overnight incubation, E. coli transformants were subjected to differential oligonucleotide
hybridization (51, 52) to analyze for mutations in the adducted region.
This method permits the detection of specific sequences using
oligonucleotide probes. G, T, A, C, and D probes (Fig. 2C)
determine coding specificity at the site of
-OH-PdG. The S probe
hybridizes to the complementary SnaBI-containing strand. L
and R probes confirm the presence of the 13-mer insert. Automated DNA
sequence analysis was performed as necessary.
 |
RESULTS |
To understand the mechanism of the translesion synthesis across
-OH-PdG in human cells, we first conducted in vitro
experiments to select candidate polymerases whose translesion synthesis
activity and fidelity are consistent with the in vivo
results, and we then examined the role of one (pol
) of the
candidates in human cells.
Pol
-catalyzed Translesion Synthesis--
A running start
experiment (Fig. 3) using a 16-mer primer
and a 28-mer template showed that pol
bypassed
-OH-PdG very
weakly only in the presence of PCNA. Extended products were not
observed opposite the adduct, and the majority of the extension was
terminated at one base before the adduct site. These results suggest
that nucleotide insertion opposite
-OH-PdG and the subsequent
extension are poor. When the read-through experiment was catalyzed by
exo+ Klenow enzyme, full-length products were rarely
observed, and some extended products were observed opposite the adduct.
These results suggest that the full-length products observed in the pol
-catalyzed reaction were generated by true bypass synthesis across
the adduct. No stable insertion of a nucleotide opposite
-OH-PdG by
pol
was confirmed by nucleotide incorporation experiments using 10 and 100 µM dNTP (Fig. 4).
The results of these experiments indicate that pol
/PCNA catalyzes
bypass of
-OH-PdG very weakly. At this time the fidelity of this
bypass synthesis is not known.

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Fig. 3.
Translesion synthesis catalyzed by pol
/PCNA or exo+ Klenow enzyme.
32P-5'-end-labeled 16-mer primer/28-mer template complex
(40 nM) was incubated with various amounts of calf thymus
pol or exo+ Klenow enzyme in the presence of 100 µM each of four dNTPs at 30 °C for 30 min for pol and 37 °C for 10 min for Klenow. Where indicated, PCNA (140 ng) was
added to 10-µl reaction mixture. Reaction products were analyzed in
denaturing 20% polyacrylamide gel. X indicates the position
of -OH-PdG. The activity of pol decreased during prolonged
storage but was not re-determined, and the original unit value was
used.
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Fig. 4.
Incorporation of a nucleotide opposite
-OH-PdG by pol /PCNA.
32P-5'-end-labeled 18-mer primer/28-mer template complex
(40 nM), 0.75 units of pol , 140 ng of PCNA, and 10 µM (left) or 100 µM
(right) dNTP were used. The other conditions were the same
as those in Fig. 3.
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In subsequent experiments designed to determine the nucleotide distance
between the adduct and the primer terminus at which pol
/PCNA
recovered efficient synthesis, we found that exonucleolytic proofreading prevailed over polymerization when the primer terminus was
located three nucleotides or less 5' to the adduct (Fig.
5). When the terminus was five
nucleotides away, net polymerization efficiency increased. At seven
nucleotides, proofreading became marginal, and polymerization was
predominant. Therefore, if a translesion polymerase catalyzes DNA
synthesis
7 nucleotides past
-OH-PdG, the subsequent synthesis can
be performed efficiently by pol
.

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Fig. 5.
Resumption of DNA synthesis by pol
/PCNA. 32P-5'-end-labeled primers
of various lengths (19-26) were annealed to a modified 28-mer
template, and the primer extension reaction was performed using pol (0.75 units) and PCNA (140 ng) as described in the legend to Fig 3. X
represents -OH-PdG.
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Translesion DNA Polymerase-catalyzed Bypass Synthesis--
As
-OH-PdG inhibits DNA synthesis strongly, it is conceivable that
translesion polymerases participate in bypassing this adduct. To
determine which polymerase(s) plays a role in the accurate and
inaccurate bypass syntheses, we first examined the abilities of pol
and pol
to catalyze a bypass synthesis. The running start
experiments revealed that both polymerases could bypass this adduct
(Fig. 6). Qualitative nucleotide
incorporation experiments (Fig.
7A) showed that pol
inserted predominantly dAMP and, weakly, dGMP and dTMP opposite
-OH-PdG, whereas it preferentially inserted dCMP and, moderately,
dAMP and dTMP opposite dG. The extension experiments (Fig.
7B) with the 19-mer primer revealed that dA and dG but not
dC or dT termini were extended from opposite
-OH-PdG. These results
suggest that the bypass synthesis catalyzed by pol
predominantly
results in a G
T transversion, which is the major miscoding event
observed in human cells (34). A similar analysis with pol
(Fig.
7A) showed that this polymerase inserted dGMP weakly, dAMP
and dTMP marginally, and no dCMP opposite
-OH-PdG, whereas it
predominantly inserted correct dCMP opposite dG. Extension experiments
(Fig. 7B) showed that dA, dG, and dT termini, but not a dC
terminus, were extended weakly. These results suggest that pol
- and
pol
-catalyzed bypass syntheses are inaccurate and do not account
for the accurate synthesis in human cells.

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Fig. 6.
Translesion syntheses catalyzed by pol
and pol .
32P-5'-end-labeled 16-mer primer/28-mer template complex
(40 nM) was incubated with various amounts of a DNA
polymerase in the presence of 100 µM each of four dNTPs
at 37 °C for 10 min. Reaction products were analyzed in denaturing
20% polyacrylamide gel. X indicates the position of
-OH-PdG.
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Fig. 7.
Translesion DNA polymerase-catalyzed
nucleotide incorporation opposite -OH-PdG
(A) and extension from termini opposite
-OH-PdG (B). A,
32P-5'-end-labeled 18-mer primer/28-mer template complex
(40 nM) was incubated with a DNA polymerase in the presence
of 10 µM of one dNTP at 37 °C for 10 min.
Concentrations of polymerases were 3.63 nM pol , 3.1 nM pol , 2.5 nM pol , 14.4 nM
REV1, and 5.4 nM pol in a 10-µl reaction mixture.
B, 5' 32P-labeled 19-mer primer/28-mer template
complex (40 nM) was incubated with a DNA polymerase in the
presence of four dNTPs (100 µM each) at 37 °C for 10 min. Concentrations of DNA polymerases were the same as those used in
panel A.
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In experiments using pol
, REV1 and pol
(Fig. 7, A
and B), REV1 exclusively inserted correct dCMP, and pol
incorporated dCMP and dTMP opposite
-OH-PdG (Fig. 7A).
However, no extension from these termini was observed (Fig.
7B), suggesting that these two polymerases require another
DNA polymerase for the subsequent extension to complete accurate
translesion synthesis. As pol
is known to have this capability,
i.e. extension of a primer from a mismatched terminus and
from a terminus opposite DNA lesions (19, 53-56), we examined a pol
-catalyzed extension from four termini opposite
-OH-PdG. Although
this polymerase catalyzed extension from all four termini opposite dG,
with a dC terminus being most efficiently extended, the extension from
a dC terminus opposite
-OH-PdG was much less efficient than that
from the other three (dA, dG, and dT) termini (Fig. 7B).
This result suggests that pol
does not efficiently complete the
accurate synthesis initiated by pol
or REV1. Rather, it may
contribute to error-prone syntheses by extending from dA, dG, and dT
termini generated by other polymerases. No efficient extension was
observed as expected when REV1 and pol
were simultaneously added to
a reaction mixture (data not shown). Pol
did not efficiently insert
any nucleotide opposite
-OH-PdG (Fig. 7A). These results
suggest that the combination of pol
and pol
or REV1 and pol
does not account for the accurate translesion synthesis observed in cells.
The Role of Pol
in Mutagenic Bypass in Human Cells--
The
results of the in vitro experiments have suggested that pol
and pol
contribute to the cellular miscoding events. Using the
XPV cell line CTag, we found a lower miscoding frequency of 1.1%
(Table I) than in XPA cells (10-12%)
(34). However, as this XPV cell line and the XPA cell line do not have
an isogenic background, it may not be appropriate to compare the
results directly. To address this issue, we introduced into CTag cells
an expression plasmid containing mXPV, which was previously
shown to complement the defect in human XPV cells (49). The
introduction of the mXPV plasmid significantly (2.4-fold,
p < 0.001) increased the miscoding frequency, which
was largely ascribed to the increase in the number of G
T
transversions. A similar enhancing effect (2.3-fold, p < 0.001) of mXPV was noted when control and
mXPV-transfected cells were pretreated with mitomycin C. Pretreatment of cells with mitomycin C appears to cause a slight
increase in miscoding frequencies in these engineered XPV cells, though
the increases were not statistically significant. The fractions of
progeny derived from the modified strand were 24 and 23% for
CTag/pIRES and CTag/pIRES-mXPV, respectively, without
mitomycin pretreatment, and 27 and 22% for CTag/pIRES and
CTag/pIRES-mXPV, respectively, with pretreatment, showing no
significant differences between these two cell lines. Subsequently,
another XPV cell line, XP30RO(sv), was used to confirm the result with
CTag; a very low miscoding frequency was also noted in this cell line
(Table I). Taken together, our results indicate the following. (i) Pol
does not play a major role in translesion synthesis across
-OH-PdG. (ii) Pol
is not critical to error-free bypass. (iii)
Pol
is primarily responsible for inaccurate translesion synthesis
(
-OH-PdG
T). The latter two ideas are supported by the results of
in vitro experiments (Fig. 7, A and
B).
 |
DISCUSSION |
Acrolein is a bifunctional agent that reacts with the 1 and
N2 positions of dG to form two exocyclic propano
adducts. The exocyclic rings are formed in the region involved in
Watson-Crick hydrogen bonding to dC. Both adducts inhibit DNA
synthesis, and
-OH-PdG miscodes in human XPA cells (34). To
investigate the cellular translesion synthesis mechanism for
-OH-PdG, we conducted experiments in vitro with purified
eukaryotic DNA polymerases and compared the results with the previous
in vivo data to deduce a likely in vivo mechanism.
The Role of Pol
in Inaccurate Synthesis in Human Cells--
We
showed that pol
bypassed
-OH-PdG (Fig. 6), incorporated dAMP but
not dCMP opposite this adduct (Fig. 7A), and extended the
primer efficiently from this dA terminus (Fig. 7B). These results suggest that pol
-catalyzed synthesis can be highly
inaccurate, resulting in
-OH-PdG
T transversions and that pol
does not contribute to error-free translesion synthesis. The miscoding frequencies obtained in the two XPV cell lines (Table I) were significantly lower than those obtained in XPA cells (34), and the
lowered frequencies were complemented, though not perfectly, by the
introduction of mXPV (Table I). The mXPV did not
affect translesion synthesis efficiency. These results indicate that pol
plays a minor role in the overall process of translesion synthesis but is largely responsible for the inaccurate synthesis. The
involvement of pol
in inaccurate replication was also reported recently for
-OH-PdG (57). In S. cerevisiae, pol
has
been shown to be responsible for the accurate synthesis past 8-oxo dG
(58), the inaccurate synthesis past (6-4) thymine-thymine dimers (59),
and both accurate and inaccurate syntheses past acetylaminofluorene dG
adducts (59).
The Role of Other Translesion Polymerases in Inaccurate Synthesis
in Human Cells--
We observed miscoding events in XPV (CTag) cells,
though at reduced frequencies, suggesting that another polymerase(s)
catalyzes inaccurate translesion synthesis in the absence of pol
.
Among the polymerases examined, we found that pol
and pol
incorporate incorrect nucleotides opposite
-OH-PdG (Fig.
7A); pol
bypassed this adduct (Fig. 6) and extended from
the dA, dG, and dT termini (Fig. 7B); and pol
inserted
dCMP and dTMP opposite
-OH-PdG (Fig. 7A), but no further
extension was observed from these termini (Fig. 7B). This
extension may be catalyzed by pol
(Fig. 7B), as has been
observed in vitro for abasic sites (53, 55, 56) and (6-4)
thymine-thymine dimers (19, 54, 55).
What Mechanism Operates in Error-free Translesion
Synthesis?--
The experiments confirm and extend our previous work
(34), demonstrating that accurate translesion synthesis of
-OH-PdG is the major event in human cells, accounting for ~90% of the products. It appears unlikely that pol
or pol
contribute to a
substantial degree for the following reasons. (i) Neither polymerase inserted correct dCMP opposite
-OH-PdG (Fig. 7A), and
neither catalyzed extension from a dC terminus opposite this adduct
(Fig. 7B). (ii) XPV cells conducted error-free translesion
synthesis (Table I) with a substantial level of translesion synthesis. (iii) The introduction of mXPV did not enhance the level of
translesion synthesis. In contrast to pol
and pol
, pol
and
REV1 incorporated dCMP relatively efficiently opposite
-OH-PdG (Fig.
7A), but extension from this dC terminus was not observed
with either polymerase. Extension may be catalyzed by other polymerases
such as pol
and pol
, as has been observed for (6-4)
thymine-thymine dimers (19, 54, 55) and abasic sites (53, 55, 56). Pol
, however, catalyzed limited extension from a dC terminus opposite
-OH-PdG as compared with that from the other three termini (Fig. 7B). We did not observe any fully extended products by the
simultaneous addition of REV1, which exclusively inserted dCMP, and pol
to a reaction mixture. Thus, the 3'-terminal dC paired to
-OH-PdG was very resistant to extension by pol
as well as by pol
. It is likely, then, that pol
is not involved in the accurate synthesis, but rather may play a role in inaccurate synthesis. With all
the translesion polymerases examined, extension from purine (dA and dG)
termini appears to be more efficient than it is from pyrimidine (dT and
dC) termini, and a dC terminus is most resistant to such extension
(Fig. 7B). In conclusion, pol
and REV1 can serve to
insert dCMP, but a polymerase that can catalyze a
7 nucleotide
extension is required to propose a two polymerase-catalyzed bypass
mechanism (19-23). Thus, the mechanism for this error-free synthesis
is currently unknown.
Pol
is possibly responsible for the accurate synthesis, though its
in vitro bypass ability does not seem sufficient to account for the in vivo bypass synthesis. Another possibility is
that other DNA polymerases, such as pol
(60), pol
(60), and pol µ (60) catalyze this accurate translesion synthesis in cells. For
example, our preliminary experiments have shown that pol
, unlike
translesion polymerases, extends a primer efficiently from a dC
terminus (data not shown). We should also consider, however, that the
current in vitro system lacks critical accessory factors that mediate the activity of these polymerases. In conclusion, our
results indicate that multiple DNA polymerases are involved in the
translesion synthesis across
-OH-PdG and that accurate and
inaccurate translesion syntheses are catalyzed by different polymerases.
 |
ACKNOWLEDGEMENTS |
We thank A. P. Grollman for his
encouragement and support, F. Johnson and M. C. Torres for
synthesizing modified oligonucleotides, R. Woodgate and P.A. Fisher for
generous gifts of pol
and PCNA, respectively, and M. Cordeiro-Stone
and J. E. Cleaver for providing XPV cell lines.
 |
FOOTNOTES |
*
This work was supported by NCI, National Institutes of
Health, United States Public Health Service Grants CA76163 (to M. M.), CA47995 (to A. P. G.), and CA92528 (to Z. W.), and grants from the
Core Research for Evolution Science and Technology, Japan Science and
Technology Corporation (CREST, JST) and the Bioarchitect Research
Project of RIKEN (to F. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 631-444-3082;
Fax: 631-444-7641; E-mail: maki@pharm.sunysb.edu.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M212535200
 |
ABBREVIATIONS |
The abbreviations used are:
pol, DNA polymerase;
-OH-PdG, the 6R and 6S isomers of
3H-6-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one;
BSA, bovine serum albumin;
DTT, dithiothreitol;
exo, 3'
5'
exonuclease;
-OH-PdG, the 8R and 8S isomers of
3H-8-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one;
PCNA, proliferating cell nuclear antigen;
XPV, xeroderma pigmentosum
variant;
mXPV, mouse XPV cDNA.
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