From the Laboratories for Organismal Biosystems,
Graduate School of Frontier Biosciences, Osaka University and
§ Core Research for Evolutional Science and Technology
(CREST), Japan Science and Technology Corporation, 1-3 Yamadaoka,
Suita, Osaka 565-0871, Japan, and the ¶ Faculty of Bioscience and
Biotechnology and the Frontier Collaborative Research Center, Tokyo
Institute of Technology, Yokohama 226-8501, Japan
Received for publication, August 8, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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The blockage of transcription elongation by RNA
polymerase II (pol II) is thought to be a trigger for
transcription-coupled repair in the pathway of nucleotide
excision repair. Purified pol II and oligo(dC)-tailed templates
containing a single non-bulky DNA lesion on the transcribed strand such
as an apurinic/apyrimidinic (AP) site, uracil, or 8-oxoguanine (8-oxoG)
were used for transcription elongation assays. In this system pol II
could bypass both the AP site and uracil without pausing and insert
cytosine opposite the AP site and either guanine or adenine opposite to
uracil. Thus, the AP site on the DNA templates could lead to correct
transcription only if depurination at guanine occurred, whereas uracil
generated either the correct transcriptional product or an incorrect
one with a G:C to A:T transition. In the case of 8-oxoG, pol II stalled at the lesion, but sometimes bypassed it and inserted a cytosine residue or the incorrect adenine residue leading to a G:C to T:A transversion. These findings indicate that 8-oxoG lesions caused a
blockage of transcription elongation and/or the misincorporation of a
ribonucleotide by pol II, implying the initiation of
transcription-coupled repair of 8-oxoG and/or transcriptional mutagenesis.
The genome as a carrier of genetic information in living cells is
vulnerable to DNA-damaging agents of both endogenous and environmental
origins. Once the DNA has lesions, essential DNA-dependent processes are interfered with and mutation or cell death can occur. Thus, DNA repair removes lesions from DNA to maintain genomic integrity. To remove DNA lesions, cells have two major repair pathways;
nucleotide excision repair
(NER)1 operating primarily on
bulky helix-distorting damage caused by environmental mutagens and base
excision repair (BER) for non-bulky and non-helix-distorting DNA
modifications caused by endogenous and some chemical carcinogen-induced
damage (1-3). When RNA polymerase II in the elongation phase
encounters DNA damage that blocks transcription, transcription-coupled
repair (TCR), a specialized pathway that efficiently removes lesions on
the transcribed strand, operates to counteract the immediate and
cytotoxic response of the interference (4-6). The transcribed strand
is repaired much faster by TCR than is the non-transcribed strand or
inactive regions by global genome repair (GGR) (7). TCR is found not
only in eukaryotes but also in prokaryotes (5). Its importance in
humans has been suggested by studies of autosomal recessive human
inherited diseases: xeroderma pigementosum (XP) and Cockayne's
syndrome (CS) (8). NER-deficient XP is classified into seven genetic
complementation groups (XP-A to XP-G). Both the TCR and GGR subpathways
of NER are defective in all these groups except for XP-C and XP-E, in which only GGR is impaired. CS is mostly classified into two genetic complementation groups (CS-A and CS-B). XP-B patients and certain individuals with XP-D or XP-G show features of CS in addition to XP
symptoms (XP-B/CS, XP-D/CS, and XP-G/CS). CS-A and CS-B cells are
deficient in TCR but proficient in GGR.
Although TCR was first thought to be a specific subpathway of NER, it
was recently reported that 8-oxoguanine (8-oxoG) and thymine
glycol are also repaired by a TCR subpathway of BER (TCR of
oxidative damage), and this subpathway is specifically deficient in
CS-A, CS-B, XP-B/CS, XP-D/CS, and XP-G/CS but proficient in XP-A. These
results suggest that the symptoms of CS, such as postnatal growth
failure and neurological complications, result from a defect in the TCR
of oxidative damage, the repair requiring XPG and TFIIH as well
as CSA and CSB (9, 10).
8-oxoG is an important premutagenic lesion due to its potential to
mispair with adenine, thus generating G:C to T:A transversions (3). Its
biological significance is revealed by the existence of a three-tiered
defense system composed of the proteins MutT (8-oxodGTPase), Fpg (DNA
glycosylase/AP lyase), and MutY (DNA glycosylase) in Escherichia
coli. Inactivation of any of the genes of these three generates a
mutator phenotype attributed to the persistence of 8-oxoG in DNA or in
the pool of deoxynucleoside-triphosphates (11, 12). In human cells, the
hOGG1 gene encodes a DNA glycosylase/AP lyase that
catalyzes the removal of 8-oxoG and incises DNA at the resulting AP
site. However, it was reported that the TCR of 8-oxoG was proficient in
mOGG1 ( Although the mechanism of TCR is unknown, the process is thought to be
initiated by a blockage of transcription elongation by RNA polymerase
II at DNA lesions (6, 23). Considering that 8-oxoG is neither a bulky
nor a helix-distorting lesion (3), it is unlikely that it blocks RNA
polymerase II. Moreover, it was reported that DNA polymerases
preferentially insert an adenine residue opposite 8-oxoG, leading to
A:8-oxoG mispairs (14, 15) and that E. coli or T7 RNA
polymerase efficiently bypasses such a lesion without pausing or
arresting during elongation by inserting either an adenine or a
cytosine residue (16, 17). To assess the contribution of RNA polymerase
II to the TCR of 8-oxoG in mammalian cells, we investigated whether the
purified RNA polymerase II stalls at the AP site, uracil, and 8-oxoG on
the transcribed strand during the elongation reaction.
Enzymes and Chemicals--
RNA polymerase II (pol II) was
prepared from HeLa nuclear pellets as described (18). E. coli formamidopyrimidine-DNA glycosylase (Fpg) and human
apurinic/apyrimidinic endonuclease (AP endonuclease) were purchased
from Trevigen. Uracil DNA glycosylase (UDG) was obtained from
USB. NTPs were from Amersham Biosciences, and restriction enzymes were from New England Biolabs and TOYOBO.
Construction of DNA Templates Containing a Site-specific DNA
Lesion--
The 24-mer oligodeoxyribonucleotides containing an AP site
analogue (3-hydroxy-2-(hydroxymethyl)-tetrahydrofuran) (dSpacer), uracil, or 8-oxoG shown in Fig. 1A were synthesized and
purified by Qiagen. To generate a substrate suitable for transcription elongation by mammalian RNA pol II on a lesion, oligonucleotides containing a lesion were 5'-phosphorylated by T4 polynucleotide kinase
using ATP and incorporated into covalently closed circular DNA as
described (19). The plasmid pBlueScript II KS-GTG (pBSII KS-GTG) was
constructed by replacing the ApaI-KpnI fragment
of pBlueScript II KS- with the synthetic 99-bp DNA duplex
containing the damage site (Fig. 1A). The purity of each DNA
substrate was assessed by agarose gel electrophoresis. To confirm the
proper insertion of a lesion into pBSII KS-GTG, each DNA substrate was incubated with the indicated enzyme and analyzed by 1% agarose gel
electrophoresis. For the elongation reaction by pol II, an oligo(dC)-tailed template was prepared as described (20) from the
covalently closed circular DNA containing a site-specific DNA lesion.
The pBSII KS-GTG containing a lesion at a specific site was digested
with PstI, and poly(dC) tails of 35-40 nucleotides were
added to the 3' ends by terminal deoxynucleotide transferase using
dCTP. After digestion of the DNA with SmaI to generate two fragments, the DNA substrate fragment with a lesion was purified on an
agarose gel and used as a template.
RNA Polymerase II Elongation Reactions--
For the
transcription elongation assay (21), 20 µl of reaction mixtures
containing 50 ng of dC-tailed template and 0.5 µl of pol II in a
buffer (10 mM HEPES (pH 7.9), 50 mM KCl, 10%
glycerol, 0.1 mM EDTA, 3.2% polyethylene glycol, 0.25 mM dithiothreitol, 6 mM MgCl2, and
8 units RNase inhibitor (Promega)) were preincubated for 30 min at
30 °C. Elongation was started by adding 5 µl of NTP mixture (50 µM each of ATP, CTP, and GTP, 10 µM UTP,
and 1 µCi [ RT-PCR and RNA Sequencing--
RNA transcripts for sequence
analysis were extracted from mixtures of the elongation reaction in the
presence of a cold NTP mixture (50 µM each of ATP, CTP,
GTP, and UTP) with an RNA purification kit (Qiagen) and then treated
with RNase-free DNase I (TAKARA) as recommended by the supplier.
Purified RNAs were employed to synthesize PCR products with a One-step
RT-PCR kit (Qiagen) or a cMaster RT-PCR system and RT kit
(Eppendorf) and primers (751-770) 5'-GCCCTGCTGCCATGCGCGG-3' and
(1003-984) 5'-ACTCATTAGGCACCCCAGGC-3'. The PCR products (253 bp)
were purified with a PCR purification kit (Qiagen), analyzed by
electrophoresis on a 2% agarose gel or on a 6% non-denatured
polyacrylamide gel, and then sequenced using the BigDye Terminator kit
(Applied Biosystems) with PCR primers.
DNA Templates Containing DNA Lesions at Specific Sites--
To
analyze the transcription elongation at DNA lesions (AP site, uracil,
and 8-oxoG) by human RNA polymerase II, oligo(dC)-tailed templates were
generated from purified closed circular duplex DNA substrates
containing a lesion at a specific site (Fig.
1A). A 3054-bp substrate,
designated pBSII KS-GTG, contains these DNA lesions within the
recognition sequence (5'-GTGCAC-3') of the restriction enzyme
Alw44I (Fig. 1A). When control DNA substrate (with no damage) was digested with Alw44I, three DNA
fragments (1246 bp, 1078 bp, and 730 bp) were observed (Fig.
1B, lanes 2). DNA substrates containing either an
AP site or uracil were also completely digested by Alw44I
(Fig. 1B, lanes 4 and 6), while DNA
substrate with 8-oxoG was completely resistant to the cleavage (Fig.
1B, lane 8), indicating that ~100% of the
substrate contained 8-oxoG lesions. When closed circular DNA substrates
containing either uracil or 8-oxoG were incubated with DNA repair
enzymes (uracil DNA glycosylase and HAP endonuclease, or Fpg
glycosylase), those with the lesion were cleaved to an open circular
form but the control substrate was not. The partial digestion of the
template with DNA repair enzymes was due to an insufficient
concentration of enzymes available from the supplier (Fig. 1,
C and D). The AP site was not tested because the
dSpacer, a (3-hydroxy-2-(hydroxymethyl)-tetrahydrofuran) that was used
as an analogue of the AP site in the experiments, is known to be
resistant to the nicking activity of AP lyase. These results, as well
as those from the sequence analysis of RT-PCR products (Fig.
3C), indicate the presence of site-specific DNA damage in
the DNA substrate.
RNA Polymerase II Elongation Reaction--
Transcription
elongation by pol II was carried out with oligo(dC)-tailed templates
containing no damage (control template), AP site, uracil, or 8-oxoG.
(The lesions were built on the template strand.) pol II could
synthesize transcripts using the control template as expected (Fig.
2B, lanes 1 and
2). Using control template digested with either
Alw44I or N.BstNBI, which partially generates double or single strand breaks under the conditions, pol II produced 129 nt (from the PstI site to the Alw44I site)
and 101 nt (from the PstI site to the N.BstNBI
site) transcripts (Fig. 2, A and B, lanes
3-6). When the template containing either the AP site or uracil
was used, pol II could catalyze the synthesis of the transcript (Fig.
2B, lanes 7-10). On the other hand, using the template with 8-oxoG, pol II synthesized 128-nt transcripts from the
PstI site to 8-oxoG (Fig. 2B, lanes 11 and 12, arrow) and bypassed the lesion as well
(Fig. 2B, bracket), suggesting that some pol II
stalled at the 8-oxoG site while some bypassed the lesion. When these
templates containing a base lesion were incubated with T7 RNA
polymerase (T7 RNA pol), instead of human pol II, T7 RNA Pol did
not stall at the lesion (data not shown).
To confirm the pausing of 8-oxoG on transcription elongation by pol II
at 8-oxoG, time course experiments were carried out (Fig.
2C), and the transcripts (shown by brackets in Fig.
2C) were quantified (Fig. 2D). In the case of the
templates containing no damage, AP site, or uracil, pol II generated
similar amounts of transcripts beyond the lesion in a
time-dependent manner. In contrast, the amount of bypassed
transcripts that was synthesized for 12 min by pol II using the
oligo(dC) template containing 8-oxoG, was less than one-third that of
the others. These results indicate that 8-oxoG lesions partly inhibited
the transcription elongation by pol II.
RT-PCR and RNA Sequencing--
To examine the nucleotide
preference for incorporation opposite a lesion, we next investigated
the sequences of DNA fragments produced by RT-PCR using bypassed
transcription products. The RT-PCR products (253 bp) were observed only
when a purified RNA fraction was incubated with RT (Fig.
3A, upper panel).
Incubation of the RNA fraction with RNase I generated no RT-PCR
products (Fig. 3A, lower panel). These results
indicated that under our experimental conditions, the PCR product was
generated by the transcripts but not by the template DNA. The RT-PCR
products were digested with Alw44I. If there are mutations
at a restriction site (GTGCAC) containing a lesion, PCR products from
the transcripts should be resistant to cleavage. The results (Fig.
3B) showed that the PCR products from the uracil template
were partially resistant, but the products derived from templates with
no damage, AP site, or 8-oxoG were not resistant. Sequence analyses
(Fig. 3C) indicated that in the transcripts from the
template with no damage or AP site, no change of sequence was observed.
In contrast, the uracil lesion partially generated G:C to A:T
transitions. The results are consistent with the finding that
UDG-deficient E. coli cells have more G:C to A:T transitions
in DNA and mRNA (3, 17, 22). As for 8-oxoG, pol II inserted not
only cytosine but also adenine opposite the lesion, although less
adenine was incorporated than cytosine.
Effects of Adding Nucleotides on pol II-stalled Transcription at
8-oxoG--
It is known that the 8-oxoG lesion is mutagenic because of
its ambiguous pairing with cytosine and adenine (15, 16). To examine
which pairing causes pol II to stall, the product of transcription stalled at the 8-oxoG site was quantified at different concentrations of CTP, ATP, or GTP. As the CTP concentration increased, the amount of
transcript at pausing sites increased (Fig.
4B, lanes 1-3 and C, circle), whereas as the ATP concentration
increased the amount decreased (Fig. 4B, lanes
5-7 and C, square). When the concentration of GTP or UTP was increased, no effect was observed (Fig.
4A, lanes 1 and 4, and data not
shown). In addition, sequence analyses of the bypassed transcription
products (Fig. 4D) revealed that G:C to T:A transversions
were increased at high ATP concentrations, but decreased at high CTP
concentrations (Fig. 4E and see the panel for
8-oxoG in Fig. 3C). Considering that the flanking sequence of the lesion is very rich in TC, high levels of ATP may indirectly improve read-through by facilitating elongation around the lesion. However, this is not the case because high levels of GTP did not affect
the read-through. Although we did not directly examine the inserted
nucleotide of the paused transcripts, our results indicate that pol II
stalled at 8-oxoG lesions due to the insertion of a cytosine, while it
bypassed the lesions by inserting adenine.
In this study, we found that elongating pol II can efficiently
bypass AP sites by inserting cytosine residues and bypass uracil lesions by inserting either adenine or guanine residues. On the other
hand, a significant fraction of pol II stalled at 8-oxoG lesions by
inserting the correct base, while the rest bypassed the site using
either correct or incorrect base insertions. The results are
summarized in Table I.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) cells, suggesting that the TCR of oxidative
damage occurs independent of OGG1 (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP) and terminated by adding 100 µl of Stop buffer (7 M urea, 0.35 M NaCl, 10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 1% SDS) at the times indicated. The purified transcripts were resuspended in
formamide loading dye (98% deionized formamide, 25 mM Tris borate-EDTA, 0.025% bromphenol blue, and 0.025% xylene cyanol) and
separated on a denatured 6% polyacrylamide gel. The dried gels were
analyzed using a FUJIFILM BAS 2500 bio-image analyzer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Covalently closed circular duplex DNA
containing a single DNA base lesion. 24-mer oligonucleotides
containing a DNA base lesion and the plasmid pBSII KS-GTG are shown
diagrammatically. Four Alw44I restriction sites are
indicated. One Alw44I site (underlined) overlaps
the region containing a lesion. Unique PvuI and
SmaI restriction enzyme sites are also indicated.
B, 1% agarose gel demonstrating the presence of the 8-oxoG
lesion. pBSII KS-GTG containing a single lesion at a specific site was
digested with Alw44I. C, DNA substrates
containing uracil were incubated with UDG and HAP endonuclease
(APE). D, DNA substrates containing 8-oxoG were
incubated with or without Fpg glycosylase. The mobility of the
covalently closed circular (CCC) and nicked circular
(NC) pBSII KS-GTG is indicated along the side of the
gel.
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Fig. 2.
Elongation reaction of pol II with oligo
dC-tailed template containing a single DNA base lesion. A,
oligo(dC)-tailed template containing a lesion. The lesions have been
built on the template strand. Alw44I and N.BstNBI
generate double and single strand breaks, respectively. B,
autoradiograph after denaturing polyacrylamide gel electrophoresis of
the transcripts demonstrating the elongation reaction through the
region containing a DNA base lesion. Oligo(dC)-tailed templates were
incubated with pol II and radioactively labeled nucleoside
triphosphates (see "Experimental Procedures"). Control templates
without DNA base lesion (lanes 1 and 2).
Templates partially digested with Alw44I or
N.BstNBI (lanes 3-4 and 5-6).
Template containing an AP site (lanes 7 and 8),
uracil (lanes 9 and 10), or 8-oxoG (lanes
11 and 12). M, size markers (MspI
digest of pBR322 labeled with [3'-32P]). The band
labeled with an asterisk is a natural pausing site.
C, time course of pol II elongation reactions with templates
containing no lesion (lanes 1-4), AP site (lanes
5-8), uracil (lanes 9-12), or 8-oxoG (lanes
13-16). Pausing site and elongation products are indicated by an
arrow and brackets, respectively. The band
labeled with an asterisk is a natural pausing site.
D, quantification of the bypass products. The elongation
products in brackets in C were quantified using a FUJIFILM
BAS 2500 bio-image analyzer. Photo-stimulated luminescence
(PSL) was used as counts of products.
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Fig. 3.
Analysis of transcript from template
containing a base lesion. A, upper panel: 2%
agarose gel analysis of the PCR products (253 bp) with (+) or without
( ) RT. Lower panel: 2% agarose gel analysis of the RT-PCR
products from RNA treated with (+) or without (
) RNase I at 37 °C
for 120 min. B, 6% polyacrylamide gel electrophoresis of
the RT-PCR products derived from bypassed transcripts with (+) or
without (
) Alw44I digestion. M:
HindIII digest plus øx174-HaeIII digest.
C, sequence analysis of bypassed transcripts. Signal peaks
of sequence correspond to the Alw44I site (GTGCAC).
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Fig. 4.
Effect of ATP or CTP concentration on
transcription elongation reaction using a template containing an 8-oxoG
lesion. A, oligo(dC)-tailed templates containing an 8-oxoG
lesion were preincubated with pol II at 30 °C for 30 min, and then
the NTP mixture (50 µM each of ATP, CTP, and GTP, 10 µM UTP, and 1 µCi [ -32P]UTP)
(A, lane 1) or the NTP mixture supplemented with
200 µM of ATP, CTP, or GTP (A, lanes
2-4) was added. The transcript paused at a lesion is indicated by
an arrow. Lane M, size markers (MspI
digest of pBR322 labeled with [3'-32P]).
B, the NTP mixture (lane 4) or the NTP mixture
supplemented with several concentrations of ATP or CTP (lane
1 and 7:400 µM, lane 2 and 6:200
µM, lane 3 and 5:100 µM) was
added after preincubation of the template with pol II. The mixtures
were further incubated at 30 °C for 12 min for transcription
elongation. The transcript paused at a lesion is indicated by an
arrow. Lane M, size markers (MspI
digest of pBR322 labeled with [3'-32P]). C,
quantification of stalled transcripts. The density of the bands
corresponding to the stalled transcript in B was measured
using a FUJIFILM BAS 2500 bio-image analyzer and divided by the value
corresponding to the transcript obtained in the presence of the control
level of nucleotides (B, lane 4, 50 µM each) to give the relative amounts of stalled
transcripts. D, sequence analysis of the bypassed
transcription products. Upper panel: 2% agarose gel
demonstrating the presence of the PCR products from bypassed
transcripts at high concentrations of ATP (400 µM) or CTP
(400 µM) with (+) or without (
) RT. Lower
panel: 6% polyacrylamide gel electrophoresis demonstrating the
presence of mutation at the 8-oxoG site in the bypassed transcripts
produced by RT-PCR at high concentrations of ATP (400 µM)
or CTP (400 µM). The products were incubated with (+) or
without (
) Alw44I. The mutation rendered the RT-PCR
product resistant to the digestion with Alw44I.
E, sequence analysis of bypassed transcripts at high
concentrations of ATP(400 µM) or CTP (400 µM). Signal peaks of sequence correspond to the
Alw44I site (GTGCAC).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Stall of RNA polymerase II and inserted base in transcripts on damaged
templates
AP sites are the most frequent DNA base lesions in cells and potentially genotoxic and mutagenic (1, 3). These lesions are generated by spontaneous depurination at guanine at an estimated rate of ~9000/day/genome in humans (24). Our results indicate that pol II could insert cytosine residues opposite AP sites without pausing. When depurination occurred at guanine, no mutant proteins were generated through a bypass elongation reaction at the AP site. The mechanism by which pol II inserts cytosine opposite AP sites without any Watson-Crick pairing information is unknown as yet. It is known that E. coli RNA or DNA polymerase exhibits the preferential insertion of adenine opposite AP sites (4, 30). Therefore, in mammals, bypass transcription at the AP site might play a role in maintaining genome integrity. However, we cannot rule out the possibility, due to the limitation of the sensitivity of our assay that pol II inserts bases other than cytosine at low frequency.
Uracil in DNA is frequently generated by the deamination of cytosine in a pH- and temperature-dependent manner (3, 24). Uracil can pair with adenine without blocking DNA replication and therefore is very mutagenic. In fact, UDG-deficient E. coli shows a high frequency of spontaneous G:C to A:T transitions. However, the present study indicates that in transcription, pol II inserts guanine and adenine opposite uracil without blocking transcription elongation. E. coli RNA polymerase also can insert guanine and adenine opposite lesions in vitro (17). Thus, uracil in DNA would cause transcriptional infidelity and produce mutant proteins. In fact, it has been reported that the transcriptional bypass of uracil by E. coli RNA polymerase results in the production of mutant proteins in vivo (22). These results suggest that uracil has to be removed before pol II meets the lesion to avoid the production of a mutant protein. In mammalian cells, four types of uracil DNA glycosylase exist to remove uracil lesions (1). Although the exact role of each of these uracil DNA glycosylases in the cells is not known, it is likely that they remove uracil lesions on transcribed DNA and thus prevent transcriptional mutagenesis.
8-oxoG on the template strand of DNA directs the incorporation of
non-cognate dAMP as well as dCMP through DNA replication leading to G:C
to T:A transversions (14, 15). It has been shown that CS cells are
deficient in the TCR of oxidative damage such as 8-oxoG and thymine
glycol as well as TCR in NER. Moreover, the frequency of G:C to T:A
transversions at 8-oxoG in CS cells was 30-40%, while that in normal
cells was 1-4%. The results indicate that the unrepaired 8-oxoG
blocked transcription by pol II in CS/XP-G cells but not in normal
cells (9). Their results are consistent with the present findings that
pol II stalled at 8-oxoG on the transcribed strand in the in
vitro transcription elongation assay using oligo(dC) template. In
addition, some pol II stalled at 8-oxoG lesions upon inserting
cytosine, while others could bypass the site by inserting cytosine.
What mechanisms are involved in the preference for either stalling or
bypassing? 8-oxoG forms base pairs with both cytosine and adenine,
depending on whether the base adopts a syn or an
anti conformation about the glycosidic bond (25, 26). The
syn conformations, which 8-oxoG favors, can pair with
adenine, while the anti forms can pair with cytosine. It is
speculated that when pol II inserts an adenine opposite the
syn conformation or cytosine opposite the anti
form it might be able to bypass the lesion because of the expected base
pairing. However, when pol II inserts a cytosine opposite an 8-oxoG
syn form, cytosine can not pair in the proper manner and pol
II stalls at the lesion. Our results indicate that higher
concentrations of adenine decreased the pausing, while higher
concentrations of cytosine led to the stalling of pol II at the lesion,
indicating that adenine can properly pair with 8-oxoG but cytosine can
not (Fig. 4). These findings would support the idea that the
syn conformation of 8-oxoG lesions blocks pol II elongation.
However, in regard to the blocking of pol II by 8-oxoG, we cannot rule
out the possibility that additional factors may be required to ensure
the stalling of pol II at 8-oxoG (27). On the other hand, we obtained
mutant products of bypassed transcription from the 8-oxoG templates as well as the uracil templates. It has been reported that MutT-deficient strains of E. coli that lack the sanitization of
8-oxo-dGTP, produced such mutant proteins (28, 29). These results
suggest that mutant transcripts generated by bypass transcription
produce mutant proteins. Since 8-oxoG is not repaired in CS cells,
more mutant proteins might be produced in CS cells, which could
contribute to the symptoms of CS.
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ACKNOWLEDGEMENT |
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We thank Aya Yanagida for technical assistance.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and CREST of Japan Science and Technology (JST).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.:
81-6-6879-7971; Fax: 81-6-6877-9136; E-mail:
ktanaka@fbs.osaka-u.ac.jp.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M208102200
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ABBREVIATIONS |
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The abbreviations used are: NER, nucleotide excision repair; BER, base excision repair; TCR, transcription-coupled repair; GGR, global genome repair; XP, xeroderma pigementosum; CS, Cockayne's syndrome; pol II, RNA polymerase II; 8-oxoG, 8-oxoguanine; Fpg, formamidopyrimidine-DNA glycosylase; AP, apurinic/apyrimidinic; UDG, uracil DNA glycosylase; NTP, ribonucleoside triphosphate; RT, reverse transcriptase.
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