From the Department of Carcinogenesis, University of
Texas M. D. Anderson Cancer Center, Science Park-Research
Division, Smithville, Texas 78957 and the ¶ Department of
Environmental Medicine, New York University School of Medicine, Tuxedo,
New York 10987
Received for publication, December 5, 2000, and in revised form, February 5, 2001
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ABSTRACT |
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Both prokaryotic and eukaryotic cells have the
capacity to repair DNA damage preferentially in the transcribed strand
of actively expressed genes. However, we have found that several types
of DNA damage, including cyclobutane pyrimidine dimers (CPDs)
are repaired with equal efficiency in both the transcribed and
nontranscribed strands of the adenine phosphoribosyltransferase
(APRT) gene in Chinese hamster ovary cells. We further
found that, in two mutant cell lines in which the entire
APRT promoter region has been deleted, CPDs are still
efficiently repaired in both strands of the promoterless APRT gene, even though neither strand appears to be
transcribed. These results suggest that efficient repair of both
strands at this locus does not require transcription of the
APRT gene. We have also mapped CPD repair in exon 3 of the
APRT gene in each cell line at single nucleotide
resolution. Again, we found similar rates of CPD repair in both strands
of the APRT gene domain in both APRT
promoter-deletion mutants and their parental cell line. Our findings
suggest that current models of transcription-coupled repair and global
genomic repair may underestimate the importance of factors other than
transcription in governing the efficiency of nucleotide excision repair.
The original findings of Hanawalt and colleagues (1, 2), which
demonstrate that ultraviolet (UV) light-induced cyclobutane pyrimidine
dimers (CPDs)1 are repaired
much more efficiently in an actively transcribed gene, such as
dihydrofolate reductase (DHFR) than in noncoding regions,
and that repair is more efficient in the transcribed strand of
such genes than in the nontranscribed strand, have been extended to a
number of gene loci, in a wide range of prokaryotic and eukaryotic
cells (3-8).
In mammalian cells, nucleotide excision repair (NER) is thought to
involve two distinct subpathways: transcription-coupled repair (TCR),
which selectively and very efficiently repairs transcription-blocking damage in the transcribed strand of actively expressed genes, and
global genomic repair (GGR), which is responsible for repairing damage
in the nontranscribed strand and the rest of the genome (2, 5-8). TCR
occurs only in genes that are transcribed by RNA polymerase II. Cells
from individuals with Cockayne's syndrome (CS) are competent in GGR
but deficient in TCR (9-11). Interestingly, even though CS cells are
deficient in TCR, repair in actively transcribed genes is still
significantly more efficient that repair in inactive regions of the
genome. Cells from xeroderma pigmentosum complementation group C (XPC)
patients are competent in repair of CPDs in transcriptionally active
genes (with repair occurring primarily in the transcribed strand) but
are defective in GGR (12, 13). Chinese hamster ovary (CHO) cells are
profoundly deficient in global genomic repair of CPDs, typically
showing efficient TCR of CPDs in the transcribed strand of actively
transcribed genes, but little repair in the nontranscribed strand, or
in nontranscribed regions of the genome (2, 14, 15).
Not all genes, however, show the characteristic pattern of
preferential, TCR of transcription-blocking damage on the transcribed strand, originally described for the DHFR gene. Venema,
Troelstra, van Hoffen, and colleagues (9-13) have found that, in
normal human cells, CPDs in both the template and nontemplate strands
of the transcriptionally active adenine deaminase (ADA) gene
appear to be "preferentially" repaired. In XPC cells, CPDs in the
5'-portion of the ADA gene were found to be repaired much
more efficiently in the transcribed strand than in the nontranscribed
strand, but CPDs in the 3'-portion of the ADA gene were
efficiently repaired in both strands (12, 13). Furthermore, in a
patient with severe combined immune deficiency, in which the
ADA promoter region has been deleted and the gene is not
expressed, the efficiency of CPD repair was found to be only slightly
reduced compared with that normally seen for the transcribed strand of
an intact ADA gene (16, 17). Efficient repair of CPDs on
both strands of an actively transcribed mammalian gene has been
reported for two other gene loci, the human In this study, we have carefully examined the kinetics, strand
specificity, and transcription dependence of NER of UV-induced CPDs at
the endogenous Chinese hamster APRT gene locus, in
hemizygous CHO cell lines that contain only a single copy of this gene.
In contrast to the preferential repair of CPDs in the transcribed strand, which is observed in the CHO DHFR gene, we have
found that CPDs on both strands of the CHO APRT gene are
repaired with equal efficiency. We observe a similar lack of strand
specificity for repair of BPDE and CC-1065-induced DNA damage at the
APRT locus. We have further found that, in two mutant cell
lines in which the entire APRT promoter region has been
deleted, CPDs are still efficiently repaired in both strands of the
promoterless APRT gene, even though neither strand appears
to be transcribed. These results, which were initially obtained by
Southern analysis using a relatively small DNA fragment that includes
the entire APRT gene, have been confirmed by
ligation-mediated polymerase chain reaction (LMPCR) analyses in which
rates of repair were determined for each CPD site along each DNA strand
in exon 3 of the APRT gene, in all three cell lines.
Together, these results demonstrate that the highly efficient repair
observed on both strands of the Chinese hamster APRT locus
is not dependent on transcription of the APRT gene.
Cells, Cell Culture, and Carcinogen Treatment--
The Chinese
hamster CHO-AT3-2 cell line is hemizygous for the endogenous
APRT gene locus (20); these cells contain a single, actively
transcribed APRT gene, which is located on the CHO Z7 chromosome. ATS-88 and T2S-24 are two spontaneous APRT
promoter deletion mutants, which were derived from CHO-AT3-2. For these experiments, cultures were grown to 50-70% confluence in 150-mm dishes, in DNA Isolation--
Cells were washed three times with DPBS, and
lysed with lysing buffer (0.5% SDS, 10 mM Tris, pH 7.8, 10 mM EDTA, 10 mM NaCl, 100 µg/ml proteinase K)
at room temperature for 1 h. The proteinase K was removed by
phenol extractions followed by diethyl ether extractions, and the DNA
was ethanol-precipitated and resuspended in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA). RNA was removed by
treatment with RNase A (0.4 µg/ml) followed by phenol and diethyl ether extractions and ethanol precipitation. For Southern blotting, DNAs were digested overnight with either Asp718 or
BamHI (10 unit/µg of DNA) and checked for completeness of
digestion by agarose gel electrophoresis. Replicated and nonreplicated
DNAs were separated by the CsCl gradient centrifugation in a Ti 50 rotor (37 × 103 rpm for 64 h at 21 °C)
(23). Only the nonreplicated DNA was used for repair kinetics analysis.
UvrABC Nuclease Treatment--
The UvrA, UvrB, and UvrC proteins
were purified as described previously (23, 24). The UvrABC reaction was
the same as previously described (21, 23). Briefly, an aliquot
containing 3 µg of DNA was reacted with UvrA, UvrB, and UvrC (330 nmol) each in reaction buffer (50 mM Tris, pH 7.5, 100 mM KCl, 1 mM ATP, 10 mM
MgCl2, and 1 mM dithiothreitol), at a final
volume 100 µl at 37 °C for 90 min. The protein-DNA mixture was
then ethanol-precipitated (with 15 µg of tRNA, as a carrier) and
resuspended in 10 µl of TE buffer.
Thermal-alkaline Treatment--
The method was the same as
previously described (22). To induce DNA strand breakage at the
CC-1065-DNA adducts site, 3 µg of DNA was added to 40 µl of
solution containing 10 mM NaOH, 75% formamide, 2.5 mM Tris, and 0.25 mM EDTA. The mixtures were
heated at 90 °C for 20 min and quenched in an ice bath.
Cleavage of CPDs--
The DNA was incubated with T4 endoV
(protein/DNA molar ratio 6:1, assuming the average DNA length is 14 kb)
in a solution of 100 mM NaCl, 10 mM Tris, pH
7.5, and 0.5 mM EDTA at 37 °C for 60 min. For Southern
blotting, the protein-DNA mixture was ethanol-precipitated with 15 µg
of tRNA and resuspended in 10 µl of TE buffer. For LMPCR,
dithiothreitol was added to a final concentration of 10 mM,
and the mixtures were preincubated with the Escherichia coli photolyase (0.5 µg/µg of DNA) at room temperature under yellow light for 3 min. The mixtures were then irradiated with 366-nm UV light
(Sylvania 15-watt F15T8) for 60 min at room temperature at a distance
of 5 cm to generate strand breakage (25). After enzyme treatment, the
DNA was purified by phenol and diethyl ether extraction,
ethanol-precipitation, and then dissolved in TE buffer.
DNA Denaturation and Gel Electrophoresis--
DNA was denatured
in 90% formamide at 37 °C for 60 min. Immediately after
denaturation, the samples were electrophoresed at 5 V/cm for 3 h
in a 0.5% agarose gel in TBE buffer (25 mM Tris, pH 8.0, 25 mM boric acid, and 2.5 mM EDTA) with 0.5 µg/ml ethidium bromide. After electrophoresis, the samples were
transferred to an Oncor membrane. The DNA on the membrane was then
hybridized with strand-specific DNA probes.
Strand-specific DNA Probes--
pGEM-zf11(+)-APRT and
pGEM-zf11( RNA Isolation and Electrophoresis--
Total RNA was isolated
using the guanidinium method (26). In brief, cells were lysed with a
guanidinium solution (4 M guanidinium isothiocyanate, 20 mM sodium acetate, pH 5.2, 0.1 mM
dithiothreitol, 0.5% N-lauroylsarcosine) and genomic DNA
was sheared with a 20-gauge needle. Total RNA was isolated by
centrifugation through a 5.7 M CsCl gradient, at 35 × 103 rpm for 12-20 h. The pelleted RNA was then
dissolved in TES (10 mM Tris, pH 7.4, 5 mM
EDTA, 1% sodium dodecyl sulfate). Messenger RNA was isolated with the
Invitrogen FastTrack 2.0 mRNA kit. The RNA was then run in a
glyoxal-Me2SO-NaPO4 gel, transferred to an
Oncor membrane, and hybridized with strand-specific probes.
Ligation-mediated Polymerase Chain Reactions--
The sequencing
reactions were performed as described previously (27).
Ligation-mediated polymerase chain reactions (LMPCR) were performed as
described (25, 28) with two modifications. T4 polymerase from Oncor,
Inc. instead of Sequenase was used to perform the first primer
extension reaction in LMPCR. A specific amount of
32P-labeled linearized pBR322 plasmid DNA (about 20,000 dpm) was added to each sample at the beginning of the reaction as an
internal standard. After LMPCR, equivalent counts of 32P,
representing equivalent amounts of template DNA for each time point of
the reaction, were loaded into each lane of the sequencing gel. Primers
APRT 3-1 to 3-3 were used for detecting CPDs in the transcribed strand of APRT exon 3, and primers
APRT 3-4 and 3-5 were used for detecting CPDs in the
nontranscribed strand. Because it was difficult to identify a third
primer for the nontranscribed strand due to the sequence of the exon 3 region, primer APRT 3-5 was also used for probe
synthesis. The sequences of the primers were: APRT 3-1, 5'-TCCTTACACCTCAGCCCTAAC-3', Tm = 50.3 °C;
APRT 3-2, 5'-CAGCCCTAACACGCCCCCTCTC-3',
Tm = 62.7 °C; APRT 3-3, 5'-GCCTAGACTCCAG GGGATTCTTGTT-3', Tm = 59.1 °C; APRT 3-4, 5'-TGACCACCACCCCTAGCTTCT-3', Tm = 55.1 °C; APRT 3-5, 5'-CCACCACCCCTAGCTTCTCCATGTTTC-3', Tm = 64.5 °C.
Quantitation--
Autoradiographs were scanned with a Bio-Image
Analyzer, using a 100 Visage whole-band analysis software program. The
average number of damage sites per APRT fragment was
calculated by the Poisson distribution equation: P(0) = e Lack of Strand Specificity of Repair in a Transcriptionally Active
APRTGene--
The CHO cell line AT3-2 contains a single, actively
transcribed APRT gene (20). This APRT hemizygous
cell line was chosen for use in our experiments to avoid any potential
complications of allelic differences in transcription or DNA repair.
Exponentially growing cultures were treated with the DNA-damaging
agents: UV, BPDE, or CC-1065. To assay the removal of DNA damage, cells
were harvested at various times after treatment with damaging agents. For detection of UV-induced CPDs, DNAs isolated from UV-irradiated cells were treated with T4 endoV, which specifically cuts CPD sites
(1). UvrABC nuclease was used to detect BPDE-DNA adducts in DNAs
isolated from BPDE-treated cells (21), and DNAs isolated from
CC-1065-treated cells were subjected to thermal-alkaline treatment to
allow detection of CC-1065-DNA adducts (22). After these treatments,
the resultant DNAs were denatured and separated by electrophoresis in
an agarose gel; the separated DNAs were then transferred to a nylon membrane and
probed with 32P-labeled, APRT-transcribed (T) or
nontranscribed (NT) strand-specific probes. The results in Figs.
1-3 show that even though the kinetics for the removal of these three different types of DNA damage are quite
different, the time course of CPD photoproduct, BPDE-DNA adduct, or
CC-1065-DNA adduct removal is similar for both strands of the
APRT gene. As a control to confirm that these cells have not
lost their capacity for transcription-coupled repair, we also examined
CPD repair in the T and NT strands of the DHFR gene. It has
been previously shown that UV-induced CPDs are preferentially repaired
in the transcribed strand of the DHFR gene in CHO cells (2,
21). Our results in Fig. 4 confirm that
this is also the case in CHO-AT3-2 cells. Although 80% of CPDs are
removed from the T strand of the DHFR gene by TCR within
24 h, only 15% of CPDs are removed from the NT strand (Fig. 4).
The inefficient repair of CPDs in the NT strand of the DHFR
gene seen in Fig. 4 shows that CHO-AT3-2 cells, like other CHO sublines
(2, 14, 15), are deficient in global genomic repair of CPDs; this is further demonstrated in Fig. 5, which
shows that the majority of the CPDs in bulk genomic DNA remain
unrepaired even after 24 h of incubation. Thus, our results
demonstrate that, although repair at the CHO APRT gene locus
is much more efficient than repair in bulk genomic DNA, there is no
apparent strand specificity or preferential repair of damage on the
transcribed strand; similar rates and extents of repair are seen for
both strands of the APRT gene.
CPDs in Both Strands of a Promoter-deleted APRT Gene Are Still
Efficiently Repaired--
To determine whether efficient repair of
CPDs at the CHO APRT locus is dependent upon transcription
of the APRT gene, we have examined CPD repair in two
different CHO-AT3-2-derived, APRT promoter-deletion mutants
(ATS-88 and T2S-24). In ATS-88, a 1.15-kb deletion has eliminated the
entire promoter region and first two exons of the APRT gene;
in T2S-24, a much larger (25 kb) 5'-extending deletion, with virtually
the same 3'-breakpoint as ATS-88, has eliminated the same region
(promoter and first two exons) of the APRT gene (Fig.
6). These two promoter-deletion cell
lines were UV-irradiated, and their DNAs were isolated and then treated
with T4 endoV, in the same manner as for their parental cells described
above. Because the entire APRT promoter region has been
deleted from each of these mutant cell lines, if repair of the T strand
of the APRT gene is coupled with or dependent upon
transcription, one would expect to see a specific decrease in CPD
repair in what would normally be the T strand of the APRT
gene. Our results in Fig. 7, however,
still show efficient repair of CPDs in both strands of the
APRT gene domain in both of these promoter-deletion mutants; in each case, 80% of the CPDs in each strand are repaired within 24 h. These results suggest that CPD repair at the CHO
APRT locus does not require transcription of the
APRT gene, or even the presence of the APRT
promoter region.
Only One Strand of the APRT Gene Is Transcribed in Wild Type Cells,
and Neither Strand of This Gene Is Transcribed in ATS-88 or T2S-24
Cells--
To confirm the transcription status of the APRT
gene domain in the parental AT3-2 cell line, and ATS-88 and T2S-24
APRT promoter-deletion mutants, we have used strand-specific
probes for the APRT gene to screen both total cellular RNA
and polyadenylated mRNA isolated from each cell line. The results
in Fig. 8 show that: 1) only one strand
of the APRT gene is transcribed in
APRT+ AT3-2 cells, and 2) neither strand of this
gene appears to be transcribed in either the ATS-88 or T2S-24
promoter-deletion mutants. The 0.9-kb transcript observed in Fig. 8
represents APRT mRNA. No other RNA transcripts were
detected with either T or NT strand-specific probes for the
APRT gene.
Fine Mapping of CPD Repair in Exon 3 of Either a Transcriptionally
Active or Inactive APRTGene--
To confirm our findings that CPDs
are efficiently repaired in both the T and NT strands of the Chinese
hamster APRT gene, even in the absence of transcription of
this gene, we further mapped CPD repair in each strand of the exon 3 region of the APRT gene using LMPCR techniques (Fig.
9). The same
DNAs used to characterize CPD repair at
the defined gene fragment level were used to map CPD repair at the
single nucleotide level, using the method described by Pfeifer and
Dammann (28), with one modification. To correct for differential
recovery due to multiple ethanol precipitations during sample
preparation, a fixed amount of 32P-labeled linearized
pBR322 was added to each sample before LMPCR treatment. Then, after
LMPCR, a fixed amount of 32P counts was loaded for each
sample to ensure equal loading of sample DNAs prior to gel
electrophoresis. Typical autoradiographs are shown in Fig. 9. These
results show that in APRT + AT3-2 cells (Fig.
9A), CPDs in both the T and NT strands of exon 3 of the
APRT gene are efficiently repaired. CPDs also appear to be
efficiently repaired in both strands of exon 3 in both APRT promoter deletion mutants: ATS-88 (Fig. 9B) and T2S-24 (Fig.
9C).
Quantitation of the repair kinetics for CPDs formed at sites along both
DNA strands in the exon 3 region of the APRT gene in these
three cell lines are shown in Fig. 10.
The vertical columns at each CPD site in Fig. 10 represent the time
required for removal of 50% of the CPDs formed at that particular
site. Remarkably similar patterns and kinetics of repair at CPD sites
on the NT strand were observed in all three cell lines. Times required
for 50% removal of CPDs, determined at 25 CPD sites along the NT
strand of the exon 3 region of the APRT gene, ranged from
6.0 to 13 h in AT3-2 cells, from 7.0 to 16 h in ATS-88 cells,
and from 7.0 to 12.5 h in T2S24. However, the average time
(mean ± S.E.) required for removal of 50% of the CPDs formed at
each site, calculated for 25 CPD sites along the NT strand of the
exon 3 region of the APRT gene, was identical for all three
cell lines; 9.1 ± 0.4 h in AT3-2 cells, 9.6 ± 0.5 h in ATS-88 cells, and 9.0 ± 0.3 h in T2S-24 cells.
Although a slightly faster overall rate of repair and somewhat more
sequence-dependent variation in repair rates was observed for CPD sites on the T strand in AT3-2 cells, rates of repair on the T
strand in ATS-88 or T2S-24 cells were virtually identical to the rates
seen on the NT strand. Times required for 50% removal of CPDs,
determined for 27 CPD sites along the T strand of the exon 3 region of
the APRT gene, ranged from 2.1 to 10.1 h in AT3-2 cells, whereas in ATS-88 or T2S24 cells these times ranged from 5.5 to
12 h, or from 6.9 to 11.5 h, respectively. The average time
(mean ± S.E.) required for removal of 50% of the CPDs formed at
each site, calculated for these 27 CPD sites along the T strand of the
exon 3 region of the APRT gene, was 7.7 ± 0.4 h
for AT3-2 cells, 8.9 ± 0.3 h for ATS-88 cells, and 8.9 ± 0.3 h for T2S-24 cells. Interestingly, although there seems to
be much less sequence-dependent variation in CPD repair
rates in the APRT gene than has been reported for several
other genes (28, 31, 32), we did find four CPD sites on the T strand of
exon 3 (at bases 56, 73, 129, and 134 of this exon) that appeared to be
repaired two to four times more rapidly in AT3-2 cells than in either
ATS-88 or T2S-24 cells.
NER in mammalian cells is thought to involve two distinct
subpathways: a TCR pathway, which selectively and very efficiently repairs transcription-blocking damage in the transcribed strand of
actively expressed genes, and a GGR pathway, which is responsible for
repairing damage in the nontranscribed strand and the rest of the
genome (2, 5-8). However, several discordant lines of evidence suggest
that NER in mammalian cells may not be that simple. For example, CS
cells exhibit no deficiency in the repair of helix-distorting dG-C8-AAF
adducts, which are very effective blocks to RNA polymerase II
transcription, suggesting that these adducts must be removed by GGR,
not by TCR (29). Second, recovery of RNA synthesis from the middle of
the DHFR gene following UV irradiation appears to be much
faster than can be accounted for by the kinetics of removal of
UV-induced DNA lesions from the 5'-half of this gene (30). Third, LMPCR
studies of repair in the human JUN gene (31, 32) have
found very rapid repair of CPDs on both strands in the vicinity of the
transcription initiation site (between nucleotides Efficient repair of CPDs on both DNA strands has been previously
reported for two human genes, ADA and In this study we demonstrate that the highly efficient repair observed
on both strands of the APRT locus does not require transcription of the APRT gene and is unaffected by deletion
of the entire APRT promoter region. CHO cells are profoundly
deficient in expression of p48 UV-DDB (14, 15), and GGR of CPDs in
these cells is extremely inefficient, as evidenced by the very slow repair of CPDs in the nontranscribed strand of the DHFR
locus and in bulk genomic DNA. Thus, the patterns of CPD repair we have observed at the APRT gene locus are not readily explainable
by either TCR or enhanced levels of GGR. Our findings of similar CPD
repair efficiencies in both strands of the intact APRT gene, as well as in transcriptionally inactive, promoterless APRT
genes, suggest the possibility that efficient repair of some actively expressed genes may not necessarily depend on transcription, per se, but may be due to unique chromatin structure or to chromatin changes that precede transcription.
A variety of modifications of chromatin structure, including histone
acetylation, phosphorylation, ubiquitination, or methylation might
affect the rate of NER (8, 33-35). Changes in the chromatin structure
of active genes can involve both cis-acting sequences (such
as enhancer, silencer, or locus control regions), and
trans-acting transcription factors or nonhistone proteins
such as HMG14 and HMG17. However, the involvement of these factors in
NER has yet to be established. Because nucleosome-free DNA is known to
be repaired much more rapidly than nucleosomal DNA (35, 36), we
wondered whether efficient repair of damage on both DNA strands of the
APRT gene might reflect a lack of nucleosome formation at
this locus. However, we have obtained preliminary evidence, based on
micrococcus nuclease digestion, that the APRT gene in AT3-2
cells contains nucleosome structure (data not shown).
Interestingly, deletion of the APRT promoter region has no
apparent effect on replication timing; we have recently obtained results suggesting that in all three cell lines examined in this study,
the APRT gene domain is replicated early in S-phase. In contrast, Dijkwel and Hamlin (37) have reported that replication of the
DHFR locus in a DHFR promoter-deletion mutant
occurs 5-6 h later in S-phase than replication of the normal, actively
transcribed DHFR locus. To our knowledge, no one has
examined CPD repair in the DHFR domain of their
DHFR promoter-deletion mutant.
In summary, the results presented in this paper clearly demonstrate
that the highly efficient repair observed on both strands of the
APRT locus does not require transcription of the
APRT gene or even the presence of the APRT
promoter region. Our findings suggest that current models of
transcription-coupled and global genomic nucleotide excision repair may
underestimate the importance of factors other than transcription in
governing the efficiency of repair and their contribution to the
heterogeneity of NER that has been observed in mammalian cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin gene (18) and the
Chinese hamster adenine phosphoribosyltransferase (APRT)
gene (19); however, in both of these studies, the large size of the
genomic fragment used to assay CPD repair, in relation to the size of the transcribed region of the gene, complicated interpretation of the results.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium supplemented with 10% fetal calf serum. Prior to UV irradiation, or treatment with BPDE or CC-1065,
the medium was removed and cells were washed with Dulbecco's phosphate-buffered saline (DPBS). For UV treatments, cells were irradiated at a fluency rate of 1 J/m2/s, using GE1518
germicidal lamps (predominate emission, 254 nm) as the UV source. For
BPDE or CC-1065 treatments, either BPDE (4 µM) or CC-1065
(60 nM) was added to cell cultures in 15 ml of DPBS, and
the cultures were incubated at 37 °C for 30 min (21, 22). After
three rinses with fresh DPBS to remove the DNA-damaging agent, the
cells were incubated in fresh medium with 5-bromo-2'-deoxyuridine (10 µM) and 5-fluorodeoxyuridine (1 µM). After
incubation at 37 °C for various periods of time, the treated cells
were lysed for DNA isolations.
)-APRT vectors were constructed by inserting a
3.9-kb BamHI fragment containing the CHO APRT gene into the pGEM-zf11(+) or (
) vectors. These two constructs allowed us to isolate either the transcribed (T) or the nontranscribed (NT) strand of the APRT gene. Single-stranded
pGEM-zf11(+)-APRT or pGEM-zf11(-)-APRT DNA phages
containing either the transcribed or nontranscribed strand of the
APRT gene, respectively, were isolated by infecting the
vector-containing cells with carrier phage M13 K07. To generate
32P-labeled DNA probes for either the T or NT strand, 0.1 µg of template DNA was added to reaction buffer containing 50 pmol of the appropriate strand-specific primer, 5 µM
dGTP/dTTP/dATP, 5 units of Klenow fragment, and 16.7 pmol of
[
-32P]dCTP, and the mixture was incubated at 18 °C
overnight. For generation of the T strand-specific APRT
probe, we used a primer with the sequence: 5'-TGCGAGAAGCGGGACTGAAAA-3'.
For generation of the NT strand-specific probe, we used a primer with
the sequence: 5'-TGATCCATGCAAGACAAC-3'. To produce strand-specific
probes for exon 3 of the APRT gene, a PCR-amplified fragment
of the exon 3 region was used as the template and either T or NT
strand-specific primer was used to perform separate linear PCR reactions.
n, where n is the number
of UvrABC nuclease-sensitive sites (UNSS), or thermal-alkaline labile
sites (TALS), or T4 endoV-sensitive sites (ESS). The ratio of labeling
intensity of the full-length fragment from the enzyme (or heat)-treated
sample to that of the untreated sample is equal to P(0). For
the results from LMPCR experiments, the intensities of all CPD-site
bands were compared after subtraction of background values obtained
from T4 endoV-treated, nonirradiated control lanes. A repair
kinetics curve was generated for each CPD position, and the time
required for removal of 50% of the initial CPDs at each site was then
determined from this curve. Statistical analyses of the quantitated
repair rate data were carried out using StatView 4.01 (Abacus Concepts.
Inc.)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Removal of CPDs from the
APRT gene in UV-irradiated (20 J/m2) AT3-2 cells. DNAs isolated from cells
after various post-irradiation incubation periods were digested with
BamHI then treated with T4 endoV (protein/DNA molar ratio of
6:1). These DNAs were then denatured, separated by electrophoresis,
transferred to an Oncor membrane, and hybridized with double-stranded
(DS), transcribed (T) strand-specific, or
nontranscribed (NT) strand-specific 32P-labeled
probes for the APRT gene, as described under
"Experimental Procedures." The symbol (±) represents DNA with or
without T4 endoV treatment. At the top (a) is a
typical autoradiograph; at the bottom (b) are the
quantitative results. The numbers of ESS (T4 endoV-sensitive sites) in
the T or NT strands were calculated by the Poisson distribution, based
on densitometric scanning of the autoradiographs (23). The average
number of CPDs formed per 3.9-kb fragment of DNA in these experiments
after 20 J/m2 UV irradiation was calculated to be
~0.8.
View larger version (51K):
[in a new window]
Fig. 2.
Removal of BPDE-DNA adducts from the
APRT gene in AT3-2 cells. DNAs isolated from
BPDE-treated (4 µM) AT3-2 cells were digested with
Asp718 and treated with UvrABC nuclease (6x and
8x represent protein/DNA molar ratios of 6 and 8). The
resultant DNAs were then denatured, separated by electrophoresis,
transferred to an Oncor membrane, and hybridized with double-stranded
(DS), transcribed (T) strand-specific, or
nontranscribed (NT) strand-specific 32P-labeled
probes for the APRT gene, as described under "Experimental
Procedures." The symbol (±) represents DNA with or without UvrABC
nuclease treatment. At the top (a) is a typical
autoradiograph; at the bottom (b) are the
quantitative results. The numbers of UNSS (UvrABC nuclease-sensitive
sites) in the T or NT strands were calculated by the Poisson
distribution, based on densitometric scanning of the autoradiographs
(23). The average number of BPDE-DNA adducts formed per 9.4-kb
Asp718 fragment of DNA in these experiments after 4 µM BPDE treatment was calculated to be ~1.6.
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Fig. 3.
Removal of CC-1065 induced of
thermal-alkaline labile sites (TALS) from the
APRT gene in AT3-2 cells. DNAs from
CC-1065-treated (60 nm) cells were digested with Asp718
followed by thermal-alkaline treatment (22). The resultant DNAs were
then denatured, separated by electrophoresis, transferred to an Oncor
membrane, and hybridized with double-stranded (DS),
transcribed (T) strand-specific, or nontranscribed
strand-specific (NT) 32P-labeled probes for the
APRT gene, as described under "Experimental
Procedures." The symbol (±) represents DNA with or without
thermal-alkaline treatment. At the top (a) top is
a typical autoradiograph, and at the bottom (b)
are the quantitative results. The numbers of TALS in the T or NT
strands were calculated by the Poisson distribution, based on
densitometric scanning of the autoradiographs (22). The average number
of CC-1065-DNA adducts formed per 9.4-kb Asp718 fragment of
DNA in these experiments after CC-1065 treatment was calculated to be
~0.8.
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Fig. 4.
Removal of CPDs from the DHFR
gene in UV irradiated AT3-2 cells (20 J/m2). The
same DNAs described in Fig. 1 were first digested with
Asp718, then treated with T4 endoV, denatured, separated by
electrophoresis, transferred to an Oncor membrane, and hybridized
with double-stranded (DS), transcribed (T)
strand-specific, or nontranscribed (NT) strand-specific
32P-labeled probes for the DHFR genes, as
described under "Experimental Procedures." The symbols,
abbreviations, methods, and quantitation are the same as in Fig.
1.
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Fig. 5.
CPD repair in the genomic DNA of
UV-irradiated CHO AT3-2 cells. Cells were grown to 50-70% of
confluency, irradiated with (lanes 3-10) or without
(lanes 1 and 2) UV light (20 J/m2)
and incubated for different time in growth medium. The genomic DNAs
were isolated from cells, and treated with T4 endoV (protein/DNA molar
ratio of 6:1). Samples isolated from each time point and the
nonirradiated control were cut with T4 endoV (lanes 2,
4, 6, 8, and 10) or mock
treated (lanes 1, 3, 5, 7,
and 9). These DNAs were then denatured, separated by
electrophoresis in a 0.5% agarose gel, and stained with ethidium
bromide (0.5 µg/ml). Note: DNAs isolated from UV-irradiated cells
after different incubation times show similar sensitivity to cutting by
T4 endoV, suggesting that there is very little global genomic repair of
CPDs in these cells.
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Fig. 6.
Restriction maps of the APRT
gene domain in the CHO AT3-2, ATS-88, and T2S-24 cell lines,
showing the extents of the deleted regions in ATS-88 and T2S-24.
Restriction sites: BamHI, B; EcoRI,
E; EcoRV, Ev; KpnI,
K; NdeI, Nd; PstI,
Ps; XbaI, Xb; XhoI,
X.
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Fig. 7.
Removal of CPDs from the APRT
domain in UV irradiated (20 J/m2) ATS-88 cells
(A) and T2S-24 cells (B). The
symbols, abbreviations, methods, probes, and quantitation are the same
as in Fig. 1.
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Fig. 8.
Detection of APRT gene
transcripts or mRNAs from different CHO cell lines. Total RNA
10 µg (A) or mRNA (B) isolated from AT3-2,
ATS-88, T2S-24 cells were separated by gel electrophoresis, transferred
to an Oncor membrane, and hybridized with APRT
strand-specific probes, as described under "Experimental
Procedures"; T, hybridizations using a probe specific for
APRT template strand transcripts; NT,
hybridizations using a probe specific for transcripts from the opposite
strand.
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Fig. 9.
Fine mapping of CPD repair in exon 3 of the
APRT gene in UV-irradiated (20 J/m2) CHO
cells. A, AT3-2 cells; B, ATS-88 cells; and
C, T2S-24 cells. Genomic DNAs isolated from nonirradiated
cells (lane 4) and UV-irradiated cells after different
incubation times (0, 2, 4, 8, and 24 h in lanes 5-9)
were digested with T4 endoV, and subjected to LMPCR as described under
"Experimental Procedures." The resultant DNAs were separated by
electrophoresis in an 8% denatured polyacrylamide gel, transferred to
a nylon membrane, and then probed with 32P-labeled
APRT DNA fragments. The positions and intensities of T4
endoV incision bands represent the sites and extents of CPD
formation/repair along the sequence. Lanes 1-3 are the
Maxam-Gilbert sequencing reactions for G, AG, and TC. Left
panel, transcribed strand; right panel, nontranscribed
strand. Regions with contiguous pyrimidines are shown at the
left.
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Fig. 10.
Quantitation of CPD repair rates in the
transcribed strand (lower) and nontranscribed strand
(upper) in exon 3 of the APRT gene in
CHO cells. A, AT3-2; B, ATS-88; and
C, T2S-24 cells. Repair rates, expressed as the time (h)
required for 50% removal of CPDs, were determined for each position
with a visible signal above background, from autoradiographs such as
those shown in Fig. 9. Comparison of the intensities of CPD-site bands
after subtraction of background values obtained from T4 endoV-treated,
nonirradiated control lanes allowed us to establish time-course repair
kinetics curves for each CPD site. The time required for removal 50%
of the initial CPDs formed at each site was then determined from these
curves. Vertical columns represent repair rates at each CPD
site. These data represent the average of two experiments for each DNA
strand.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 and +100),
where >90% of the dimers are repaired within 4 h, and a gradient
of repair efficiency along the gene, with faster repair at the 5'-end
and diminished repair at the 3'-end. Interestingly, CS cells, which are
clearly deficient in repair of the transcribed DNA strand of the
JUN gene, still efficiently repair CPDs on both strands near
the transcription initiation site (31). Because TCR is presumed to be
initiated by an RNA polymerase II stalled at a helix-distorting
lesion in the transcribed strand during transcription (2, 5-8, 30), the rapid repair observed at sequence positions upstream of the initiation site and on the nontranscribed strand in the 5'-portion of
the JUN gene is unlikely to represent TCR.
-actin (9,
13, 17, 18). Although, the efficient repair of both template and nontemplate strands observed at these loci has been ascribed to the
possible occurrence of TCR on both strands, either as a result of
internal antisense transcription or overlapping convergent transcription from a downstream gene, little evidence has been presented to substantiate such suggestions. Vreeswijk and co-workers (19) have also reported a lack of strand bias of CPD repair at the
APRT locus in V79 cells. However, their study examined repair of CPDs in an 18.2-kb BclI fragment, upon which the
entire APRT gene transcript (transcription initiation site
to polyadenylation site) occupies only ~2.2 kb. They did not
determine the transcription status of the APRT alleles
present in the V79 cell line or whether any other transcribed sequences
were present within the BclI fragment that could have
contributed to the overall pattern of repair that was observed.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Aziz Sancar and Dr. Steven Lloyd for their generous gifts of E. coli photolyase and T4 endonuclease V, respectively.
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FOOTNOTES |
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* This work was supported by United States Public Health Services Grants ES03124, ES08389 (to M.-s. T.) and GM56165 (to G. M. A.) from the National Institutes of Health (NIH), and service core support from NIEHS, NIH Center Grants ES077784 and ES00260.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.: 845-731-3585; Fax: 845-351-3492; E-mail: tang@env.med.nyu.edu.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010973200
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ABBREVIATIONS |
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The abbreviations used are: CPD, cyclobutane pyrimidine dimer; APRT, adenine phosphoribosyltransferase; CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase; NER, nucleotide excision repair; TCR, transcription-coupled repair; GGR, global genomic repair; CS, Cockayne's syndrome; XPC, xeroderma pigmentosum complementation group C; ADA, adenosine deaminase; BPDE, benzo(a)pyrene diol epoxide; LMPCR, ligation-mediated polymerase chain reaction; DPBS, Dulbecco's phosphate-buffered saline; T4 endoV, T4 endonuclease V; T, transcribed; NT, nontranscribed; PCR, polymerase chain reaction; UNSS, UvrABC nuclease-sensitive sites; TALS, thermal-alkaline labile sites; ESS, T4 endonuclease V-sensitive sites; DS, double-stranded; kb, kilobase(s).
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REFERENCES |
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