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J. Biol. Chem., Vol. 275, Issue 42, 32635-32641, October 20, 2000
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From the Department of Molecular Genetics, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, 980-8575 Japan
Received for publication, May 12, 2000, and in revised form, July 25, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Although single-strand breaks (SSBs) occur
frequently, the cellular responses and repair of SSB are not well
understood. To address this, we established mammalian cell lines
expressing Neurospora crassa UV damage endonuclease (UVDE),
which introduces a SSB with a 3'-OH immediately 5' to UV-induced
cyclobutane pyrimidine dimers or 6-4 photoproducts and
initiates an alternative excision repair process. Xeroderma pigmentosum
group A cells expressing UVDE show UV resistance of almost the
wild-type level. In these cells SSBs are produced upon UV irradiation
and then efficiently repaired. The repair patch size is about seven
nucleotides, and repair synthesis is decreased to 30% by aphidicolin,
suggesting the involvement of a DNA polymerase
DNA single-strand breaks
(SSBs)1 are frequently
produced by environmental genotoxic agents and by endogenous cellular
reactions. SSBs cause double-strand breaks when replication forks
encounter SSBs and, thus, result in chromosomal rearrangements and
instability (1). Despite the potentially harmful effects of SSBs,
however, little is known about the details of the repair mechanisms and cellular responses to SSBs in mammalian cells. This may be due to the
experimental difficulty to produce SSBs alone. Genotoxic agents that
produce SSBs (ionizing radiation, oxidizing agents, and alkylating
agents) generate a variety of DNA lesions (2). For instance, ionizing
radiation and bleomycin produce not only SSBs but also base lesions and
double-strand breaks (2, 3).
One of the immediate responses to SSBs in mammalian cells is thought to
be the activation of poly(ADP-ribose) polymerase (PARP). PARP binds to
SSBs and is activated (4). Although PARP has been considered to be
involved in the repair of SSBs, especially in replicating cells (4),
its precise role is still not well understood. Another player in the
response to SSBs may be XRCC1, which binds to DNA ligase III and DNA
polymerase In addition to nucleotide excision repair (NER) for UV-induced DNA
damage, the filamentous fungus, Neurospora crassa, and the
fission yeast Schizosaccharomyces pombe, possess an
alternative excision repair mechanism, which is initiated by an
endonuclease called UV damage endonuclease (UVDE) and is referred to as
UVDE-initiated excision repair (9-14). UVDE introduces a SSB
immediately 5' to UV-induced cyclobutane pyrimidine dimers (CPDs) and
6-4 photoproducts, leaving 3'-hydroxyl and 5'-phosphoryl groups at the
site of cleavage (9-11). Until now UVDE has been found only in some
eukaryotic microorganisms and in some bacteria including Bacilus
subtilis (9) and Dienococcus radiodurans (15), but
neither a similar enzymatic activity nor any homologous genes have been
found in mammalian cells.
To understand the cellular responses and repair of SSBs in mammalian
cells, we have made use of UVDE. We introduced the N. crassa
UVDE gene into a human and a Chinese hamster ovary (CHO) cell lines and
analyzed the responses of the transfected cells to UV. Using these
unique systems, we found the following. 1) Judging from the UV
resistance of xeroderma pigmentosum group A (XPA) cell line expressing
UVDE, UVDE-initiated alternative excision repair in human cells works
almost as efficiently as NER. 2) The UVDE-initiated repair is mediated
mainly by aphidicolin-sensitive DNA polymerase(s), and the repair patch
size is about seven nucleotides. 3) XRCC1 and PARP cooperate and
contribute to cell survival after SSBs are produced.
Cell Lines, Vectors, and Transfection--
A human cell line
derived from an XPA patient, XP12ROSV, was obtained from Dr. K. Tanaka
(Osaka University) and used as the host cell for complementation of UV
sensitivity by the introduced UVDE gene. The CHO cell line EM9 was
purchased from the American Type Culture Collection. Plasmid pCY4B, a
derivative of the one described by Niwa et al. (16),
contains a chicken Immunoblot Analysis--
Whole cell extracts were prepared from
cultured cells by homogenizing cell pellets in a lysis buffer (50 mM Tris-HCl (pH 7.5), 0.3 M KCl, 0.05% Nonidet
P-40, 2 mM dithiothreitol) containing protease inhibitors
(protease inhibitor mixture tablets, Roche Molecular Biochemicals).
After centrifugation of the homogenate at 100,000 × g,
supernatants were recovered and used for immunoblot analysis as well as
for the incision assay described below. Western analysis was done by
standard methods using whole cell extracts (20 µg of protein of
each), fluorotrans membrane (PALL Gelman Laboratory), and an antibody
raised against N. crassa UVDE (18) (1:500 dilution). Immune
complexes were detected by using an ECL plus Western blotting detection
system (Amersham Pharmacia Biotech).
For detection of poly(ADP-ribose) (pADPr), cells were harvested from
35-mm culture dishes just before reaching confluence. Cells were washed
with phosphate-buffered saline (PBS) and irradiated with 2.5, 7.5, and
20 J/m2 UV. After UV irradiation, cells were incubated for
various periods of time at room temperature. After incubation, cell
extracts were prepared by adding 400 µl of SDS-polyacrylamide gel
electrophoresis sample buffer (1% SDS, 1% In Vitro Incision Assay--
Nicking activity of UVDE was
measured as described (18). The whole cell extracts (40 µg of protein
of each), and the synthetic oligonucleotides containing CPD were used.
UV Survival--
5 × 103 exponentially growing
XPA cells were plated per 100-mm culture dish (9.3 × 102 cells per 60-mm dish for CHO cells) and incubated in
culture medium. 9 h after plating, cells were washed with Hanks'
solution (Nissui) and irradiated with UV at various doses. The cells
were then cultured for 12 days (8 days for CHO cells) in either the regular medium or medium containing 2 mM 3AB.
Measurement of Repair Synthesis Using Autoradiography--
Cells
grown on glass microscope slides in culture dishes were irradiated with
7.5 J/m2 and 15 J/m2 UV and incubated at
37 °C for 1 h in medium supplemented with 10 µCi/ml of
[3H]dThd (Amersham Pharmacia Biotech). The dishes were
then washed with cold PBS. The cells on the microscope slides were
fixed with cold 5% trichloroacetic acid, washed with ethanol, and
dried at room temperature. The slides were dipped in NR-M2 emulsion
(Konica, diluted 1:1 with H2O), dried, and exposed for 7 days at 4 °C in a light-tight plastic box. The slides were then
developed and counter-stained with 3% Giemsa stain (Merck). About 150 nuclei with fewer than 100 silver grains were counted for each slide. The results were shown as a percentage histogram for each slide. Repair
synthesis was taken to be the difference between the percentage values
of corresponding irradiated and non-irradiated samples.
Measurement of DNA Repair Synthesis by BrdUrd-induced Density
Shift--
DNA repair synthesis was measured essentially as described
by Smith et al. (19). XPA[UVDE] cells were grown in 150-mm
culture dishes in medium containing 0.4 µCi/ml
[32P]orthophosphate (Amersham Pharmacia Biotech) for 5 days and then subcultured in non-radioactive medium for 2 days before
to UV irradiation. Then the cells were incubated for 2 h in the
medium with FdUrd (1 µM, Sigma), BrdUrd (10 µM, Sigma), and hydroxyurea (2.5 mM, Sigma),
washed twice with PBS, and irradiated with 20 J/m2 and 40 J/m2 UV. Cells were then incubated for 3 h in medium
containing FdUrd, BrdUrd, hydroxyurea, and 5 µCi/ml
[3H]dThd supplemented or not with 10 µg/ml aphidicolin.
Cells were then washed with PBS and lysed as described by Smith
et al. (19). DNA solutions thus obtained were subjected to
neutral CsCl gradient sedimentation and fractionated, then assayed for
radioactivity. The fractions containing unreplicated (parental-density)
DNA were pooled and used for alkaline CsCl gradient sedimentation.
Repair synthesis was taken to be specific incorporation
(3H/32P) in the parental-density DNA of the
alkaline rebanding. The ratio of repair synthesis sensitive to
aphidicolin was calculated as (repair synthesis in the presence of
aphidicolin/repair synthesis in the absence of aphidicolin).
Analysis of Patch Size by BrdUrd-induced Density
Shift--
Repair patch size was also measured as described by Smith
et al. (19). XPA[UVDE] cells were prelabeled with
32P, incubated in medium containing FdUrd, BrdUrd, and
hydroxyurea for 2 h, and irradiated with 20 J/m2 UV as
described above. Cells were then incubated in medium containing FdUrd,
BrdUrd, hydroxyurea, and [3H]dThd for 3 h, then
lysed. The parental-density DNA was purified by two successive neutral
CsCl gradient sedimentation processes. Two kinds of DNA were prepared
for use as markers as follows. 32P-Prelabeled DNA was
prepared from cells immediately before irradiation with UV. Fully
BrUra-substituted hybrid DNA was prepared from unirradiated cells that
were incubated for 3 h in medium containing FdUrd, BrdUrd, and
[3H]dThd.
The isolated parental-density DNA was sonicated. The size distribution
of the fragments of the sonicated DNA was determined by electrophoresis
in a denaturing polyacrylamide gel as described (20). From the data,
the number-average molecular size of the fragments was calculated as
described (21). The size of the repair patches was measured in alkaline
CsCl gradients using the parental-density DNA sonicated to a
number-average molecular size of 188 nucleotides. The gradients were
fractionated, and the radioactivity of each fraction was measured. The
repair patch size was calculated as follows. First, the distance
between the 32P-prelabeled DNA distribution and that of the
3H-repair label distribution was measured. This distance
was then compared with the separation between the peak of the
32P-prelabeled DNA and that of the 3H-labeled
fully BrUra-substituted DNA, which was determined from a separate
analysis of DNA markers in a similar gradient. The ratio of these two
distances was then multiplied by the average fragment size to give the
average repair patch size.
In another experiment, [3H]BrdUrd (Moravek) was used as
the isotopic label in place of [3H]dThd. Hydroxyurea was
not used. The number-average molecular size of the sonicated
parental-density DNA was 211 nucleotides in this case, and the
experiment was performed in the same way as above.
Alkaline Gel Analysis--
The relative number of SSBs in the
genomic DNA from cells collected at various periods of time after UV
irradiation was determined by using alkaline gel analysis. Briefly,
cells were prelabeled with [32P]orthophosphate, washed
with Hanks' solution, and irradiated or unirradiated with 20 J/m2 UV. Cells were incubated in medium for various periods
at 37 °C. In a separate experimental series, measurements over a
smaller time scale from 0.5 min to 40 min were done. In this case,
after UV irradiation, cells were incubated in Hanks' solution at room temperature. At appropriate periods of time, cells were lysed by
incubating in 0.5% SDS, 100 µg/ml proteinase K (Wako), 10 mM Tris, 1 mM EDTA (pH 8) at 37 °C
overnight. Genomic DNA was isolated by phenol/chloroform extraction and
ethanol precipitation. Each 5-µg DNA sample was mixed with alkaline
loading buffer (22) and electrophoresed in a 3.5% alkaline-agarose
(Agarose H, Wako) gel. A set of 5'-end 32P-labeled DNA
fragments (Marker 8GT; Nippon Gene) was prepared by using T4
polynucleotide kinase (Takara) and [ Establishment of XPA Cell Line Expressing cDNA of Neurospora
UVDE and Nicking Activity of the Cell Extracts--
cDNA of
N. crassa UVDE was introduced behind the chicken Survival of XPA Transfectants--
The colony-forming ability of
the XPA transfectants after UV irradiation was assessed. XPA[Vector]
cells were extremely sensitive to UV irradiation, whereas XPA[cXPA]
cells exhibited UV resistance (Fig. 2).
XPA[UVDE] cells showed almost the same level of UV resistance as
XPA[cXPA] cells at low UV doses (Fig. 2). As the UV dose increased, XPA[cXPA] cells became more UV-resistant than XPA[UVDE] cells (Fig.
2). Thus, the alternative excision repair found in eukaryotic microorganisms provided NER-deficient human cells with UV resistance of
almost wild-type level. Now the question is how UV-induced DNA damage
is repaired in these cells.
SSBs in Genomic DNA of XPA[UVDE] Cell after UV
Irradiation--
To examine whether SSBs are actually produced after
UV irradiation in XPA[UVDE] cells, we conducted alkaline gel
electrophoresis analysis of the genomic DNA of UV-irradiated
XPA[UVDE] cells. After 20 J/m2 UV irradiation, cells were
incubated in buffer for various periods of time before genomic DNA was
isolated and electrophoresed on an alkaline-agarose gel. Unirradiated
DNA migrated as a discrete band near the origin, whereas the DNA
isolated after UV irradiation showed a broad smeared band on the gel
(Fig. 3A). These results indicate that the SSBs are actually produced by UVDE in intact XPA[UVDE] cells immediately after UV irradiation. To quantify the
SSBs, we measured the amount of DNA between 5.6- and
14.3-kilobase DNA. The amount of smeared DNA gradually increased
up to 40 min after UV irradiation and reached a plateau level (Fig.
3A). Two hours after UV irradiation, the amount of the SSBs
was significantly decreased (Fig. 3B), indicating repair of
SSBs. Under the conditions used, no significant difference in the
extent of the smear was observed in XPA[Vector] and XPA[cXPA] cells
(Fig. 3B). These data suggest that in XPA[UVDE], SSBs are
produced by UVDE, which initiates an alternative excision repair in
human cells.
Repair Process for SSBs Produced by UVDE in XPA[UVDE]
Cells--
First we characterized the repair synthesis in XPA[UVDE]
cells after UV irradiation. Unscheduled DNA synthesis after UV
irradiation was observed in XPA[UVDE] cells but not in XPA[Vector]
cells (Fig. 4). The unscheduled DNA
synthesis in XPA[UVDE] was slightly less than unscheduled DNA
synthesis determined in HeLa cells and increased with UV doses (Fig.
4). We next examined the sensitivity of the repair synthesis to
aphidicolin, a specific inhibitor of DNA polymerase Estimation of Repair Patch Size in XPA[UVDE] Cells--
The
measurement of repair patch size is an extension of the method used to
measure the repair synthesis. 32P-Prelabeled XPA[UVDE]
cells were irradiated with 20 J/m2 UV and incubated in
medium containing [3H]dThd, hydroxyurea, and BrdUrd for
3 h. Parental-density DNA was isolated by two successive processes
of neutral CsCl gradient sedimentation. This DNA was sonicated to an
average size of 188 nucleotides and then centrifuged to equilibrium in
alkaline CsCl gradients. Under these conditions, the increase in
density of DNA fragments that contain repair patches (synthesized in
the presence of BrdUrd) is large enough to be measured and can be compared with the increase in density of DNA completely substituted with BrUra. Gradients were fractionated, and the radioactivity profiles
of 3H and 32P were determined (Fig.
5). The density of the DNA molecules
containing repair patches (shown by the profile of 3H) was
clearly larger than that of bulk genomic DNA (shown by the profile of
32P) (Fig. 5). Based on the shift between the profiles of
3H and 32P and referring to the position of
fully BrUra-substituted DNA, the patch size was determined as 8 ± 2 nucleotides. In the second experiment we did not add hydroxyurea, and
[3H]BrdUrd was used as the isotopic label. In this case,
the patch size was determined as 7 ± 2 nucleotides (radioactivity
profiles were not shown).
Involvement of PARP Activation in the UV Resistance of
XPA[UVDE]--
We further investigated whether PARP and XRCC1 are
involved in the repair process. In the presence of 3AB, a widely used
inhibitor of PARP, enhanced lethality after UV irradiation was observed in XPA[UVDE] cells (Fig. 2). By contrast, in XPA[Vector] cells and
XPA[cXPA] cells, no significant increase in sensitivity to UV was
observed in the presence of 3AB (Fig. 2). These results demonstrate the
involvement of PARP in the repair of the SSBs introduced by UVDE. By
immunoblot analysis with monoclonal antibody to pADPr, we examined
whether the activation of PARP occurs in XPA[UVDE] in response to
UVDE-induced SSBs. Thirty seconds after 20 J/m2 UV
irradiation in XPA[UVDE] cells, a significant amount of pADPr was
synthesized (Fig. 6). This is consistent
with the result that the SSBs had already been introduced in the
genomic DNA of XPA[UVDE] after 30 s of 20 J/m2 UV
irradiation (Fig. 3A). A peak for the poly(ADP-ribosyl)ation of cellular proteins was found only 2 min after UV irradiation in the
cells. After 10 min, no significant pADPr was observed (Fig.
6A) even in the presence of considerable SSBs at this time (Fig. 3). Under the conditions used, no significant pADPr was detected
in XPA[cXPA] cells (Fig. 6A) and XPA[Vector] cells (data not shown) until 10 min after UV irradiation. In the presence of 3AB,
the inhibition of pADPr synthesis in XPA[UVDE] cells occurred (Fig.
6A). Thus, these results indicate that PARP is activated in
XPA[UVDE] cells in response to the SSBs produced by UVDE and the
activation of PARP is transient in the case of irradiation with high
doses of UV. The amount of pADPr synthesis in XPA[UVDE] cells is
dependent on UV dose, and 7.5 J/m2 irradiation was
necessary to detect pADPr synthesis in our assay (Fig.
6B).
Cell Survival of EM9 Transfectants--
Since XRCC1 is thought to
be involved in the processing of SSBs, we investigated whether XRCC1 is
actually necessary for repair of SSBs introduced by UVDE. The
Neurospora UVDE gene was introduced into the CHO cell line
EM9, which is mutated in the XRCC1 gene (23). The obtained transfectant
was designated as EM9[UVDE]. Anti-Neurospora UVDE antibody
detected UVDE expression in EM9[UVDE] cells (data not shown). The
colony-forming ability of the CHO transfectants after UV irradiation
was assessed. EM9[UVDE] cells were much more sensitive to UV than
EM9[Vector] cells (Fig. 7). This
indicates the involvement of XRCC1 in the repair of UVDE-introduced SSBs. In the presence of 3AB, only a very slight increase in
sensitivity to UV was observed in EM9[UVDE] cells (Fig. 7). Thus, the
inhibition of PARP activation does not influence the survival of EM9
cells, which lack active XRCC1 protein.
We established mammalian cell lines, which enabled us to examine
the repair characteristics and cellular responses of SSBs produced by a
foreign UV endonuclease, UVDE. UVDE introduces a nick immediately 5' to
UV-induced CPDs and 6-4 photoproducts and initiates an alternative
excision repair in several eukaryotic and prokaryotic microorganisms.
The first interesting question was how much of the UV sensitivity of
NER-deficient human cells is complemented by the expressed UVDE. We
previously found that UVDE-initiated repair is a rapid global genome
repair and is effective for most UV-induced DNA damage before NER
occurs in S. pombe cells (24). As shown in Fig. 2,
NER-deficient human host cells acquired the UV resistance of the
wild-type level. This is the first example of an extensive
complementation of UV sensitivity in NER-deficient human cells by a
foreign repair protein.
As previously reported, pyrimidine dimer DNA glycosylase only partially
complements the UV sensitivity of NER-deficient human cells (25), far
less than UVDE shown here. This different efficiency of complementation
between UVDE and pyrimidine dimer DNA glycosylase is explained by the
different structures of SSBs produced by the endonucleases. UVDE
produces 3'-OH ends, which are common intermediates during DNA
replication, repair, and recombination processes and constitute
appropriate primer terminus for DNA polymerases. However, 3'-unsaturated aldehyde termini produced by pyrimidine dimer DNA glycosylases have to be removed before repair synthesis. Another explanation is that pyrimidine dimer DNA glycosylase repairs only CPDs,
whereas UVDE can repair both CPDs and 6-4 photoproducts (14). Only at
higher UV doses were XPA[cXPA] cells more UV-resistant than
XPA[UVDE]. This difference may be due to the lack of transcription coupling in UVDE-initiated repair or due to the increase of UV-induced DNA lesions other than CPDs or 6-4 photoproducts, which are not recognized by UVDE but repaired by NER. This may also be due to the
incomplete processing of the SSBs excessively produced by UVDE during
the short time span. We showed that a large number of SSBs are
introduced in XPA[UVDE] after irradiation with a high dose of UV
within half a minute, and considerable SSBs remain to be repaired even
2 h after irradiation (Fig. 3).
The next interesting question is how the UVDE-initiated repair proceeds
in human cells. The repair synthesis was shown to be mostly dependent
on aphidicolin-sensitive DNA polymerase, and the determined patch size
of the repair was about seven nucleotides. Since the repair patch size
of the proliferating cell nuclear antigen-dependent pathway
of BER has been reported as between 7 and 14 nucleotides (26), or less
than 10 nucleotides in length (27, 28), the patch size for
UVDE-initiated excision repair fits in reasonably well with that of the
BER pathway. It has also been reported that the repair synthesis of the
proliferating cell nuclear antigen-dependent pathway is not
catalyzed by DNA polymerase The third interesting question about UVDE-initiated repair of UV damage
concerns the cellular responses to the induced SSBs in mammalian cells.
Western blot analysis showed that immediately after irradiation with a
high dose of UV, cellular proteins were poly(ADP-ribosyl)ated in
XPA[UVDE] cells (Fig. 6). This is in contrast to the response in
XPA[cXPA] cells, which showed no significant synthesis of pADPr (Fig.
6A). By adding 3AB, a competitive inhibitor of PARP, to
XPA[UVDE] cells, pADPr synthesis was suppressed (Fig. 6A).
These results give additional clear evidence for PARP activation by
SSBs in human cells. 10 min after UV irradiation, pADPr was no longer
observed (Fig. 6A). This is explained by reports that, after
excessive activation of PARP, pADPr has a short half-life close to 1 min (34), and the levels of NAD, which is a substrate of PARP, are
depleted (4).
We have shown here that 3AB enhanced the UV lethality of XPA[UVDE],
whereas 3AB did not make any significant difference to survival in
XPA[cXPA] cells, indicating the involvement of PARP in the repair of
the SSBs. These results are consistent with reports that cells treated
with alkylating agents and x-rays, which are known to produce SSBs in
cells, are sensitive to 3AB (4). The lethal effect of 3AB on cells
treated with these agents is known to be maximal in S phase (35, 36),
suggesting that PARP is a survival factor playing an essential role
during recovery from SSBs in S phase. PARP is known to interact
directly with XRCC1 (7). It has been reported that S-phase-specific
repair of SSBs mediated by XRCC1 is indispensable for resistance to
alkylating agents in CHO cells (6). Therefore, to link the effect of
3AB on XRCC1, we introduced the UVDE gene into a CHO cell line, EM9, that is defective in the XRCC1 gene. We showed that EM9 cells expressing UVDE (EM9[UVDE]) are extremely sensitive to UV, indicating the involvement of XRCC1 in repair of the SSBs (Fig. 7). The addition of 3AB results in almost no increase in the UV sensitivity of EM9[UVDE] (Fig. 7). This result suggests that PARP and XRCC1 play essential roles in the same pathway, probably in the same
S-phase-specific recovery pathway for SSBs. It has been shown that
nuclear foci of XRCC1 co-localize with Rad51 (6). Thus, PARP and XRCC1
may function in concert with a homologous recombination pathway in the
processing of SSBs as well as double-strand breaks, which are produced
from SSBs during the replication process. Recent molecular and genetic
analyses of repair-deficient strains from various organisms suggest
that SSBs are one of the major risk factors for genome instability
induced by oxidative DNA damage. We consider that UVDE-expressing cell
lines offer a unique experimental system for the analysis of the
cellular response to SSBs in mammalian cells.
/
-dependent long-patch repair. Immediately after UV
irradiation, cellular proteins are poly(ADP-ribosyl)ated. The UV
resistance of the cells is decreased in the presence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase. Expression of UVDE in XRCC1-defective EM9, a Chinese hamster ovary cell
line, greatly sensitizes the host cells to UV, and addition of
3-aminobenzamide results in almost no further sensitization of the
cells to UV. Thus, we show that XRCC1 and PARP are involved in the same
pathway for the repair of SSBs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(5), and is thought to be involved in a base excision
repair (BER) pathway. Recently it was reported that XRCC1 is also
involved in an S-phase-specific repair pathway of SSBs (6). Since XRCC1
binds to PARP (7) and some phenotypic characteristics of
XRCC1-deficient cells are similar to those of PARP-deficient cells (8),
both proteins may be involved in the S phase-specific mode of SSB
repair. However, the functional relationship between PARP and XRCC1 is
unknown and remains to be elucidated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin promoter (obtained from Dr J. Miyazaki,
Osaka University). This plasmid was used for expression of the N. crassa UVDE gene in XPA cells and EM9 cells. pCY4B-UVDE was made
by inserting the EcoRI fragment carrying the UVDE-coding
region into the EcoRI site of pCY4B. XP12ROSV cells and EM9
cells were transfected with pCY4B-UVDE together with a plasmid
harboring a G418 resistance marker by Lipofectin (Life Technologies,
Inc.). Transfectants were selected in medium containing 200 µg/ml (in
the case of XPA cells) or 400 µg/ml (in the case of EM9 cells) of
G418. Stable transfectants of XP12ROSV and EM9 expressing UVDE
were obtained and named XPA[UVDE] and EM9[UVDE], respectively. The
transfectant of XP12ROSV expressing wild-type XPA cDNA (17) was
obtained from Dr. K. Tanaka. This cell line is referred to as
XPA[cXPA] in this paper. A XP12ROSV cell line transfected with the
vector pCY4B plasmid was designated as XPA[Vector]. An EM9 clone
transfected with the vector was designated as EM9[Vector].
Human cells were grown in Eagle's minimal essential medium
(Nissui) containing 10% fetal calf serum, whereas CHO cells were grown
in 10% fetal calf serum-supplemented Dulbecco's modified medium (Nissui).
-mercaptoethanol, 5%
glycerol, 25 mM Tris-HCl (pH 6.5), and 0.05% bromphenol
blue) to each dish. In another experiment, cells were preincubated in
medium containing 2 mM 3-aminobenzamide (3AB, Sigma) for
2 h before UV irradiation. The cells were then washed with PBS
containing 2 mM 3AB, irradiated with UV, incubated, and
lysed as above. The extracts thus obtained were centrifuged at 15,000 rpm (18,000 × g) for 5 min at 4 °C. The
supernatants were recovered after centrifugation, resolved by 8%
SDS-polyacrylamide gel, and transferred to the fluorotrans membrane. To
detect pADPr bound to proteins, the membrane was probed with monoclonal
antibodies to pADPr (1:500 dilution; Trevigen, Inc.) and
peroxidase-labeled goat antibodies to mouse IgG (Kirkegaard & Perry
Laboratories, Inc.). We used actin as the control (monoclonal antibody to actin, clone C4, Roche Molecular Biochemicals).
-32P]ATP (NENTM
Life Science Products, Inc.). These size markers were electrophoresed in an alkaline gel containing the DNA sample. The gel
was dried and analyzed using FLA-2000 (Fujifilm). As a measure of the
relative amount SSBs, we took the following ratio: the radioactivity
from the 5.6- to14.3-kilobase area/the total radioactivity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
promoter and was introduced into a human XPA cell line. Cell lines transfected with UVDE (XPA[UVDE]), wild-type XPA cDNA
(XPA[cXPA]), and pCY4B vector (XPA[Vector]) were obtained.
Immunoblotting using polyclonal anti-Neurospora UVDE
antibody shows a single band only in XPA[UVDE] cells (Fig.
1A). The nicking activity of
the extract prepared from XPA[UVDE] cells to UV damage is shown in
Fig. 1C. The extract introduced an incision immediately 5'
to the CPD, as judged by the decrease of a 49-mer band and concomitant
strong appearance of the 20-mer band. This incision activity is the
same as previously reported for recombinant UVDE (18). These data indicated that XPA[UVDE] cells express UVDE and retain its nicking activity.
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Fig. 1.
Expression and nicking activity of UVDE.
A, Western blot analysis of UVDE in XPA[UVDE] and
XPA[Vector]. The protein size marker is indicated at the left in kDa.
B, 49-mer oligonucleotide with a single TT dipyrimidine
(CPD) used for in vitro assay. C,
nicking activity of the cell extracts to a 5'-labeled 49-mer duplex DNA
containing a single CPD. This assay was conducted by using whole cell
extracts derived from XPA[UVDE] and XPA[Vector]. The location of
the unnicked 49-mer and nicked 20-mer is indicated at the left.
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Fig. 2.
Survival of XPA cell lines after UV
irradiation. Exponentially growing XPA[UVDE] (filled
circles), XPA[Vector] (open triangles), and
XPA[cXPA] (open squares) cells were irradiated with UV and
grown without (solid lines) or with (broken
lines) 2 mM 3AB for 12 days. Survival experiments were
repeated three times, and typical results from a single experiment are
shown.
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Fig. 3.
SSB frequencies of various XPA transfectants
irradiated with UV and incubated for various time intervals. A,
top, SSB frequencies at short periods. XPA[UVDE] cells
prelabeled with 32P were either irradiated with 20 J/m2 UV or unirradiated. They were incubated for various
periods of time in buffer at room temperature and then lysed for
collection of the genomic DNA. The samples (5 µg of each) of purified
DNA were electrophoresed through a 3.5% alkaline agarose gel. The gel
was dried, and the radioactivity of 32P was visualized as
described under "Experimental Procedures." C stands for
the unirradiated control. Bottom, the ratio (radioactivity
from 5.6- to 14.3-kilobase (kb) area/total
radioactivity) is shown on the histogram as a measure of the relative
amount of SSBs. B, SSB frequencies at longer periods
of time. XPA[UVDE] (gray), XPA[Vector]
(white), and XPA[cXPA] (black) cells were
prelabeled, irradiated, and incubated for various intervals of time in
growth medium at 37 °C. These experiments were repeated three times,
and typical results from a single experiment are shown. C
stands for the unirradiated control.
, , and .
We measured repair synthesis using the BrdUrd density shift technique
(see "Experimental Procedures"). Exponentially growing cells were
exposed to 20 J/m2 and 40 J/m2 UV. Hydroxyurea
was added to the growth medium to reduce the level of semi-conservative
DNA synthesis. At both doses used, most of UV-induced repair synthesis
was aphidicolin-sensitive (Table I).
These data suggest that repair synthesis is mediated mainly by
aphidicolin-sensitive DNA polymerase(s): presumably by DNA polymerase
and/or (see "Discussion").
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Fig. 4.
Unscheduled DNA synthesis after UV
irradiation of cells. XPA[UVDE] cells were irradiated with 7.5 J/m2 UV (light gray) and 15 J/m2 UV
(dark gray). XPA[Vector] (black) and HeLa
(white) cells were irradiated with 7.5 J/m2 UV.
After UV irradiation, cells were incubated for 1 h in medium
containing [3H]dThd, then washed, fixed, and processed
for autoradiography to detect repair synthesis. About 150 nuclei having
fewer than 100 silver grains were counted for each cell line. Repair
synthesis was taken to be the difference between the percentage values
of corresponding irradiated and non-irradiated samples. The results
were shown as a percentage histogram for each cell line.
Buoyant density shift measurement of repair synthesis after UV
irradiation in XPA [UVDE] cells in the presence or absence of
aphidicoline
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Fig. 5.
Radioactivity profiles of alkaline CsCl
gradients of sonicated DNA from XPA[UVDE] cells. XPA[UVDE]
cells prelabeled with 32P were irradiated with 20 J/m2 UV and incubated for 3 h in medium containing
[3H]dThd, hydroxyurea, and BrdUrd. DNA of
parental-density was isolated and centrifuged to equilibrium in
alkaline CsCl gradients after the DNA had been sonicated to a
number-average size of 188 nucleotides. The arrow shows the
horizontal position of fully BrUra-substituted DNA. Open
circles represent the 3H repair label, and
filled circles represent the 32P prelabel.
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Fig. 6.
Poly(ADP-ribosyl)ation of cellular proteins
after UV irradiation in XPA[UVDE] cells. A, time
course of poly(ADP-ribosyl)ation. XPA[UVDE] as well as XPA[cXPA]
cells were irradiated with 20 J/m2 UV. At the indicated
periods of time (0.5, 2, 5, and 10 min; C, unirradiated
control), cell extracts were prepared and subjected to immunoblot
analysis with antibodies to pADPr (a) or to actin
(b). B, the dependence of pADPr formation on UV
dose. XPA[UVDE] cells were irradiated with the indicated dose of UV.
The irradiation time is 30 s in each case. Then, at the indicated
times (0.5, 2, and 5 min; C, unirradiated control), cell
extracts were prepared and subjected to immunoblot analysis as
above.
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Fig. 7.
Survival of EM9 cell lines after UV
irradiation. Exponentially growing EM9[Vector] (filled
square) and EM9[UVDE] (open circle) cells were
irradiated with UV and grown without (solid lines) or with
(broken line for EM9[UVDE]) 2 mM 3AB for 8 days. Survival experiments were repeated three times, and typical
results from a single experiment are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(29) and is catalyzed by DNA polymerase
or (27, 28, 29). By in vitro assays with purified
recombinant proteins, we and other groups (30, 31) show that the SSBs
produced by UVDE became substrates for cleavage by FEN1 (flap
endonuclease 1), which has already been shown to be a factor involved
in the proliferating cell nuclear antigen-dependent BER
pathway (27, 28, 32, 33). Thus, the SSBs produced by UVDE in
XPA[UVDE] cells may be processed by DNA polymerase and/or and
components that are common with long patch repair pathway of BER.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. H. Tohda for useful discussions. We also thank Dr. S. J. McCready for critical reading of this manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by Ministry of Education, Science, Sports, and Culture of Japan Grants 08280101 and 10480131.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.: 81-22-717-8465;
Fax: 81-22-717-8470; E-mail: ayasui@idac.tohoku.ac.jp.
Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M004085200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SSB, single-strand break; PARP, poly(ADP-ribose) polymerase; BER, base excision repair; UVDE, UV damage endonuclease; NER, nucleotide excision repair; CPD, cyclobutane pyrimidine dimer; CHO, Chinese hamster ovary; XPA, xeroderma pigmentosum group A; pADPr, poly(ADP-ribose); 3AB, 3-aminobenzamide; FdUrd, fluorodeoxyuridine; BrdUrd, bromodeoxyuridine; dThd, thymidine; BrUra, bromouracil; PBS, phosphate-buffered saline.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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