(Received for publication, January 21, 1997)
From the Departments of Biochemistry and Chemistry, Center in Molecular Toxicology and the Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Repair of the exocyclic DNA adduct
propanodeoxyguanosine (PdG) was assessed in both in vivo
and in vitro assays. PdG was site-specifically incorporated
at position 6256 of M13MB102 DNA, and the adducted viral genome was
electroporated into repair-proficient and repair-deficient Escherichia coli strains. Comparable frequencies of PdG T and PdG
A mutations at position 6256 were detected following
replication of the adducted genomes in wild-type E. coli
strains. A 4-fold increase in the frequencies of transversions and
transitions was observed in E. coli strains deficient in
Uvr(A)BC-dependent nucleotide excision repair. A similar
increase in the replication of the adduct containing strand was
observed in the repair-deficient strains. No change in the frequency of
targeted mutations was observed in strains deficient in one or both of
the genes coding for 3-methyladenine glycosylase. Incubation of
purified E. coli Uvr(A)BC proteins with a duplex 156-mer
containing a single PdG adduct resulted in removal of a 12-base
oligonucleotide containing the adduct. Incubation of the same adducted
duplex with Chinese hamster ovary cell-free extracts also resulted in
removal of the adduct. PdG was a better substrate for repair by the
mammalian nucleotide excision repair complex than the bacterial repair
complex and was approximately equal to a thymine-thymine dimer as a
substrate for the former. The results of these in vivo and
in vitro experiments indicate that PdG, a homolog of
several endogenously produced DNA adducts, is repaired by the
nucleotide excision repair pathway.
Exocyclic adducts that block the Watson-Crick base pairing region
of DNA bases are produced from a range of exogenous and endogenous
chemicals (1). Numerous exocyclic adducts have been identified
including etheno and substituted ethano adducts to deoxyadenosine,
deoxyguanosine, and deoxycytidine, propeno and substituted propano
adducts to deoxyguanosine, and complex bicyclic adducts to
deoxyguanosine (1, 2). A particularly important class of cyclic adducts
include propeno and substituted propano adducts to deoxyguanosine.
These include the pyridimidopurinone adduct,
M1G,1 formed from
malondialdehyde and the hydroxy-propanodeoxyguanosine adducts
formed from crotonaldehyde and acrolein (Fig. 1) (2). These adducts have been detected at levels from 1500-5000/cell in
liver DNA from healthy human beings (3, 4) and appear to be highly
mutagenic in site-specific mutagenesis assays (5-11).
The high levels of propano adducts detected in human tissues suggests that their removal by DNA repair enzymes may be an important protective mechanism against genetic diseases that might be induced by these adducts. At present, no information is available on the enzymes involved in the repair of these adducts. Certain cyclic adducts such as N2,3-ethenodeoxyguanosine and 1,N6-ethenodeoxyadenosine are substrates for the bacterial and mammalian 3-methyladenine glycosylases (12-15), and Singer and colleagues have characterized other human proteins that remove cyclic bases from duplex oligonucleotides in vitro (16-18). Propanodeoxyguanosine (PdG), which is a model for the chemically unstable M1G and hydroxy-PdG adducts, is not a substrate for these glycosylases (18).
We have undertaken an investigation of the repair of the prototypical exocyclic deoxyguanosine adduct, PdG, in vivo and in vitro. PdG was used as a model for the M1G and hydroxy-PdG adducts because of its stability during oligonucleotide synthesis and during the construction of large duplex oligonucleotides, which are necessary for in vitro repair assays. The removal of PdG was assessed in vivo by determining the mutation frequency and strand bias observed upon replication of M13 genomes containing this adduct in wild-type and repair-deficient strains of Escherichia coli. An increase in mutation frequency and adducted template replication in a repair-deficient strain was taken as an indication of the involvement of the deleted gene in repair. These experiments indicated that PdG residues are repaired by nucleotide excision repair. The in vivo results were confirmed by using an in vitro assay for the excision of the PdG adduct by both the E. coli and hamster excinucleases. Thus, PdG, a surrogate for endogenous adducts present in healthy humans, is a substrate for both bacterial and mammalian nucleotide excision repair proteins.
BssHII and KspI restriction
endonucleases and DNA ligase were purchased from Boehringer Mannheim.
T4 polynucleotide kinase was purchased from U. S. Biochemical Corp.
Calf thymus DNA and lauryl sulfate were purchased from Sigma.
5-Bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)
and isopropyl
-D-thiogalactoside were obtained from Gold Biotechnology (St. Louis, MO). Nitrocellulose transfer membranes (BA85
82.5-mm diameter) were from Schleicher & Schuell.
[
-32P]ATP (3000 Ci/mmol for hybridization experiments
and 7000 Ci/mmol for excision assay) was from DuPont NEN. Unadducted
and PdG-adducted 8-mer oligonucleotides for mutagenesis experiments,
5
-GGTXTCCG-3
(where X is G or PdG), and 12-mer
oligonucleotide for the in vitro excision assay,
5
-GAAXCTACGAGC-3
(where X is PdG), were
synthesized by Midland Certified Reagent Co. (Midland, TX). The
oligonucleotides were purified by reversed phase HPLC and run as single
bands on denaturing polyacrylamide gels (6). Oligonucleotides used as hybridization probes were prepared using an Applied Biosystems automated DNA synthesizer in the Vanderbilt University Molecular Toxicology Molecular Genetics Core.
The E. coli strain JM105
(supE, tri, rpsL, endA, sbcB15, hsdR4,
(lac-proAB) [F
traD36, proAB,
LacIQZ,
M15]) was used as the host
bacterium for the production of M13MB102. CJ236
(dut
, ung
, thi,
relA/pCJ105, [F
Cmr]) was used as the host
bacterium for the production of uracil-containing M13MB102, with the
medium supplemented with 0.25 µg/ml uridine. AB1157
(thr-1, ara-14,
leuB6,
(gpt-proA)62,
lacY1, tsx-33,
qsr
,supE44, galK2,
, rac
, hisG4,
rfbD1, mgl-51, rpsL31,
kdgK51, xyl-5,
mtl-1, argE3,
thi-1); AB1887 (thr-1,
ara-14, leuB6,
(gpt-proA)62, lacY1,
tsx-33, supE44, galK2,
, rac
, hisG4,
rfbD1, mgl-51, rpsL31,
kdgK51, xyl-5,
mtl-1, argE3,
thi-1, uvrA6); and AB2463
(thr-1, ara-14,
leuB6,
(gpt-proA)62,
lacY1, tsx-33, supE44,
galK2,
, rac
,
hisG4, rfbD1, recA13,
rpsL31, kdgK51, xyl-5,
mtl-1, argE3, thi-1) were obtained from Barbara Bachman, Yale University. MV1161
(thr-1, ara-14,
leuB6,
(gpt-proA)62,
lacY1, tsx-33, supE44,
galK2, hisG4, rfbD1,
mgl-51, rpsL31, kdgK51,
xyl-5, mtl-1,
argE3, thi-1,
rfa-550); MV1174 (alkA1,
thr-1, ara-14,
leuB6,
(gpt-proA)62,
lacY1, tsx-33, supE44,
galK2, rfbD1, mgl-51,
rpsL31, kdgK51,
xyl-5, mtl-1,
argE3, thi-1,
rfa-550); GC4800 (tagA1,
zhb::Tn5,
(p(sfiA::lacZ)
clind
) thr-1,
ara-14, leuB6,
(gpt-proA)62, lacY1,
tsx-33, supE44, galK2,
hisG4, rfbD1, mgl-51,
rpsL31, kdgK51, xyl-5,
mtl-1, argE3, thi-1); and MV2157 (alkA1,
tagA1, zhb::Tn5,
thr-1, ara-14,
leuB6,
(gpt-proA)62,
lacY1, tsx-33, supE44,
galK2, rfbD1, mgl-51,
rpsL31, kdgK51, xyl-5,
mtl-1, argE3,
thi-1, rfa-550) were
obtained from Michael Volkert, University of Connecticut. AB1157,
AB1887, MV1161, MV1174, GC4800, and MV2157 were converted to F
derivatives as described previously (5). NR10148 (ara, thi,
prolac, zcf-117::Tn10, F
prolac(F
128-27),
(uvrB-bio)) was obtained from Roel Schaaper, National Institute
of Environmental Health Sciences.
Single-stranded DNAs were isolated as described by Maniatis and co-workers (19). Replicative form DNAs were harvested using Qiagen columns (Pasadena, CA).
Construction of Singly Adducted M13MB102M13MB102
containing an 8-nucleotide gap in the ()-strand and uracil in the
(+)-strand was prepared and transformed into bacterial strains as
described (7). Base pair substitution mutations and template strand
utilization of the progeny phage were determined by hybridization as
described (7, 20).
Substrates containing either a centrally located PdG or T<>T adduct were prepared as described (21, 22) by phosphorylating, annealing, and ligating six or eight overlapping oligonucleotides to generate a 140- (T<>T) or 156-base pair (PdG) duplex. As controls, unmodified (140-mer) or psoralen monoadducted DNA (45-mer) were prepared in a similar manner.
Excision Assay with Uvr(A)BCUvrA, UvrB, and UvrC were
purified as described (23) and kindly provided by Dr. M.-S. Tang. The
reaction mixtures contained 1.5 fmol of radiolabeled DNA, 15 nM UvrA, 15 nM UvrB, 15 nM UvrC, and 100 ng of X174 DNA in 25 µl of buffer that was 50 mM Tris, pH 7.5, 100 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol, and 1 mM ATP. Incubation was at 37 °C with the reaction being
stopped by the addition of 2 µl of a 1:1 mixture of 0.25 M EDTA and 5 mg/ml oyster glycogen, followed by ethanol
precipitation. Recovered DNA was resuspended in formamide/dye mixture
and resolved on an 8% denaturing polyacrylamide gel. Following
autoradiography to visualize the repair products, the level of excision
was quantitated by PhosphorImager analysis. For unmodified DNA, 5-µl
aliquots were withdrawn from the 25-µl reaction mixture such that
each time point was represented by ~0.3 fmol of DNA.
A repair-proficient
(wild-type) Chinese hamster ovary cell line, K1, was used to assay
excision in a mammalian system. Cells were grown to exponential phase
as a suspension culture in Eagle's minimum essential medium
supplemented with 10% fetal bovine serum. Cells were harvested and
cell-free extract (CFE) was prepared as described (24) and stored at
80 °C in buffer that was 25 mM Hepes, pH 7.9, 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM dithiothreitol, and 12.5%
glycerol (v/v). The CFE concentration was 12.1 mg/ml, and the extract
was stable for at least two cycles of thawing and refreezing.
Using an internally labeled DNA
substrate (at the fifth phosphodiester bond 5 to the PdG residue or at
the eleventh bond 5
to T<>T), this assay detects the excised
fragment resulting from both 5
and 3
incisions. The reaction mixtures
contained 25 fmol of radiolabeled DNA, 121 µg of CFE, and 70 ng of
pBR322 in 50 µl of buffer that was 35 mM Hepes, pH 7.9, 10 mM Tris, pH 7.5, 60 mM KCl, 40 mM NaCl, 5.6 mM MgCl2, 0.4 mM EDTA, 0.8 mM dithiothreitol, 2 mM ATP, and 3.2% glycerol with 20 µM each of dATP, dCTP, dGTP, and TTP, and bovine serum albumin at 0.2 mg/ml. The
reaction was carried out at 30 °C for 90 min with 10 µl of the
mixture (~5 fmol DNA) being removed at 15, 30, 60, and 90 min. DNA
was deproteinized with proteinase K (0.2 mg/ml for 15 min at 37 °C)
followed by phenol, phenol:chloroform, and ether extractions then
precipitated with ethanol in the presence of 20 µg of oyster
glycogen. Recovered DNA was resuspended in formamide/dye mixture and
resolved on a 10% denaturing polyacrylamide gel. Following autoradiography to visualize the repair products, the level of excision
(oligomers in the 23-30-nt range) was quantitated by scanning the
dried gel with an AMBIS systems scanner.
Recombinant M13 genomes containing a site-specifically positioned
PdG adduct were constructed by ligation of the PdG 8-mer (5-GGT(PdG)TCCG-3
) into a gapped duplex DNA molecule containing uracil in the (+)-strand and an 8-base gap in the (
)-strand (7). Control genomes were constructed by ligation of the unmodified 8-mer.
The oligonucleotides used for genome construction were purified by
reversed phase HPLC and were judged greater than 99.3% pure by HPLC
and denaturing polyacrylamide gel electrophoresis of
32P-labeled material. The structures of the
oligonucleotides were verified by high field NMR analysis and by HPLC
analysis of the deoxynucleosides released by nuclease digestion. The
fully ligated genomes were isolated from the remaining starting
material and incompletely ligated duplexes by agarose gel
electrophoresis in the presence of 0.5 µg/ml ethidium bromide (6).
Purified, fully ligated recombinant M13MB102 DNA was electroporated
into the SOS-induced competent E. coli strains. Following
overnight growth and elution of the entire plaque population from
primary transformations, the eluate was diluted and replated on
X-gal/isopropyl-
-D-thiogalactoside indicator plates to
give approximately 1,500 plaques/plate (6). These secondary plates were
used in the hybridization assay (7). Elution of the primary plates
enabled us to standardize the number of plaques/plate for the
hybridization assay, as well as allowed screening of a large number of
the transformants that were produced during electroporation. The
mutation frequency was not significantly different when the DNA was
screened for mutations after either the primary or secondary plating
(data not shown). The base pair substitution mutation frequency was
determined by screening for transitions and transversions at position
6256 by differential hybridization with 13-mer probes (7). Recombinant
single-stranded M13MB102 DNAs containing each of the four possible
bases at the adduct site were hybridized in parallel as assay standards
(7).
Table I lists the mutation
frequencies determined following electroporation of PdG-containing M13
genomes into strains deficient in nucleotide excision repair or one or
both of the 3-methyladenine glycosylase genes. The base pair
substitution mutation frequencies induced by the PdG adduct in M13MB102
replicated in LM102 and LM101 were similar to the mutation frequency
previously reported in JM105 (7). LM102 and LM101 are wild-type for DNA
repair. When PdG-adducted M13MB102 was transformed into LM103, there
was approximately a 4-fold increase in both PdG A transitions as well as the PdG
C and PdG
T transversions at position 6256 (Table I). The total mutation frequency induced in this strain was
11.6% as compared with the overall mutation frequency of 3.1% in the
wild-type strain LM102. In contrast, no differences in mutation
frequencies were detected when PdG-containing genomes were
electroporated into strains deficient in one or both of the two genes
that code for 3-methyladenine glycosylase activity (LM108, LM109, and
LM110).
|
In these initial in vivo experiments, uracil residues were
randomly incorporated into the (+)-strand of the recombinant M13 genomes to minimize replication of the nonadducted strand. However, we
have recently found that a significant amount of replication of the
adducted genomes occurs in which the (+)-strand is used as the template
(20). This accounts for the high percentage of progeny phage that
contain G at position 6256 because the (+)-strand contained a C residue
at this position. It is possible to estimate the percentages of
replication that occur from the ()- and (+)-strands by placing T
residues opposite the adduct in the recombinant genomes. Utilization of
the (+)-strand as template leads to the incorporation of A residues at
position 6256, whereas utilization of the (
)-strand as template leads
to incorporation of whatever bases are inserted opposite PdG. For the
experiments described below, recombinant M13 genomes were constructed
that did not contain uracil residues in the (+)-strand. This
reduces the apparent mutation frequency at position 6256 by
approximately 50% (20).
M13MB102 genomes that contained G:T or PdG:T at position 6256 were
electroporated into a wild-type strain (JM105), a
recA strain (LM104), and two nucleotide
excision repair-deficient strains (LM103 and NR10148), and the
identities of the bases incorporated were probed by hybridization
analysis (Table II). For nonadducted genomes, the
(
)-strand was used as template approximately 90% of the time, and no
G
T transversions were detected within the limit of sensitivity of
the assay (~0.1%). When PdG was present in the (
)-strand, most of
the replication occurred from the nonadducted (+)-strand. The total
amount of replication from the (
)-strand was estimated by summing the
frequencies of incorporation of G, T, and C and assuming that A was
incorporated at the same frequency as T. The assumption of equal
frequencies of PdG
A and PdG
T mutations was based on the
results obtained with M13MB102 containing PdG:C at position 6256 (Table
I). The remainder of the incorporation of A during replication of
M13MB102 containing PdG:T at position 6256 was from utilization of the
(+)-strand as template, and this clearly represented the majority of
the replication events.
|
The PdG-containing ()-strand was used as template approximately 6%
of the time in JM105 and 4% of the time in LM104. The concordance of
these results indicates that the shift in template utilization induced
by PdG was not the result of recA-mediated strand switching. Rather, it
arose from complete replication of the (+)-strand or synthesis using
the (+)-strand as template following repair of PdG. The significant
reduction in mutation frequency observed in LM104 supports prior
findings that mutagenesis by PdG is enhanced by induction of the SOS
response (7, 20).
The contribution of nucleotide excision repair to replication of the
(+)-strand was estimated by comparing JM105 with LM103 and NR10148. The
PdG-adducted strand was used as template 21% of the time in the
uvrA strain LM103. The relative frequency of
(
)-strand utilization in JM105 and LM103 (3.5-fold) was comparable
with the ratio of mutation frequencies measured with LM102 and LM103
(3.7-fold) (Table I). Because JM105 is a wild-type strain with regard
to nucleotide excision repair, whereas LM103 is uvrA
,
these results are consistent with a role for nucleotide excision repair
in removing PdG residues. Removal of PdG in JM105 and LM102 by
nucleotide excision repair would lead to increased incorporation of A
residues during repair synthesis because of the presence of T at
position 6256 on the (+)-strand.
Although the frequency of replication of PdG was higher in the
uvrA cells, the mutation frequency was similar to that
observed in wild-type cells when corrected for template utilization.
26% of the (
)-strand replication events led to mutations in JM105,
and 38% led to mutations in LM103. Electroporation of PdG-containing M13MB102 into NR10148, which is uvrB
, produced
frequencies of template utilization (18%) and mutations (39%)
comparable with those observed in LM103. Thus, mutations in two
different genes of nucleotide excision repair led to similar effects on
replication of PdG-containing M13 genomes in
vivo.2
Results from the in vivo
mutagenesis studies suggested that the Uvr(A)BC excinuclease is
involved in the repair of the PdG adduct in E. coli because
the absence of the uvrA or uvrB genes increased
the frequency of mutations induced by PdG. Repair of the PdG adduct by
Uvr(A)BC excinuclease was further investigated by use of an in
vitro assay in which duplex DNA containing the PdG adduct was
incubated with purified Uvr(A)BC repair enzymes to determine if
excision occurred. The substrate for these in vitro
experiments was a 156-mer duplex DNA bearing PdG near the center of the
duplex. It was constructed by ligating a 5-labeled 12-mer containing
the PdG adduct at position 4, with seven other oligomers to obtain a
156-mer duplex (21). The 12-mers were purified by HPLC as described
above for the 8-mers used for recombinant genome construction. The
fully ligated DNA was purified from the partially ligated product by
running the ligation mixture on a polyacrylamide gel and excising the
band corresponding to the 156-mer. The Uvr(A)BC excinuclease repairs
damaged DNA by excision of a 12-nucleotide fragment of DNA by
hydrolysis of the eighth phosphodiester bond 5
and the fourth
phosphodiester bond 3
to the adducted nucleotide (25, 26). By
phosphorylating the oligonucleotide containing the adduct with
32P prior to ligation, we were able to determine the extent
of excision by the appearance of a 12-nucleotide band on an
autoradiogram of a polyacrylamide gel following incubation with the
repair enzymes.
A 12-nucleotide fragment of DNA was excised when the 156-mer substrate
adducted with PdG was incubated with the Uvr(A)BC excinuclease. The
extent of excision increased linearly up to 40 min, at which point it
plateaued (Fig. 2). In addition to the 12-mer product, a
band corresponding to a 74-mer was detected, which represents uncoupled
excision at only the 3 end of the duplex. There was no excision in the
samples incubated with boiled enzyme or the samples terminated
immediately after addition of the enzyme. DNA that did not contain an
adduct was also not excised by these enzymes (27). Approximately 1% of
the PdG-containing 156-mer substrate was cut by the excinuclease. A
45-mer substrate containing a psoralen mono adduct was used as a
positive control for these experiments, because psoralen is excised
very efficiently by the Uvr(A)BC excinuclease (27). Approximately 50%
of the psoralen-adducted DNA was cut. Thus, by comparison with
psoralen, PdG appears to be a relatively poor substrate for the
E. coli Uvr(A)BC excinuclease.
The ability of a mammalian nucleotide excinuclease to excise the PdG
adduct was also determined. The mammalian excinuclease hydrolyzes the
fifth phosphodiester bond 3 and twenty-second to twenty-fourth
phosphodiester bond 5
to the lesion, releasing a 27-29-nucleotide
fragment containing the adduct (28). When the PdG-modified 156-mer was
incubated with a Chinese hamster ovary CFE, 23-28-mer fragments were
released (Fig. 3, lane 2-4) that shortened
with time due to nuclease action. Parallel experiments were conducted
with a 140-mer containing a thymine-thymine cyclobutane dimer (Fig. 3,
lanes 3-8). The extent of cleavage of the cyclobutane dimer-containing duplex was approximately the same as the
PdG-containing duplex. Thus, PdG appears to be a much better substrate
for the mammalian excinuclease than for the bacterial excinuclease.
Three separate lines of evidence suggest that PdG is a substrate for nucleotide excision repair. First, the frequency of targeted base pair substitution mutations induced by PdG is higher in nucleotide excision repair-deficient strains than in wild-type strains in vivo. Second, the extent of PdG-containing template utilization is higher in nucleotide excision repair-deficient strains than in wild-type strains in vivo. Third, oligonucleotides of the expected size are removed from a PdG-containing 156-mer following incubation with reconstituted E. coli or hamster excinuclease complexes in vitro.
The studies of template utilization summarized in Table II are
particularly informative in that they reveal a dramatic switch in
template utilization when PdG is incorporated into M13 genomes. The low
percentage of ()-strand replication indicates that PdG is either a
strong block to replication or is very efficiently repaired. Moriya
et al. have shown that PdG is an effective block to
replication when incorporated into single-stranded vectors (8).
Incorporation of adducts that are strong blocks to replication into the
(
)-strand of M13MB102 could lead to an apparent shift in template
utilization because of the consecutive rather than bidirectional
mechanism of strand replication employed by M13 genomes (29). Once the
lesion is bypassed during the initial stage of DNA replication, the
(+)-strand released can be rapidly copied to a duplex. The (
)-strand
of this newly synthesized duplex will dominate viral DNA synthesis
because the duplex produced in the initial phase of genome replication
still contains the adduct in the (
)-strand.
The data in Table II indicate that a portion of the utilization of the
(+)-strand as template occurs during DNA synthesis subsequent to
nucleotide excision repair because replication of the adducted
()-strand increases in cells that are deficient in either the
uvrA or uvrB genes. This shift in template
utilization masks the mutagenic potency displayed by PdG in duplex
vectors. For example, the percentage of mutations induced by PdG in
M13MB102 increases from approximately 1.5 to 28% when correction is
made for the strand that is actually used as the template. The
magnitude of the increase in the use of the adduct-containing strand as template is approximately the same as the magnitude of the increase in
the mutation frequency. However, even in nucleotide excision repair-deficient cells, the nonadducted strand is still the preferred template for M13MB102 replication. This is likely due to replication blockade by the PdG residue on the (
)-strand, but we cannot rule out
the possibility that another repair pathway such as base excision repair contributes to the utilization of the (+)-strand as template. Interestingly, deletion of the 3-methyladenine glycosylase gene (alkA), which has been reported to participate in the repair
of etheno adducts to deoxyguanosine and deoxyadenosine residues
(12-15), does not increase the mutation frequency measured following
in vivo replication of PdG-adducted M13MB102.
Repair of PdG was further studied in an in vitro excision assay in which a 156-base pair substrate containing PdG was incubated with either the purified E. coli enzymes or a mammalian CFE. Removal of the adduct was detected by the appearance of the excised oligonucleotide fragments when reaction mixtures were analyzed by polyacrylamide gel electrophoresis. The E. coli enzymes excised approximately 1% of the adducted substrate. This low level of in vitro excision is similar to the amounts detected with other small adducts such as O6-methylguanine and DNA mismatches (27). This is considerably less than the extent of excision of larger adducts such as psoralen- or aflatoxin-derived adducts or thymine dimers (27, 30, 31). However, in contrast to excision by the E. coli excinuclease, PdG is a good substrate for removal by a mammalian excinuclease complex. The extent of excision of PdG-containing oligonucleotides by a hamster CFE is approximately the same as the extent of excision of thymine dimer-containing oligonucleotides, which are classic substrates for mammalian nucleotide excision repair.
The present findings establish that PdG is repaired by bacterial and mammalian nucleotide excision repair complexes. Although PdG is a relatively poor substrate for the bacterial excinuclease in vitro, the higher frequency of PdG-induced mutations in excision repair-deficient strains of E. coli indicates that repair by Uvr(A)BC is important in vivo. PdG appears to be a good substrate for repair by the mammalian excinuclease. This is interesting because several exocyclic deoxyguanosine adducts, including some hydroxy-PdGs, have been detected in DNA from healthy human beings (3, 4, 32). The levels of adducts detected in DNA reflect the steady-state balance between adduct generation and adduct removal, so if these exocyclic adducts are removed as efficiently as PdG by nucleotide excision repair, their contribution to endogenous DNA damage is significantly higher than one would estimate by determination of absolute adduct levels.
We are grateful to D. S. Hsu, D. Mu, J. T. Reardon, and A. Sancar for assistance in the execution of the in vitro repair assays and for helpful discussions.