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J Biol Chem, Vol. 274, Issue 38, 26822-26827, September 17, 1999
From the Institute of Development, Fission yeasts Schizosaccharomyces
pombe possess two types of excision repair systems for UV-induced
DNA damage, nucleotide excision repair (NER) and UV-damaged DNA
endonuclease (UVDE)-dependent excision repair (UVER).
Despite its high efficiency in damage removal, UVER defects have less
effect on UV survival than NER defects. To understand the differential
roles of two pathways, we examined strand-specific damage removal at
the myo2 and rpb2 loci. Although NER removes
cyclobutane pyrimidine dimers from the transcribed strand more rapidly
than from the nontranscribed strand, UVER repairs cyclobutane
pyrimidine dimers equally on both strands and at a much higher rate
than NER. The low rate of damage removal from the nontranscribed strand
in the absence of UVER indicates inefficient global genome repair (GGR)
in this organism and a possible function of UVER as an alternative to GGR. Disruption of rhp26, the S. pombe homolog
of CSB/RAD26, eliminated the strand bias of NER almost
completely and resulted in a significant increase of UV sensitivity of
cells in a uvde Research on mammalian cells and budding yeast Saccharomyces
cerevisiae has allowed us to draw a general picture of nucleotide excision repair (NER)1 for
UV- or chemical-induced damage on DNA in eukaryotes. In NER in these
species, DNA damage on the transcribed strands of transcriptionally active genes is repaired more rapidly than damage on the opposite strand or in transcriptionally silent regions of DNA (1). This strand-specific damage removal is considered to be due to the existence
of two modes of NER, one acting specifically on transcribed regions and
the other acting uniformly (at macroscopic level) throughout the
genome. These two modes of NER are called transcription-coupled repair
(TCR) and global genome repair (GGR), respectively. Several gene
products have been reported to be specifically involved in either one
of the two modes (2-4).
Cockayne syndrome (CS) is a human genetic disorder in which patients
show photosensitivity and impaired physical and mental development. Two
genes, CSA and CSB are responsible for the
classical form of CS (5, 6). Cells from the patients show a deficiency in TCR but apparently no defect in GGR (2). CSB gene is also known as ERCC6, which can complement the UV sensitivity of
rodent UV-sensitive mutants belonging to complementation group 6 (6). General conservation of the core mechanisms of NER between mammalian cells and budding yeast prompted researchers to search for yeast counterparts of these TCR-related genes, and RAD28 and
RAD26 have been identified as CSA and
CSB homologs, respectively (7, 8). Strangely, however, Rad26
but not Rad28 is involved in TCR of this organism. In addition, the
defect in TCR in S. cerevisiae caused by the
rad26 mutation does not significantly increase the UV
sensitivity and does not bring about any other remarkable phenotypes (8). Although strong similarity between CSB and Rad26 suggests homology
and conservation of this phenomenon between mammals and budding yeasts
or even throughout eukaryotes, defective TCR somehow leads to different
consequences in two organisms.
NER in fission yeast Schizosaccharomyces pombe has not been
extensively studied, although several genes, such as rad13,
rad15, rad16, swi10,
ercc3sp., and rhp16, have been identified that
are considered to be involved in NER mainly based on their sequence similarity to mammalian and budding yeast counterparts (for a review,
see Ref. 9). In addition, there has been no report of strand-specific
damage removal in S. pombe. Like the filamentous fungus,
Neurospora crassa, S. pombe possesses the second
excision repair pathway initiated by an enzyme called UVDE (10-13).
This enzyme specifically cleaves a phosphodiester bond 5' adjacent to a
UV-induced cyclobutane pyrimidine dimer (CPD) or a 6-4 photo product
((6-4) PP). After the nicking of damaged DNA, repair is thought to be
completed by a base excision repair-like process partially dependent on
the structure-specific endonuclease Rad2 (13), which is the S. pombe homolog of mammalian FEN-1 and S. cerevisiae Rad27.
Our previous work showed that although UVDE-dependent excision repair
(UVER) removes UV-induced lesions more rapidly from bulk DNA than NER,
NER defect results in a greater effect on UV survival. We have been
interested in the relationship between the two repair pathways in
S. pombe. The present work reports the transcription
dependence of NER at two loci of S. pombe and demonstrates
remarkably inefficient GGR. We have also identified the Rad26/CSB
homolog of S. pombe and showed its involvement in TCR of
this organism. We have examined several possibilities that would
explain the differential contribution of the two excision repair
systems to UV survival. Our result suggests that the existence or the
absence of transcription dependence at least partially accounts for the
differential effect of NER and UVER. We also show that UVER acts as an
alternative to GGR, especially in growing phase.
Strains, Media, and Transformation of S. pombe--
Strains used
in this study are listed in Table I. All
strains were grown in either YES or minimal medium at 30 °C (14). Double mutants were made by crossing each of the single mutants. Transformation of S. pombe cells was done by electroporation
with ECM600 Electro Cell Manipulator (BTX) following the
manufacturer's protocol.
Growth Conditions--
For experiments with exponentially
growing cells, a preculture was diluted 100-500-fold in fresh YES
medium, and cells were grown for 10-14 h with shaking to a cell
density of 1-2 × 107/ml. For experiments with
stationary phase cells, a similarly diluted culture was incubated for
72 h. The small and round shape of the cells was confirmed microscopically.
UV Survival Experiment--
Exponentially growing or stationary
phase cells in YES liquid medium were appropriately diluted with
distilled water and 500 or 10,000 cells/plate were seeded on YES agar
plates. 254-nm UV light was administered using a set of germicidal
lamps (GL-10, Toshiba, Japan) at a dose rate of 0.02-3.3
J/m2/sec. After incubation at 30 °C for 3 days, colonies
were counted.
Strand-specific CPD Assay--
Exponentially growing cells in
YES medium were washed twice with distilled water and resuspended in
phosphate-buffered saline at 5 × 106 cells/ml.
Approximately 30 ml of cell suspension was poured into a 15 cm plastic
dish (Falcon) and irradiated with 100 J/m2 of 254-nm UV
light. During the washing and UV irradiation processes cells were kept
on ice, and all materials were pre-chilled. For each strain, a total of
300 ml of UV-irradiated cell suspensions were prepared and divided into
10 aliquots. Each aliquot was centrifuged, and the cells were
resuspended in 30 ml of prewarmed YES medium and incubated at 30 °C
for 0, 20, 40, 60, or 120 min (two aliquots for each time point). The
cells were pelleted again, frozen with liquid nitrogen, and stored at
Preparation of myo2 and rpb2 Strand-specific Probes--
A part
of myo2 gene ( (16) nucleotides 1,496-2,302 of GenBankTM
accession number U75357) was amplified by PCR with primers SY78
(5'-CCCCCGGATCCCAAAGCTACTTTATTGGTATTTT-3') and SY79
(5'-CCCCCGGATCCATTATCTCATATCTGACTCTAAA-3') using Y4 strain genomic DNA
as a template and cloned into pBluescriptII SK+ (Stratagene).
Strand-specific probes were generated by asymmetric PCR using the
cloned fragment as a template and either SY78 or SY79 as a primer (17).
After the PCR reaction, the probe was purified by passing through G-50
column. Approximately 3 × 106 cpm/ml was used in
hybridization. For rpb2 strand-specific probes, a part of
the gene ( (18) nucleotides 2,623-3,138 of GenBankTM accession number
D13337) was PCR-amplified with primers SY49 (5'-GAACTGGATCCTGCACAAAGAGTTAAGCCA-3') and SY50
(5'-CCCCCGGATCCGTCGTACGTACCGTGTTTCATCCT-3') and similarly labeled.
Cloning of the rhp26 Gene--
A pair of PCR primers, SY99
(5'-GGGGGGAATTCTGACGGACTTGCACGATTTGTTTAC-3') and SY100
(5'-GGGGGGGATCCGTCGGGCAAGAAGAGTGCTTGAAGT-3') were designed based
on a partial sequence found in the course of the S. pombe
genome project conducted in Genome Research Group at National Institute
of Radiological Sciences (Chiba, Japan) that is highly similar to
S. cerevisiae RAD26 and human CSB. The PCR
fragment (~0.5 kb) obtained thus from Y4 genomic DNA was used as a
probe to screen a partial S. pombe genomic library
constructed in the EcoRI site of pBluescriptII SK+, and the
positive plasmid clone pSY69 was sequenced on both strands using
DSQ-1000 DNA Sequencer (Shimadzu, Japan).
Computer Analysis of the Rhp26 Sequence--
Open Reading Frame
(ORF) search and exon/intron structure predictions were conducted by
POMBE running on a SUN SPARC workstation (19). Subcelluar localization
of Rhp26 was predicted by PSORTII, which is available on the Internet
(20). Phylogenetic cluster analysis was done using a MOLPHY2.3b3
package with a maximum-likelihood method (21).
Reverse Transcription-PCR of rhp26 mRNA--
cDNA was
synthesized by priming of 40 µg of DNaseI-treated total RNA with
oligo(dT) (mixture of SY127-129: 5'-T20(A/C/G)-3'). The
presence of rhp26 cDNA was checked by PCR with a pair of
primers, SY119
(5'-CGCCAGGGTTTTCCCAGTCACGACGTGTTGTTGTTCTTCATG-3') and
SY126 (5'-GAGCGGATAACAATTTCACACAGGGATTCCAATCAGGATCA-3').
Disruption of rhp26--
The consecutive 1.7-and 0.1-kb
BglII fragments within rhp26 ORF of pSY69 were
replaced with an ura4 BamHI cassette. The vector sequence
was removed by EcoRI digestion, and the obtained fragment was used to transform the haploid S. pombe strain Y4. Five
stable Ura+ transformants were checked for targeted
integration of the ura4 marker by colony PCR, and three
rhp26-mutated clones were obtained.
Hydrogen Peroxide Sensitivity Assay--
Exponentially growing
cells were appropriately diluted with distilled water, and 10 to
104 cells in 5 µl were spotted on
H2O2-containing YES plates. The plates were
incubated for 3 days and photographed.
TCR of UV-induced CPDs in S. pombe--
We and others have
previously reported that S. pombe has two different excision
repair pathways for UV-induced DNA damage, NER and UVER (12, 13). By
using an immunological damage assay, we found that UVER removes CPDs
and (6-4) PPs, which are the major DNA damage induced by UV, from bulk
DNA more rapidly than does NER, although in terms of UV survival, the
contribution of NER is greater (Ref. 13 and Fig. 5a of this
paper). We have been interested in the relative roles of these two
repair systems in repair of UV-induced damage and what distinguishes
them. Since there have been no reports of TCR in S. pombe,
we first studied the transcription dependence of CPD removal by NER or
UVER using a T4 endonuclease-based endonuclease-sensitive site assay.
We decided to measure the repair rate of CPDs in the myo2
gene, which encodes a type II myosin heavy chain of S. pombe, taking advantage of its relatively high transcription level
(16)2 and the availability of
an appropriately sized restriction fragment when digested with
HindIII (~4.1 kb).
In wild type cells, CPDs were removed rapidly from both transcribed and
nontranscribed strands with essentially equal rates (Fig.
1, WT). NER-deficient
rad13
We also studied TCR at the less transcriptionally active
rpb2 locus (18), a homologous locus of which has been
commonly used in S. cerevisiae for studies of TCR (8, 22,
23). The result obtained was essentially the same as in myo2
locus (data not shown). This suggests that within this range, a
difference in transcription level does not significantly affect the
efficiency of TCR.
Cloning of the S. pombe CSB/RAD26 Homolog--
CSB and Rad26
belong to Swi/Snf2 protein family, which is a subgroup of a vast
helicase superfamily (24). We identified a short sequence in our
S. pombe genome data base that shows the highest similarity
with both S. cerevisiae Rad26 and human CSB and also lower
similarity with other Swi/Snf2 family members. Southern blot
analysis of S. pombe genomic DNA indicated that this
sequence hybridized to a 7-kb EcoRI fragment under stringent conditions. We constructed a partial genomic library from
EcoRI-digested and size-selected genomic DNA and screened it
with the same probe. Sequencing of the consecutive 4.3 kb out of the
7-kb EcoRI insert of the positive clone revealed an ORF of
2922 base pairs, encoding a 973-amino acid protein with an estimated
molecular weight of 110,900. Scanning of the sequenced region with the
POMBE program (19) did not identify either any introns within this ORF
or any other significant ORFs. Expression of this ORF was checked by a
reverse transcription-PCR experiment. The expected size of PCR product
was observed only from the RNA sample reacted with reverse
transcriptase (data not shown). Fig.
2a shows a schematic alignment
of the ORF with S. cerevisiae Rad26 and Homo
sapiens CSB. The middle part of the ORF contains the eight
helicase motifs (25), showing a high degree of conservation with the
corresponding regions of the other two homologs. Examination of this
ORF with PSORTII programs picked up one putative nuclear localization
signal and predicted it to be a nuclear protein (20). We concluded that
this ORF encodes the S. pombe ortholog of S. cerevisiae Rad26 and H. sapiens CSB based on the
following two observations. First, the deduced amino acid sequence
shows the highest similarity with Rad26 and CSB among Swi/Snf2
family members. Second, phylogenetic analysis put this ORF in the same
clade as Rad26 and CSB, leaving other Swi/Snf2 family members
such as S. cerevisiae Mot1 in an out-group (data not shown).
We propose to designate the gene rhp26 following the
standard nomenclature for the S. pombe orthologs of S. cerevisiae genes.
Effect of rhp26 Disruption on TCR--
CSB/Rad26 is known to be
specifically involved in the TCR mode of NER. In budding yeast and
mammals, disruption of the gene eliminates most of the transcription
dependence of NER. To examine the presumed involvement of Rhp26 in the
homologous pathway in S. pombe, we constructed a
rhp26 disruption mutant by replacing the N-terminal
two-thirds of the endogenous ORF with a ura4+
marker in the haploid wild type strain Y4 (Fig. 2b). The
resultant strain did not show any abnormal morphology nor growth
deficiency, suggesting no essential function of Rhp26. To assay the
effect of the disruption on TCR, we also generated a rhp26
uvde double mutant by crossing each of single mutant, since TCR of
CPDs was detected only in uvde
In the double mutant, the difference in repair rate between transcribed
and nontranscribed strands disappeared almost completely (Fig.
3a). Just as NER on the
nontranscribed strand was hardly observed in the uvde single
mutant (Fig. 1c), the rhp26 uvde double mutant
did not show any detectable CPD repair on either strand similar to a
rad13 uvde double mutant.
Effect of rhp26 Disruption on UV Survival--
As mentioned above,
our previous genetic data showed that NER is more relevant to UV
resistance than UVER. One possibility to explain the differential
effect of the two repair systems is the existence or the absence of
transcription dependence. We first examined if a defect in TCR affects
the cell survival of S. pombe. rad26 Repair of Thymine Glycol Does Not Account for the Differential
Effects of NER and UVER--
Although both NER and UVER are able to
repair the two major types of UV-induced DNA lesions, CPDs and (6-4)
PPs, NER can also repair other types of DNA damage induced by non-UV
agents. If there are any minor types of UV-induced damage that are
repaired only by NER and that are also relevant to cell survival, this would explain the differential effects of NER and UVER. Thymine glycol
probably is the best candidate to fit this possibility. This DNA damage
is known to be induced by UV irradiation as minor lesions and mostly
repaired by base excision repair but, at least in vitro, can
be repaired by NER also (26). We next examined the cell sensitivity to
hydrogen peroxide, which is known to induce thymine glycol on DNA
effectively, by a spot assay. Under the conditions used no difference
in sensitivity was observed among the wild type and NER- or
UVER-deficient strains (Fig.
4a).
Checkpoint Control-related Defects Do Not Account for the
Differential Effects of NER and UVER--
A repair intermediate in
NER, but not unexcised damage, is believed to trigger a checkpoint
control induced by UV irradiation (27, 28). This means that a defect in
NER may interfere with checkpoint signaling. In S. pombe, a
checkpoint signal is considered to be bifurcated to the mitotic arrest
and damage tolerance subpathways (29). Although mitotic arrest probably
acts only to allow time for repair and is therefore significant only
when repair systems are functional, damage tolerance may provide cells
with a means for cell survival besides damage repair. This suggests the
possibility that rad13 disruption may actually disrupt both
NER and the checkpoint-dependent damage tolerance pathway,
thus resulting in a more serious effect on UV survival than
uvde disruption. We checked this possibility by examining
the cell survival in a checkpoint-minus background. The result
indicated that even in a rad3 UVER in Stationary Phase Cells--
Our data have shown that UVER
plays the role of GGR in S. pombe. In other words, GGR of
NER may have only a minor role when UVER is functional. We hypothesized
that rapid damage removal by UVER may have less significance in cells
in stationary phase. To examine this possibility, we studied the UV
sensitivity of wild type and UVER- or NER-deficient cells in stationary
phase. Unexpectedly, wild type cells in stationary phase turned out to be more UV-sensitive than those in the exponential phase of growth under our conditions (Fig. 5a
and b). uvde disruption led to a much smaller
effect in cells in stationary phase, where NER defect caused as a
dramatic reduction in UV survival as in the growing phase.
In the present work we examined transcription dependence of two
types of excision repair systems, NER and UVER in S. pombe. It has been generally believed that transcription dependence is a
property peculiar to NER. However, it is not self-evident that other
types of excision repair systems do not have transcription dependence,
since there has been accumulating evidence that some types of oxidative
damage not usually repaired by NER are also removed preferentially from
the transcribed strands of active genes (30). Our study showed that in
S. pombe the UVER pathway operates equally on both
transcribed and nontranscribed strands at two loci, myo2 and
rpb2, which differ in transcription levels, whereas the NER
pathway has a clear strand bias. Probably due to the predominance of
UVER pathway over NER in repair of CPDs, this strand bias was not
observed in wild type cells, where both pathways were functional.
Notably, at least in our assay conditions, CPD removal from the
nontranscribed strand by NER was almost undetectable. A possible
explanation is that GGR for CPDs of S. pombe may not be as
efficient as in S. cerevisiae. We speculate that it is
because UVER plays the role of GGR in repair of UV-damage in S. pombe. This is consistent with the fact that disruption of
rhp16 gene dose not increase UV sensitivity in S. pombe as dramatically as disruption of RAD16 gene does
in S. cerevisiae (31), both of which are considered to play
a role specifically in GGR in respective organisms. On the other hand,
rhp16 disruption does increase the sensitivity of cells to
cisplatin, probably because UVER is not involved in repair of this type
of damage (31).
The present work identified Rhp26, the S. pombe homolog of
Rad26/CSB. As a member of Swi/Snf2 family, Rhp26 possesses a
highly conserved helicase domain in its middle part. Despite its name, many of the proteins having this domain do possess
DNA-dependent ATPase activity but do not really show
helicase activity, and this applies to CSB and Rad26 (32, 33). Based on
these facts, Rhp26 is not considered to have a helicase activity, but
still its helicase domain is likely to be crucial for the function.
van Gool et al. (8) report that both Rad26 and CSB have a
stretch of acidic amino acids and discuss that this motif might also be
involved in their function. Rhp26, however, does not have such an
obvious acidic region at its N terminus. This suggests either that this
region is not relevant to CSB/Rad26 function or another part of this
protein or that another protein(s) compensates for the absence of the
acidic region in S. pombe.
Disruption of rhp26 increased UV sensitivity slightly but
reproducibly, and this effect was more pronounced in a
uvde We have been concerned with why a UVER defect causes a weaker effect on
cell survival than a NER defect. In general, when two pathways are
apparently redundant for a single function, and the disruption of one
results in a weaker phenotype than the disruption of the other, the
former pathway is regarded as a backup system to the latter and is not
relevant when the latter pathway is functional. However our present
work showed that even when both NER and UVER operate, UVER is
predominant in removal of CPDs.
UV survival experiments with rad3 Disruption of rhp26 reduced the difference between NER and
UVER but did not erase it completely. This residual difference might be
attributed to Rhp26-independent TCR. Brouwer and co-workers report
that there is a Rad26-independent TCR in S. cerevisiae at the short regions downstream of the promoters of
active genes (34, 35). The contribution of this Rad26-independent TCR
was expressed as the difference in UV survival between a NER Assuming that the difference between NER and UVER is explained by the
existence or the absence of transcription dependence, why is highly
efficient UVER only equally effective in UV survival to GGR, which is
almost undetectable in our assay? This again is a difficult question to
answer. One point is that in (6-4) PP repair, NER is very efficient and
is almost comparable to UVER (13). In fact in most organisms NER
repairs (6-4) PPs at a much higher rate than CPDs. There is a general
belief that (6-4) PPs are stronger barriers against replication or
transcription and, thus, more toxic than CPDs and a major determinant
in cell killing by UV. Taken together, this might explain the apparent
irrelevance of CPD repair rates by UVER or GGR for UV survival.
If UVER has less significance than NER in UV survival due to the lack
of transcription dependence or other reasons, what is its advantage? We
speculate that the high efficiency of UVER is advantageous in rapidly
growing cells. To support this possibility, disruption of
uvde leads to an even weaker effect on the survival of
stationary phase cells. It is an attractive hypothesis that in
stationary phase, even inefficient GGR may be able to compensate for
the UVER defect taking advantage of plenty of time for damage removal.
However, it should be noted that other explanations such as
down-regulation or some toxic effect of UVER in stationary phase are
also possible. Taking into account the fact that wild type cells in
stationary phase are more UV-sensitive than those in exponential phase,
these possibilities should be examined in the future studies.
Nevertheless, UVER certainly plays a important role in the UV
resistance of growing cells.
Cells generally possess multiple repair pathways for a single type of
DNA damage. Despite this apparent redundancy, each pathway may have
some special condition in which it is more advantageous than others,
and this probably has enabled multiple pathways to be maintained during evolution.
We thank Dr. C. Kitayama for information on
transcription level of myo2 and Dr. A. M. Carr for
providing a rad3 *
This work was supported by grants-in-aid from the Ministry
of Education, Science, Sports, and Culture of Japan.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.
The nucleotide sequence reported in this paper has been
submitted to the DDBJ/GenBankTM/EBI Data Bank with
accession number AB022912.
2
C. Kitayama, personal communication.
3
S. Yasuhira and A. Yasui, unpublished result.
The abbreviations used are:
NER, nucleotide
excision repair;
TCR, transcription-coupled repair;
GGR, global genome
repair;
CS, Cockayne syndrome;
UVDE, UV-damaged DNA endonuclease;
UVER, UVDE-dependent excision repair;
CPD, cyclobutane pyrimidine
dimer;
(6-4) PP, 6-4 photo product;
ORF, open reading frame;
PCR, polymerase chain reaction;
kb, kilobase(s).
Transcription Dependence and the Roles of Two Excision Repair
Pathways for UV Damage in Fission Yeast Schizosaccharomyces
pombe*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
background. We suggest that the
combination of transcription-coupled repair of NER and rapid UVER
contributes to UV survival in growing S. pombe cells, which
is accomplished by transcription-coupled repair and GGR in other organisms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains used in this work
80 °C until DNA preparation. Genomic DNA was isolated as described
previously (14) with a slight modification. After digestion with
HindIII, DNA was treated with T4 endonuclease or
mock-treated and electrophoresed under alkaline condition (15). DNA was
transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech).
The blots were prepared in duplicate with the same DNA sample, and one
was hybridized with a sense strand-specific myo2 or
rpb2 probe; the other was hybridized with an
antisense-specific probe (see below) using ExpressHyb hybridization solution (CLONTECH) following the manufacturer's
protocol. The blots were analyzed using FLA-2000 Fluoroimage Analyzer
(Fuji, Japan). The number of CPDs in the target fragment was calculated assuming Poisson distribution. After the first hybridization and the
analysis, the blots were incubated at 100 °C in 0.5% SDS for 5 min
to remove probes and rehybridized reciprocally with the opposite
strand-specific probes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells were also able to repair CPDs on both strands
with a comparable rate to wild type cells. This implies that UVER is
the major component of CPD removal in wild type S. pombe
cells, which is consistent with our previous result (13), and also that
there is no clear transcription dependence in UVER (Fig. 1,
rad13). When the UVER pathway was eliminated by the
disruption of uvde, a clear strand bias of CPD removal emerged (Fig. 1, uvde). In fact, under our experimental
conditions, CPD removal on the nontranscribed strand was hardly
detected in this strain, whereas CPDs on the transcribed strand were
removed efficiently. Cells deficient in both NER and UVER were not able to repair UV-induced CPDs during the period of time studied, suggesting that there is no third excision repair system (Fig. 1,
rad13 uvde).
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Fig. 1.
Strand bias of NER and UVER on
myo2 locus. Cells with or without functional NER
or UVER were irradiated with 100 J/m2 of 254-nm UV and
allowed to repair damage for 0-120 min at 30 °C. Each point stands
for a mean value calculated from four hybridizations with two
independent UV irradiations and DNA isolations. WT, wild
type. 
, transcribed strand; 
, nontranscribed strand.
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Fig. 2.
a, schematic comparison of S. cerevisiae Rad26, S. pombe Rhp26, and H. sapiens CSB amino acid sequences. Eight helicase motifs (25) and
the conserved region were shown. b, disruption of the
rhp26 gene. Internal BglII fragments were
replaced with a ura4 BamHI cassette and used for
transformation of S. pombe. a.a., amino
acids.
background (Fig. 1).
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Fig. 3.
a, strand bias of CPD removal in
rhp26 uvde strain. , transcribed
strand; , nontranscribed strand. b and c,
effect of rhp26 disruption on UV survival of the strain with
(b) or without (c) UVER. Panel b: , wild type; 
, rhp26; |,
rad13. Panel c: 
, uvde; ,
rhp26 uvde; |, rad13; ,
rad13 uvde. Survival experiments were
repeated at least three times, and the typical result from a single
experiment is shown. The effect of rhp26 disruption is
stronger when UVER is absent. The differential contribution of NER and
UVER to UV survival, which is expressed as the difference of the
survival curves of uvde and rad13 cells, is
reduced by an additional rhp26 disruption of
uvde cells.
strains turned out
to be slightly, but reproducibly more sensitive than wild type cells
(Fig. 3b). This reduction of cell survival by the
rad26 mutation was more dramatic in a
uvde background (Fig. 3c). In other words, the
difference between NER and UVER was reduced when the
transcription-dependent component was removed from NER. The
residual difference of UV resistance between rhp26 uvde and rad13 might be attributed to
Rhp26-independent TCR (see "Discussion").
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Fig. 4.
a, hydrogen peroxide sensitivity of wild
type (WT) and UVER- and/or NER-deficient S. pombe
cells. A UVER or NER defect does not result in an increased
sensitivity. b, UV sensitivity of S. pombe
strains with a rad3 background. , rad3,
, rad3 uvde; +, rad3 rad13; ,
rad3 rad13 uvde. The difference between NER and
UVER still persists without functional checkpoint control.
background,
rad13 disruption leads to a stronger effect on UV survival
than uvde disruption (Fig. 4b). This suggests
that possible checkpoint control-related effects caused by
rad13 disruption is not the major reason for the apparent
difference between NER and UVER.
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Fig. 5.
Contribution of NER and UVER to UV-resistance
of S. pombe in growing phase (a) or
in stationary phase (b). uvde
disruption causes a larger increase of UV sensitivity in growing phase.
Also note that wild type cells in exponential phase are more
UV-sensitive than those in stationary phase. , wild type; ,
uvde; |, rad13; , uvde
rad13.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
background. This situation is somewhat similar to
that between TCR and GGR in S. cerevisiae. Although
rad26 disruption hardly increases the UV sensitivity of wild
type S. cerevisiae, it causes a dramatic effect in the GGR
minus rad16 strain (31). This also supports the idea that
in S. pombe UVER acts as an alternative to GGR, and that it
partially (but importantly not completely) compensates TCR defect (see below).
background showed that
a checkpoint control-related process is not involved in the difference between NER and UVER. Differential ability to repair thymine glycol, which is known to be repaired by NER in vitro, but not by
UVER, is also shown not to be able to explain the difference. This
result, however, does not exclude the possible existence of other minor UV damage, which is differentially repaired by NER and UVER and relevant to UV survival. NER is likely to act on a broader range of
damage substrate than UVER, and it is reasonable to suppose such UV
damage exists, although we do not have any positive evidence for this
hypothesis at the moment.
strain and a rad16 rad26 double mutant. Our preliminary work showed
that also in S. pombe, rhp16
rhp26 uvde is much more UV-resistant than
rad13 uvde, suggesting that
Rhp26-independent TCR significantly contributes to UV
survival.3 It would be
interesting to find mutants lacking both Rhp26-dependent and -independent TCR and to study how this affects the UV survival. Such a mutant would be also very useful to clarify the molecular mechanism of TCR.
ACKNOWLEDGEMENTS
strain. We also thank Dr. S. McCready
for a critical reading of the manuscript and helpful comments. We have
been informed by Dr. J. A. Brandsma and Professor Dr. J. Brouwer
that they analyzed TCR of S. pombe independently. We
appreciate their communication of the results before publication.
FOOTNOTES
To whom correspondence should be addressed: Div. of Radiation Life
Science, Research Reactor Institute, Kyoto University, 1010 Ooaza-Noda,
Kumatori-Cho, Sennan-Gun, Osaka 590-0494, Japan. Tel.: 81-724-51-2392;
Fax: 81-724-51-2628; E-mail: shinji@rri.kyoto-u.ac.jp.
ABBREVIATIONS
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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