From the Medical Genetic Centre, Department of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
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ABSTRACT |
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Transcription-coupled DNA repair (TCR) is
responsible for the preferential removal of DNA lesions from the
transcribed strands of RNA polymerase II transcribed genes.
Saccharomyces cerevisiae rad26 mutants and cells from
patients suffering from the hereditary disease Cockayne syndrome
display a TCR defective phenotype. Whether this lack of preferential
repair has to be explained by a defect in repair or in general
transcription is unclear at present. To discriminate between both
possibilities, we analyzed repair of UV-induced cyclobutane pyrimidine
dimers at single base resolution in yeast cells lacking
RAD26, the homolog of the Cockayne syndrome B gene.
Disrupting RAD26 affects nucleotide excision repair of transcribed DNA irrespective of the chromatin context, resulting in
similar rates of removal for individual cyclobutane pyrimidine dimers
throughout the transcribed strand. Notably, repair of transcribed sequences in between core nucleosomal regions is less efficient compared with nontranscribed DNA at these positions, pointing to a
nucleotide excision repair impediment caused by blocked RNA polymerase.
Our in vivo data demonstrate that the TCR defect in rad26 mutant cells is not due to a general transcription
deficiency but results from the inability to release the transcription
complex trapped at sites of base damage.
Ever since the first observation of transcription-coupled DNA
repair (TCR),1 it has been
suggested that RNA polymerase II (RNAPII) stalled at the site of base
damage acts as a molecular beacon to signal repair proteins toward the
damaged strand (1, 2). Studies in Escherichia coli have
provided a frame of reference for such a mechanism by the
identification of a molecular matchmaker that physically links RNAP
transcription and nucleotide excision repair (NER). It was shown that
the mfd encoded transcription-repair coupling factor (TRCF)
is able to release blocked RNAP and via interaction with UvrA
stimulates efficient targeting of the E. coli NER machinery
to the damaged template (3). Consequently, cells lacking TRCF fail to
remove cyclobutane pyrimidine dimers (CPDs) preferentially from
transcribed strands (4). In human cells, a TCR-deficient phenotype has
been associated with Cockayne syndrome (CS), whereas in yeast Rad26,
the Cockayne syndrome B (CSB) homolog has been implicated in TCR (5,
6). Primarily based on the resemblance in the molecular defect and the
limited sequence similarity between CSB/RAD26 and
mfd, it has been proposed that the encoded proteins operate
as the eukaryotic analogs of the TRCF either to disrupt or to modify
the RNAPII-DNA interaction at the site of base damage and to recruit
NER proteins providing a molecular basis for the enhanced repair rate
of the transcribed strand.
An alternative hypothesis that implicates CSB/Rad26 as an intrinsic
component in the transcription machinery has been suggested to explain
the phenotypic complexity of patients suffering from CS (7).
Hypothetically, any protein required for efficient transcription by
RNAPII, e.g. transcription-initiation or elongation factors,
could instigate a TCR deficiency when mutated. In such a scenario, a
failure to efficiently transcribe through a template molecule will
automatically reduce or abrogate the damage-signaling function of RNAP
simply because the complex does not encounter the damage within a
certain time frame. Although not easily reconciled with the notion that
both yeast and human can cope with the complete absence of Rad26/CSB,
this hypothesis has recently gained impetus by the observations that
CSB cells display a reduced level of transcription (8) and that
purified CSB enhances elongation by RNA polymerase in vitro
(9).
Based on the present biochemical and genetic data the question of
whether Rad26/CSB is a bona fide repair factor or a general transcription efficiency factor is unanswered, especially because both
scenarios predict a reduction in the rate of lesion removal from
transcribed DNA when the encoding genes are mutated. However, we
reasoned that the two scenarios can be distinguished by the characteristics of repair when analyzed at nucleotide resolution. If a
TCR defect in rad26 cells results from impaired
transcription, in other words from the absence of RNAPII-mediated
damage detection, then lesions in the transcribed strand are repaired
by the same mechanism that operates on lesions in the nontranscribed
strand i.e. global genome repair (10). Because this mode of
repair in the URA3 nontranscribed strand is highly
influenced by the chromatin environment of the damage (11, 12), this
should also be observed for repair of lesions in the transcribed strand in rad26 mutant cells. However, if a TCR defect results from
a deficiency to act upon stalled RNAPII molecules at the site of base
damage, the stalled polymerase will still be the substrate to act upon
in subsequent steps of NER. Because uniform repair rates for individual
damages in the transcribed strand in wild type cells indicates that RNA
polymerase II-mediated damage recognition is not profoundly influenced
by the sequence or chromatin context (12, 13), this model predicts that
individual lesions will be equally affected by a Rad26 deficiency, and
similar repair rates for differently positioned dinucleotides will be observed.
Strains--
The Saccharomyces cerevisiae NER
proficient (RAD+) strain used for this study is
W303-1B, genotype: MAT CPD Analysis--
Cells diluted in chilled phosphate-buffered
saline were irradiated with 254 nm UV light (Philips T U V 30W) at 70 J/m2, collected by centrifugation, resuspended in complete
medium, and incubated for various times in the dark at 28 °C prior
to DNA isolation. DNA samples (25 µg) were digested with appropriate endonucleases and, precipitated, and URA3 fragments were
isolated and end-labeled as described previously (14) using
fragment-specific oligonucleotides (sequences available upon request).
CPDs were identified using T4endoV. DNA samples were divided in two
equal parts. One was incubated with T4endoV, the other was mock
treated. Samples were subjected to spin column chromatography and
lyophilized to small volumes. Approximately equal amounts of cpms were
loaded on 6% denaturing acrylamide gels alongside Maxam-Gilbert
sequencing reactions to identify the CPD positions. After drying,
autoradiograms were prepared from the gels.
Quantification of Repair Rates--
Multiple autoradiograms were
obtained with different exposure times to allow signal determination
within the linear range of Kodak X-OMATTM-AR scientific imaging films
for each individual photoproduct. Autoradiograms were scanned (UMAXTM,
Astra 1200S) at 600 dpi and analyzed using Image masterTM software
(Pharmacia). Background levels were subtracted, and gel band
intensities were corrected for loading variations. OD values were
plotted against repair time for lesions that gave sufficient signal to
background ratios. Repair half-times (t1/2), defined
as the time at which 50% of the initial damage (signal at
t = 0) was removed, were derived from these plots.
We first analyzed whether the absence of Rad26 had any effect on
the removal of DNA damage from the nontranscribed strand of the yeast
URA3 locus. Fig. 1A
shows an example of repair analysis of UV-induced CPDs performed on the
URA3 nontranscribed strand in rad26 mutant cells.
Slow repair coincides with the internal protected region of positioned
nucleosomes, whereas lesions in linker DNA or at the boundaries of the
nucleosome are removed more efficiently. For example, lesions induced
at sequences occupied by the core of nucleosome U2 (exemplified by the
arrows in Fig. 1A) persist even after 2 h of
repair, whereas most flanking sequences are repaired after 80 min. When
the kinetics of individual dimer sites were determined from multiple
independent repair experiments, no significant difference was detected
between repair of the nontranscribed strand in
RAD+ versus rad26 cells
(Table I). This indicates that (i)
nontranscribed DNA is repaired independent of Rad26, in line with
previously published data (6, 13) and (ii) the chromatin architecture of the URA3 locus, as measured by the influence of
positioned nucleosomes on NER, is not changed in the absence of
Rad26.
INTRODUCTION
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Abstract
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Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
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References
ho can1-100 ade2-1 trp1-1 leu2-3,112
his3-11,15 ura3-1, which was rendered URA3 by transformation of a linear polymerase chain reaction fragment containing the complete locus. Uracil prototrophs were checked by
Sanger sequencing for proper recombination at its chromosomal position.
Subsequently the rad26 disruption was introduced into this
background by one-step gene replacement. Strains were kept on selective
YNB (0.67% yeast nitrogen base, 2% glucose, 2% bacto agar)
supplemented with the appropriate markers. Cells were grown in complete
YEPD medium (1% yeast extract, 2% bacto peptone, 2% glucose) at
28 °C under vigorous shaking.
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
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Fig. 1.
Repair of UV-induced CPDs at single
nucleotide resolution along the URA3 locus.
A, CPD removal from the nontranscribed strand (nucleotides
60 to 275 relative to the start codon ATG designated +1) in
rad26 mutant cells irradiated with 70 J/m2 and
assayed at 0, 40, 80, and 120 min after irradiation. Samples mock
treated or treated with the CPD-specific enzyme T4endoV are
denoted and +, respectively. A black box indicates
the internal protected regions of nucleosomes U2 (15). B,
CPD removal from the transcribed strand (nucleotides 387 to 601) in
repair proficient cells (RAD+) compared with
repair in rad26 mutant cells. The black box
indicates the internal protected region of nucleosome U4 (15).
C, graphic representation of quantified CPD repair rates
along part of the nontranscribed (upper panel) and
transcribed strand (lower panel) of the URA3
locus in rad26 mutant cells. Repair half-time values,
defined as the time at which 50% of the initial CPD signal was
removed, were calculated for each individual CPD position with a
sufficient signal to noise ratio and are plotted above their
corresponding dipyrimidine position.
The URA3 nontranscribed strand is repaired independently on the RAD26
gene product
mutant
cells irradiated with 70 J/m2 UV light. Repair half/time
values, defined as the time at which 50% of the initial CPD signal was
removed, were calculated for individual CPD positions. The values shown
are the average of two independent experiments for each genetic
background.
We then monitored repair of CPDs in the URA3 transcribed strand. Fig. 1B shows, as expected, that the rate at which CPDs are removed from the transcribed strand is severely affected when RAD26 is disrupted. Whereas CPDs are removed with a t1/2 of approximately 8 min in RAD+ cells, the t1/2 in rad26 cells increases to 52 min, when individual dimer sites are averaged. However, in contrast to repair of the nontranscribed strand where lesions only at core nucleosomal regions persist after 120 min, here, all dimer sites are still visible after 120 min of repair. To allow a visual comparison between the repair patterns of the nontranscribed and the transcribed strands in rad26 cells, we determined the repair kinetics along both strand over an approximately 550-base pair DNA region, occupied by nucleosomes U2-U4 (15), and plotted the repair half-times above the corresponding dinucleotide sequence (Fig. 1C).
In contrast to the high level of repair heterogeneity on the
nontranscribed strand, repair of individual dimer sites in the transcribed strand is markedly more homogeneous and not influenced by
the chromatin environment of the damaged DNA and in this respect resembles repair in NER proficient cells. This indicates that the
transcribing polymerase, also in the absence of Rad26, is capable of
suppressing the inhibitory effect of chromatin at the core regions of
positioned nucleosomes and thus favors an explanation in which RNAP II
transcription proceeds normally and is blocked when it encounters a CPD
in the transcribed strand, irrespective of the exact position of the
lesion. As this blocked transcription complex constitutes an equal
obstacle for each individual dimer site in subsequent steps of NER,
similar repair kinetics for individual lesions will result.
Importantly, Fig. 1C also demonstrates that at certain
positions in the transcribed strand, for instance nucleotides 50 to
10, 220 to 250, and 540 to 570, CPDs are removed significantly slower
than at neighboring sequences in the opposite, nontranscribed strand.
The observation that CPDs positioned in between core nucleosomal regions, where global genome repair can operate efficiently, are removed faster from the nontranscribed strand as compared with the
transcribed strand indicates that NER at these in principle more
accessible sequences is obstructed in the transcribed strand.
Our in vivo data strongly suggest that Rad26 is required for the efficient processing of NER blocking transcription complexes. Whether this feature resides in the Rad26 protein itself or in additional factors that are required/recruited remains elusive. Purified CSB, the human homolog of Rad26, on its own was not able to dissociate stalled RNAPII in a defined in vitro system (19). However, Rad26 and CSB contain DNA-dependent ATPase activity (16, 17) and belong to the Swi2/Snf2 family of DNA-dependent ATPases. The notion that some members of this gene family are implicated in remodelling specific DNA-protein interactions (reviewed in Ref. 18) supports a function of Rad26/CSB in modifying DNA/RNAPII contacts at the site of transcriptional arrest. In such a scenario, alternative routes to destabilize (or displace) RNA pol II might compensate for the loss of CSB/Rad26 in TCR.
Previously, we have reported that efficient removal of CPDs from a small but distinct region immediately downstream of transcription initiation does not depend on Rad26, because these lesions are repaired strand-specifically in rad26 mutants (13). A similar observation has been made for human CSB and more recently CSA cells (20, 21), demonstrating that Rad26/CSB is required for efficient TCR only during the elongating stages of RNAPII transcription. In the light of the results presented here, these data could indicate that Rad26/CSB is not essential for the alleviation of NER inhibition due to stalled RNAPII complexes prior to the point where the transition from transcription-initiation to competent transcription-elongation occurs. The question of whether this is a direct result of the presence of TFIIH in the transcription machinery during the first steps of nascent mRNA synthesis (as discussed in Ref. 13) or alternatively results from a less stable RNAPII-damaged DNA complex when the polymerase is still in its initiation mode is unanswered at present time and probably awaits the development of an in vitro TCR system that has not been established yet with eukaryotic factors.
Finally, one can envisage that the processing of stalled RNAPII
complexes is not confined to NER substrates but can also extend to base
excision repair substrates and even to naturally occurring pause sites.
The observation that some forms of oxidative damage (which are
generally not repaired by NER but by base excision repair) are not
repaired strand specifically in CS cells (22) indicates that the
function of CS extends to a broader substrate range that might
ultimately be just a stalled RNA polymerase, irrespective the cause of
stalling. Consequently, a subtle transcription-elongation defect could
result when Rad26 in yeast or CSB in humans is mutated. Maybe more
important than the inefficient removal of lesions from transcribed DNA,
a failure to recover trapped RNA polymerase II complexes as suggested
by our in vivo repair analysis might contribute to the
complex clinical phenotypes observed in Cockayne syndrome (7, 23).
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ACKNOWLEDGEMENTS |
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We appreciate comments and discussion from Richard Verhage and Judith Tasseron-de Jong.
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FOOTNOTES |
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* This work was supported by the J. A. Cohen Institute for Radiopathology and Radiation Protection.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.: 31-071-5274755;
Fax: 31-071-5274537; E-mail: Brouwer{at}chem.leidenuniv.nl.
The abbreviations used are: TCR, transcription-coupled DNA repair; RNAP, RNA polymerase; RNAPII, RNA polymerase II; NER, nucleotide excision repair; TRCF, transcription-repair coupling factor; CPD, cyclobutane pyrimidine dimer; CS, Cockayne syndrome; CSB, CS group B homolog.
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REFERENCES |
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