From the Laboratory of Chemical Biology, Department
of Pharmacological Sciences and ¶ Department of Chemistry,
State University of New York at Stony Brook, Stony Brook, New York
11794-8651
Received for publication, September 29, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Acrolein, a reactive Exocyclic base adducts are formed when various endogenous and
exogenous bifunctional agents react with DNA. Such lesions include etheno, ethano, propeno, propano, and bicyclic dG, dA, and/or dC
adducts (1, 2). Etheno and ethano adducts are formed when DNA reacts
with chloroacetaldahyde, 1-substituted oxiranes, and/or enal epoxides;
propeno and propano adducts, in reaction with malondialdehyde and
enals, respectively; and bicyclic adducts, in reactions with
p-benzoquinone, a stable metabolite of benzene (1). Etheno,
propeno, and propano base adducts have been detected in DNA isolated
from human and animal tissues (3-5). The reaction of peroxidation
products of polyunsaturated fatty acids with DNA is suspected to be the
major source of these endogenous adducts (3-5).
The ring structure of exocyclic DNA adducts prevents the formation of
normal Watson-Crick hydrogen bonds. These adducts miscode in in
vitro primer extension studies conducted with purified DNA polymerases (1, 6-9) and induce point mutations in Escherichia coli and mammalian cells (1, 10-17). Exocyclic etheno adducts are
thought to be responsible for the mutations observed in the tumor
suppressor p53 gene of humans and animals exposed to vinyl chloride
(18, 19).
Acrolein, the simplest member of the 1,N2-Propanodeoxyguanosine adducts appear to be
ubiquitous in cellular DNA and may contribute to so-called spontaneous
mutagenesis, thereby playing a role in aging and cancer. In this paper,
we establish a genotoxic mechanism for We have developed a novel experimental approach that allows us to
explore cellular responses to DNA adducts at a mechanistic level (23).
The plasmid vector used in this study employs strand-specific markers
tagged with mismatches. An oligonucleotide containing a single DNA
adduct is incorporated into heteroduplex (HD) DNA containing short
stretches of mismatches at several locations. The modified DNA is
introduced into mismatch repair-deficient hosts, and adduct-related
events are measured. Progeny plasmid are analyzed for their marker
sequences. Linkage analysis of marker sequences allows us to group
progeny according to in vivo processing events, including
excision repair, translesion DNA synthesis, and recombination repair.
Enzymes--
Restriction enzymes, T4 polynucleotide kinase, and
T4 DNA ligase were purchased from New England BioLabs.
[ Oligodeoxyribonucleotides--
The synthesis of
oligodeoxyribonucleotides containing E. coli Strains and Vector--
E. coli strains used
are shown in Table I; MO strains were
constructed by P1 transduction (27) and are mismatch repair-deficient. Plasmids pS and pA have been described (23). These plasmids differ in
DNA sequence at three regions but are otherwise identical; hence, HD
DNA prepared from these plasmids contains three mismatched regions (see
Fig. 3). A mismatched region involving a BamHI site is
located 150 nucleotides upstream from the adduct site, the others,
containing NheI and SpeI/AatII sites,
are located ~220 and 3100 nucleotides, respectively, downstream from
the adduct (see Fig. 3). As described later, these mismatches serve as
strand-specific markers.
Construction of HD DNA Containing a Single
Single-stranded (ss) pA106 was mixed with EcoRV-digested ds
pS (Steps III and IV), treated with NaOH to denature ds pS, then neutralized to form ds DNA. Circular ss pA106 and its complementary strand (derived from ds pS) were annealed to form HD DNA containing a
13-nucleotide gap. An unmodified or modified 13-mer
(3'-d(TCCATAXCTCCTC), where X is dG or
In this HD construct, six and three base mismatches are formed at the
SpeI/AatII and SnaBI sites,
respectively. At the BamHI and NheI sites, one
strand has six extra bases. These four mismatched regions serve as
strand-specific markers: A/a (BamHI site), B/b (SnaBI site), C/c (NheI site), and D/d
(AatII/SpeI site). The unmodified complementary
strand derived from ss pA106 contains the A-B-C-D linkage (Fig.
3). Transformation of E. coli and Analysis of Progeny
Plasmid--
Unmodified or modified DNA (12 ng) was introduced into
mismatch repair-deficient (mutS) MO strains (50 µl of
electrocompetent cells) by electroporation. The mutS
mutation assures that the mismatches will not be repaired. 2× YT
medium 16 g of tryptone, 10 g of yeast extract, 5 g/1000 ml
NaCl, pH 7) (950 µl) was added to the electroporation mixture, then
incubated for 20 min at 37 °C. A portion (10-50 µl of a 100×
dilution) of the mixture was plated onto a 1× YT-ampicillin (100 µg/ml) plate to determine the number of transformants in the mixture.
The remaining mixture was incubated for additional 40 min and then
added to 10 ml of 2× YT containing ampicillin. After culturing
overnight, progeny plasmid was prepared by the alkaline lysis method
and used to transform E. coli DH5
In several experiments, mitomycin C was used to induce SOS functions in
E. coli. Overnight cultures of MO strains were diluted 20-fold with prewarmed 2× YT and cultured for 2 h. Mitomycin C (1-10 µg/ml) was added to the cultures and incubated at 37 °C for
30 min with shaking. Cells were prepared for electroporation by
repeated washings with H2O.
Interpretation of Results--
When a HD construct bearing a
single In Vitro Primer Extension Studies--
Oligonucleotides used for
primer extension studies are as follows: 28-mer templates,
5'-CTGCTCCTCXATACCTACACGCTAGAAC, where X is dG,
Reaction mixtures (10 µl) contained 50 mM Tris-HCl, pH
7.4, 5 mM MgCl2, 65 nM
primer/template, 200 or 300 µM dNTP, and 0-850 nM KF exo TLS across DNA Polymerase III and/or pol I Catalyze Accurate TLS--
To
address the question of which DNA polymerase(s) is (are) responsible
for the TLS, we constructed MO233, a strain that lacks all "SOS DNA
polymerases" such as pol II (polB), pol IV
(dinB), and pol V (umuDC), in addition to the
mutS and uvrA genes. MO234 is a control strain
that expresses all SOS DNA polymerases. The degree of TLS (number of
progeny II) is not significantly different in MO233 and MO234 in the
presence or absence of induced SOS functions (Table
III). The analysis of targeted events
revealed a single DNA Polymerase I-catalyzed TLS Is Diminished and
Error-prone--
Incorporation studies using pol I (KF
exo
Extension of primers beyond Blockage of DNA Synthesis Is Rescued by Recombination
Repair--
When the In this report, we studied the genotoxicity of The frequency of progeny II derived from the TLS pathway accounts for
>30% among all progeny in the strain (MO233) lacking all the SOS DNA
polymerases (pol II, pol IV, and pol V), and the SOS induction did not
significantly increase the number of progeny II in MO233 or MO234, a
control strain containing the SOS DNA polymerases (Table III). These
results suggest that one or more constitutive DNA polymerases, pol III
and/or pol I, play the major role in this TLS, which is highly
accurate. Only two Results of this study are very different from those reported for
the model acrolein adduct PdG, which strongly blocks DNA synthesis and
efficiently induces targeted PdG In summary, our results show that the combination of nucleotide
excision repair, accurate TLS, and daughter strand gap repair protects
well E. coli from the genotoxicity of ,
-unsaturated aldehyde
found ubiquitously in the environment and formed
endogenously in mammalian cells, reacts with DNA to form an
exocyclic DNA adduct,
3H-8-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (
-OH-PdG). The cellular processing and mutagenic potential of
-OH-PdG have been examined, using a site-specific approach in which
a single adduct is embedded in double-strand plasmid DNA. Analysis of
progeny plasmid reveals that this adduct is excised by nucleotide
excision repair. The apparent level of inhibition of DNA synthesis is
~70% in Escherichia coli
recA, uvrA. The block to DNA synthesis can be overcome partially by
recA-dependent recombination repair. Targeted G
T transversions were observed at a frequency of 7 × 10
4/translesion synthesis. Inactivation of
polB, dinB, and umuD,C genes coding
for "SOS" DNA polymerases did not affect significantly the
efficiency or fidelity of translesion synthesis. In vitro primer extension experiments revealed that the Klenow fragment of
polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite
-OH-PdG. We conclude from this
study that DNA polymerase III catalyzes translesion synthesis across
-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine
to protect E. coli from the potential genotoxicity of this
DNA adduct.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated aldehyde
family, is found widely in the environment and is formed in cells via
lipid peroxidation (3). Acrolein was shown to initiate urinary bladder
carcinogenesis in rats (20). Acrolein reacts with dG residues in DNA to
form two sets of stereoisomeric propano adducts (see Fig. 1): 8
and
8
isomers of
3H-8-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (
-OH-PdG)1 (I)
and 6
and 6
isomers of
3H-6-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (
-OH-PdG) (II). The
-OH-PdG isomers are in greatest
abundance (3, 21). Chung et al. (3, 21, 22) detected
background acrolein- and crotonaldehyde (methylacrolein)-derived
adducts in experimental animals and human tissues at levels ranging
between 0.01 and 7.53 µmol/mol of guanines. These authors also
reported that oxidative stress, enhanced lipid peroxidation, and
decreased levels of glutathione increase markedly the tissue level of
propano adducts (3).
-OH-PdG in E. coli
and describe the processing of this adduct in bacterial cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol) and polymerase chain
reaction-grade dNTPs were obtained from Amersham Pharmacia Biotech and
Roche Molecular Biochemicals, respectively. The
3'
5'-exonuclease-deficient Klenow fragment (KF exo
)
was purified from E. coli CJ374 containing pCJ141, an
expression vector (gift from C. Joyce, Yale University), as described
previously (24).
-OH-PdG or
1,N2-(1,3-propano)-2'-deoxyguanosine (PdG)
(IV in Fig. 1) has been
described previously (25, 26). Oligonucleotides containing the
precursor of
-OH-PdG
(N2-[3,4-dihydroxybutyl] dG) or PdG were
purified twice by high pressure liquid chromatography using a Waters
µBondapak C18 column (3.9 × 300 mm) and acetonitrile in 100 mM triethylammonium acetate buffer, pH 6.8, at a flow rate
of 1 ml/min with the dimethoxytrityl group (DMT) on and off. The
acetonitrile gradient was 16-33% with DMT-on DNA and 0-20% with
DMT-off DNA and applied over 40 min. To generate
-OH-PdG,
oligonucleotides containing
N2-[3,4-dihydroxybutyl] dG were incubated in
100 mM sodium periodate for >6 h at room temperature (25).
-OH-PdG-containing oligonucleotides were separated from the parental
oligonucleotides by high pressure liquid chromatography as described
above for DMT-off DNA. All oligonucleotides were subjected to
electrophoresis in a denaturing 20% polyacrylamide gel. Bands were
detected by UV shadowing. Oligonucleotides were excised from the gel
then purified over a Sep-Pak column (Waters). Purified oligonucleotides
were subjected to electrospray mass spectrometry analysis. The results
were as follows: 13-mer with
-OH-PdG m/z: observed,
3901.8 ± 0.47; calculated, 3901.6; 28-mer with
-OH-PdG
m/z: observed, 8510.15 ± 0.23; calculated, 8509.6; and
28-mer with PdG m/z: observed, 8494.37 ± 0.58;
calculated, 8493.6.
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Fig. 1.
Exocyclic propano-dG adducts formed by the
reaction of
,
-unsaturated aldehydes
with DNA. I,
-OH-PdG; II,
-OH-PdG;
III, M1G, a
malondialdehyde-derived dG adduct; IV, PdG, a model for
I, II, and III
E. coli strains
-OH-PdG
Adduct--
The scheme for this construction is shown in Fig.
2. Detailed procedures have been
described previously (19). In brief, double-stranded (ds) pA was
digested with EcoRV (Step I), and the linearized plasmid DNA
was ligated to a blunt-ended duplex 13-mer,
5'-d(AGGTACGTAGGAG)/ 3'-d(TCCATGCATCCTC), containing a SnaBI site (5'-TACGTA) (Step II). Two constructs, each
containing a single insert with opposite orientation, were isolated;
one of these, pA106, was used in this study.
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Fig. 2.
Construction of HD DNA containing a
single -OH-PdG (X) residue. I,
EcoRV digestion of pA and pS; II, ligation of a
duplex 13-mer containing a SnaBI site (5'-TACGTA);
III, preparation of ss DNA; IV, preparation of
gapped HD DNA; and V, ligation of modified 13-mer. There are
three contiguous base mismatches (highlighted) in the
SnaBI site. There are also mismatches at the SpeI
(d)/AatII (D), BamHI
(a/A), and NheI (c/C) sites (the
latter two are not shown; see Fig. 3 for details). The mismatched
regions serve as strand-specific markers.
-OH-PdG) phosphorylated at the 5'-termini using T4 polynucleotide
kinase and ATP, was annealed into the gap and ligated by T4 DNA ligase.
The 13-mers are not fully complementary to the gap sequence, because
they form mismatches at and adjacent (5' and 3') to the adduct site
(Fig. 3). These mismatches, located opposite the SnaBI site, also serve as a strand-specific
marker. The ligation mixture was treated with SpeI and
EcoRV to remove residual ds pS. Closed circular ds DNA was
purified by ultracentrifugation in a CsCl/ethidium bromide solution.
DNA was concentrated by Centricon 30 (Amicon, Beverly, MA), and the
concentration was determined spectrophotometrically.
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Fig. 3.
Nucleotide sequence of regions containing
strand-specific markers and probes used for oligonucleotide
hybridization. The top strand is ss pA106, and the
bottom strand is derived from EcoRV-treated ds
pS. Marker sequences in the sequence-specific probes A-D
(overscored) and a-d (underlined) are
highlighted. L and R probes
(underlined) were used to identify progeny containing the
13-mer insert. Probes SG, ST, SA,
SC, and SD were used to determine which base
replaced -OH-PdG.
, direct connection of bases: e.g.
G-G is GG; ~, sequence interruption. Approximate distances between
markers are shown in parentheses.
-OH-PdG was incorporated into the site of b on the strand
bearing the a-b-c-d linkage; this strand is the template for leading
strand synthesis.
(Life Technologies,
Inc.). This second transformation segregates progeny plasmid derived
from each strand of the HD DNA. Transformants were inoculated
individually in 96-well plates and cultured for several hours.
Bacterial cultures were stamped onto filter paper placed on a 1×
YT-ampicillin plate and cultured overnight. The filter was treated with
0.5 M NaOH for 11 min, neutralized in 0.5 M
Tris-HCl, pH 7.4, for 7 min, washed with 1× SSC (150 mM
NaCl, 15 mM Na3 citrate, pH 7.2) then with ethanol, and baked at 80 °C for 2 h. Differential
oligonucleotide hybridization (16, 23, 28) using the
32P-labeled probes shown in Fig. 3 was employed to detect
strand-specific marker sequences and the base located at the position
of the adduct. L and R probes were used to confirm the presence of the
13-mer insert.
-OH-PdG residue is introduced into a host cell, various
events may occur (Fig. 4). If the adduct
is removed by excision repair before being replicated, the immediate
5'- and 3'-flanking mismatches also are removed. Gap-filling DNA
synthesis converts 5'-CXA (b) to 5'-ACG (B); therefore, progeny derived from the repaired strand contain the linkage of a-B-c-d
(progeny III) (Fig. 4, step 1). When the construct is replicated in the absence of DNA repair, progeny produced from the
unmodified strand contain the A-B-C-D linkage (step 2),
whereas those from the modified strand have a-b-c-d (step 4)
following translesion synthesis (TLS). When DNA synthesis is blocked by the adduct, the block may be overcome by
UmuD'2C/RecA-assisted TLS or recombination repair (daughter
strand gap repair) (29). In recombination repair, the ss gap is filled
by strand transfer from the unmodified parental strand (step
5). The 3'-end of the blocked nascent strand is used to replicate
the transferred region (step 6). These processes create a
Holliday junction. When the Holliday junction is migrated by the
RuvA·RuvB complex, a Holliday junction-specific helicase, and
resolved by RuvC, a Holliday junction-specific endonuclease (step
7), progeny with the linkage of A-B-C-D (progeny I), a-B-C-d
(progeny IV), a-B-C-D (progeny V), and A-B-C-d (progeny VI) are
created.
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Fig. 4.
Pathways involved in the processing of DNA
damage in E. coli. Input DNA (X
represents -OH-PdG) is shown in a box. The ColE1 origin
of replication (short vertical bars) and the direction of
replication (short arrow) are shown. A-D and
a-d correspond to those shown in Fig. 3. Progeny
I-VI correspond to those listed in Tables II and III.
Excision repair of the adduct (step 1) converts the sequence
of the adducted region to B; replication of a repaired molecule
produces progeny I and III. When modified DNA replicates (steps
2-4), the unmodified strand produces progeny I and the modified
strand yields progeny II following TLS (step 4). When the
adduct inhibits DNA synthesis, daughter-strand gap repair (steps
5-7) operates to overcome the inhibition. This mechanism,
involving strand transfer (step 5), formation of a Holliday
junction, gap-filling synthesis (step 6), branch migration,
and resolution of a Holliday junction by RuvC resolvase (step
7), produces progeny I, IV, V,
and VI.
-OH-PdG, or PdG; an 18-mer primer for incorporation experiments,
5'-GTTCTAGCGTGTAGGTAT; 19-mer primers for extension experiments,
5'-GTTCTAGCGTGTAGGTATN, where N is dA, dG, dC, or dT; and a 16-mer
primer for read-through experiments, 5'-GTTCTAGCGTGTAGGT. All
oligonucleotides were purified by electrophoresis in a denaturing 20%
polyacrylamide gel. The 5'-32P-end-labeled primers were
hybridized to templates at a molar ratio of 1:1.2 in a buffer
containing 50 mM Tris-HCl, pH 7.5, 5 mM EDTA,
and 500 mM NaCl. Annealing reactions were conducted by
heating at 70 °C for 5 min followed by slow cooling.
. The concentrations of dNTP were
200 µM for incorporation and extension experiments and
300 µM for read-through experiments. KF exo
was diluted in a solution containing 50 mM Tris-HCl, pH
7.5, 0.5 mg/ml bovine serum albumin, and 10% glycerol. Reactions were conducted at 25 °C for 30 min and stopped by adding 10 µl of
formamide dye mixture (90% formamide, 0.001% xylene cyanol, 0.001%
bromphenol blue, 20 mM EDTA). Samples were heated at
95 °C for 5 min, and aliquots (1.5 µl) were subjected to
electrophoresis in a denaturing 20% polyacrylamide gel (0.4 mm thick,
40 cm long) at 2600 V for 3 h. Gels were analyzed by a
PhosphorImager using ImageQuaNT software (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-OH-PdG Is a Substrate for Nucleotide Excision
Repair--
Pathway 1 depicted in Fig. 4 predicts that progeny III
derived from plasmids subjected to excision repair will contain the a-B-c-d linkage. The unmodified control construct, which contained three base mismatches and no adduct, yielded <1% of progeny III in
the uvrA (MO937) and uvr+ (MO939)
strains (Table II), indicating that the
mismatches were not subjected to nucleotide excision repair. When the
adducted construct was introduced into the uvrA strains
(MO937 and MO934), progeny III accounted for <3% of plasmids
analyzed. When the same construct was introduced into uvr+
strains (MO939 and MO933), fractions of progeny III ranged from 32 to
35%. Thus,
-OH-PdG is a substrate for UvrABC-catalyzed nucleotide
excision repair in the presence of the 5'- and 3'-flanking
mismatches.
Linkage analysis of progeny plasmid
-OH-PdG Adduct Inhibits DNA Synthesis in E. coli--
The HD
constructs were introduced into MO937 (
recA,
uvrA, alkA1, tag1). With the
unmodified control construct, more than 96% of progeny were derived
from replication of both strands (progeny I and
II, Table II). The ratio of progeny I to II, however, was not 1:1 but twice that of progeny I. This result is consistent with
that of a previous study (23). Using the modified construct, the number
of progeny I derived from the unmodified strand (78%) markedly
exceeded that of progeny II derived from TLS (17%). This result
indicates that
-OH-PdG is a strong but not complete block to DNA
synthesis. The apparent efficiency of TLS is estimated to be 27%
(17/64). The induction of SOS functions did not increase significantly
the fraction of progeny II in MO934. The fractions of progeny II
obtained in the uvr+ strains (MO939 and MO933) were lower
than those in the uvrA strains (MO937 and MO934) due to efficient repair of the adduct by nucleotide excision repair.
-OH-PdG Is Essentially Error-free--
The
adducted HD construct was introduced into MO933 ("wild"), MO934
(uvrA), MO220 (mutD5), and MO221
(mutD5,
umuDC) in the presence or
absence of induced SOS functions. Initial numbers of transformants in
the transformation mixtures were determined by plating a portion of the
transformation mixture. This analysis revealed >1 × 106 and >1 × 104 transformants per
transformation in the absence and presence of induced SOS functions,
respectively. The lower transformation efficiency of mitomycin
C-treated cells is thought to be due to DNA damage. Progeny plasmid DNA
was purified following overnight culture of the transformation mixture
in the presence of ampicillin and then digested with SnaBI,
the site for which is located in marker B (Fig. 3). This
digestion removes progeny derived from the unmodified strand and
plasmids subjected to excision repair or recombination repair (see Fig.
4), facilitating analysis of TLS events. Targeted events were analyzed
by differential oligonucleotide hybridization and DNA sequencing. This
analysis revealed that almost all targeted events were
-OH-PdG
dG, indicating accurate TLS. Only one
-OH-PdG
dT transversion
was observed among 282 transformants of SOS-uninduced MO933, yielding a
miscoding frequency of 0.35%. The numbers of transformants analyzed
were 144 for SOS-induced MO933; 190 each for SOS-induced and -uninduced
MO934 and MO220; and 144 each for SOS-induced and -uninduced MO221.
Targeted mutations were not observed in these strains. When data for
the four strains are combined, the frequencies of targeted mutations
are 0.12% and <0.15% in the absence and presence of induced SOS
functions, respectively. Thus, TLS across
-OH-PdG is highly accurate
in E. coli.
-OH-PdG
dT transversion in SOS-induced MO233,
suggesting that pol III or pol I is responsible for this mutation.
These results suggest that non-SOS DNA polymerase(s) conduct(s) TLS
across
-OH-PdG with high fidelity.
Linkage analysis in SOS DNA polymerase-deficient E. coli
), in which a primer terminus was located one base 3'
from the adduct site and a single dNTP was present, showed that
this polymerase inserts dAMP and dGMP and, to a much lesser extent,
dCMP and dTTP opposite
-OH-PdG: Frequencies of incorporation were
93, 88, 7, and 5% for dATP, dGTP, dCTP, and dTTP, respectively, in a
reaction using 85 nM KF exo
(Fig.
5A). Primer extension studies,
in which a primer terminus was located opposite
-OH-PdG and all four
dNTPs were present, showed that dAMP, dCMP, and dGMP termini were
extended similarly and the dTMP terminus was extended poorly: Yields of
extended products were 63, 57, 44, and 18% with the dA, dC, dG, and dT termini, respectively, in the reaction containing 85 nM
enzyme (Fig. 5B). These results suggest that pol I promotes
error-prone DNA synthesis across
-OH-PdG. When the read-through
experiments were conducted using a 16-mer primer in the presence of all
four dNTPs, doublet bands, caused by differing migration rates due to
the difference in the nucleotide inserted, were observed opposite the
adduct (X) and the next base (C) (Fig. 5C). This result
indicates that different nucleotides are inserted opposite this adduct
and then extended. Effects of the nucleotide difference is obscured as
the length increases. This result is consistent with those of
incorporation and extension studies described above. Because dCMP is
not the preferred nucleotide inserted, TLS catalyzed by this polymerase
is likely to be error-prone.
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Fig. 5.
In vitro primer extension
catalyzed by exonuclease-deficient Klenow fragment. A,
incorporation of a deoxyribonucleotide opposite -OH-PdG;
B, extension from primer terminus located opposite
-OH-PdG; C, translesion DNA synthesis with 16-mer
primer.
-OH-PdG or PdG was observed at 85 and
170 nM KF exo
(Fig. 5C). The
fractions of primers extended beyond these modified bases are 36% at
85 nM and 69% at 170 nM for
-OH-PdG and
11% at 85 nM and 23% at 170 nM for PdG. These
results indicate that PdG is more inhibitory than
-OH-PdG.
-OH-PdG-containing HD construct was introduced
into MO933, MO934, MO233, or MO234, the relative fractions of progenies IV, V, and VI increased. The sum of these three types of progeny ranged
from 8 to 19% in the uvrA strains (MO934, MO233, and
MO234). In the uvr+ strain (MO933), percentages
were 7 and 5% in the absence and presence of induced SOS functions,
respectively. The lower values may reflect the substantial repair of
the adduct in this uvr+ strain. When the
modified HD construct was introduced into the
recA strains (MO937 and MO939), fractions of
the three types of progeny were low: 2.3% in MO937 and 1% in MO939.
In a previous report (23), mismatches were shown not to increase the
number of recombinants. Therefore, the increases observed can be
ascribed to a RecA-dependent recombination repair mechanism
(daughter strand gap repair) in response to the DNA synthesis block
caused by the adduct (Fig. 4). These increases are consistent with the
general finding that
-OH-PdG inhibits DNA synthesis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-OH-PdG in
E. coli and the response of this organism to the presence of
the adduct. Our results indicate that
-OH-PdG is a good substrate for nucleotide excision repair; in this respect, it is similar to the
structurally related PdG and malondialdehyde-derived propeno-dG adduct,
pyrimido[1,2-a]purin-10(3H)-one
(M1G) (III in Fig. 1) (30).
-OH-PdG inhibits
DNA synthesis; the apparent efficiency of TLS is 27% in the absence of
induced SOS functions. The inhibition of DNA synthesis created by
-OH-PdG is partly overcome by daughter strand gap repair
(recombination repair) as determined by the increase in the number of
recombinants of progenies IV, V, and VI (Tables II and III). Because
the parental unmodified strand is used to fill in a gap generated as a
result of the synthesis block (refer to Fig. 4), this repair is
mechanistically accurate and therefore contributes to an error-free
recovery from DNA synthesis block.
-OH-PdG
T transversions were observed among
the total of 2687 transformants of various E. coli strains
obtained in the presence or absence of SOS induction. The overall
targeted mutation frequency is 0.07%. Marnett and his colleagues also
have observed the lack of mutagenicity of this adduct (accompanying
article (39)). Because pol I is necessary for the replication of ColE1
origin-based plasmids (31), inactivation of this gene is not possible.
Our in vitro primer extension studies show that this
polymerase appears to catalyze error-prone TLS across
-OH-PdG (Fig.
5). pol I is believed to catalyze the filling of small gaps and the
formation of DNA primers in E. coli. Therefore, we speculate
that pol III, but not pol I, is responsible for this highly accurate
TLS. The accurate TLS was also observed in the strain (MO220) carrying
a mutation in the mutD (dnaQ) gene that codes for
the pol III-associated 3'
5'-exonuclease (
subunit) (31). This
exonuclease removes nucleotides from the 3' terminus when incorrect
nucleotides are inserted during pol III-catalyzed DNA synthesis.
Therefore, if this TLS is catalyzed by pol III, the result of the
accurate TLS in this strain suggests that correct dCMP is inserted
almost exclusively opposite
-OH-PdG and extended: The accurate TLS
can be ascribed to the selection of the correct deoxyribonucleoside
triphosphate, dCTP. Another possibility is that only
-OH-PdG:dC
pair, but not other incorrect pairs, can be extended by pol III.
T and PdG
A mutations in
E. coli and simian kidney cells (12, 15). PdG presents a
stronger block than
-OH-PdG when tested by primer extension
experiments in vitro. When a 25-mer containing
-OH-PdG was incubated at room temperature with its complementary strand, two
species migrating more slowly than the starting material appeared, accounting eventually for 5% of the total
oligonucleotide.2 This
observation suggests that interstrand cross-linking has occurred, an
event that would take place only if
-OH-PdG exists in a ring-open
form similar to that reported for M1G paired with dC in
duplex DNA (32). An NMR structural study (see accompanying article
(40)) shows unequivocally that ring opening of
-OH-PdG occurs when
the adduct is located opposite dC in duplex DNA, in a conformation
allowing formation of Watson-Crick bonds. Because
-OH-PdG is located
in a mismatched region in our HD DNA, the structure of the adduct
cannot be stated with certainty; however, Watson Crick hydrogen bonding
generated by ring opening of
-OH-PdG would account fully for the
enhanced translesion synthesis and relative lack of miscoding of the
natural acrolein adduct.
-OH-PdG.
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ACKNOWLEDGEMENTS |
---|
We thank M. Berlyn, M. F. Goodman, R. Kolodner, T. Nohmi, and M. Volkert for bacterial strains; R. Rieger for electrospray mass spectrometry analysis; and C. Torres for synthesizing PdG-containing oligonucleotides.
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FOOTNOTES |
---|
* This research was supported by United States Public Health Services Grants CA76163 (to M. M.) and PO1CA47995 (to A. G.).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.
§ Both authors contributed equally to this work.
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, December 21, 2000, DOI 10.1074/jbc.M008918200
2 I.-Y. Yang, M. Hossain, H. Miller, S. Khullar, F. Johnson, A. Grollman, and M. Moriya, unpublished studies.
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ABBREVIATIONS |
---|
The abbreviations used are:
-OH-PdG, 8
and
8
isomers of
3H-8-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one;
-OH-PdG, 6
and 6
isomers of
3H-6-hydroxy-3-(
-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one;
DMT, dimethoxytrityl group;
ds, double-strand(ed);
dA, 1,N6-ethenodeoxyadenosine;
KF exo
, 3'
5'-exonuclease-deficient Klenow fragment;
M1G, pyrimido[1,2-
]purin-10(3H)-one;
PdG, 1,N2-(1,3-propano)-2'-deoxyguanosine;
ss, single-strand(ed);
HD, heteroduplex;
TLS, translesion DNA synthesis;
pol, polymerase.
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