Lack of Transcription-coupled Repair of Acetylaminofluorene
DNA Adducts in Human Fibroblasts Contrasts Their Efficient
Inhibition of Transcription*
Michiel F.
van Oosterwijk
,
Ronald
Filon
§,
Anton J. L.
de Groot
§,
Albert A.
van Zeeland
§, and
Leon H. F.
Mullenders
§¶
From the
Department of Radiation Genetics and
Chemical Mutagenesis, Medical Genetics Center, Leiden University
and the § J. A. Cohen Institute, Interuniversity Research
Institute for Radiopathology and Radiation Protection, 2333 AL
Leiden, The Netherlands
 |
ABSTRACT |
The
N-(deoxyguanosine-8-yl)-2-acetylaminofluorene (dG-C8-AAF)
lesion is among the most helix distorting DNA lesions. In normal fibroblasts dG-C8-AAF is repaired rapidly in transcriptionally active
genes, but without strand specificity, indicating that repair of
dG-C8-AAF by global genome repair (GGR) overrules transcription-coupled repair (TCR). Yet, dG-C8-AAF is a very potent inhibitor of
transcription. The target size of inhibition (45 kilobases) suggests
that transcription inhibition by dG-C8-AAF is caused by blockage of
initiation rather than elongation. Cockayne's syndrome (CS) cells
appear to be extremely sensitive to the cytotoxic effects of dG-C8-AAF
and are unable to recover inhibited RNA synthesis. However, CS cells
exhibit no detectable defect in repair of dG-C8-AAF in active genes,
indicating that impaired TCR is not the cause of the enhanced
sensitivity of CS cells. These and data reported previously suggest
that the degree of DNA helix distortion determines the rate of GGR as
well as the extent of inhibition of transcription initiation. An
interchange of the transcription/repair factor TFIIH from promoter
sites to sites of damage might underlie inhibition of transcription
initiation. This process is likely to occur more rapidly and
efficiently in the case of strongly DNA helix distorting lesions,
resulting in a very efficient GGR, a poor contribution of TCR to repair
of lesions in active genes, and an efficient inhibition of
transcription.
 |
INTRODUCTION |
Nucleotide excision repair
(NER)1 constitutes a
versatile process capable of recognizing and eliminating a broad
spectrum of DNA lesions including UV-induced photolesions, chemically
induced bulky DNA adducts, and certain types of DNA cross-links (1). Two NER subpathways have been identified: the transcription-coupled repair (TCR) pathway and the global genome (GGR) repair pathway. The
TCR pathway is confined to the transcribed strand of transcriptionally active genes and is dependent on ongoing transcription (2, 3), whereas
the GGR pathway removes lesions from both nonexpressed DNA and
transcriptional active genes. The contribution of TCR and GGR to the
repair of bulky DNA lesions in active genes varies remarkably,
demonstrated most strikingly for the two main UV-induced photolesions
i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4
pyrimidone photoproducts (6-4 PP); briefly 6-4 PP are removed predominantly by GGR, whereas CPD are removed predominantly by TCR.
Currently there is only limited knowledge concerning the factors that
determine the efficiency of DNA damage recognition by the NER proteins.
Studies with the purified Escherichia coli UvrABC
excinuclease complex have revealed that structural determinants such as
localized unwinding of the DNA helix or DNA bending or kinking
influence the efficiency of NER recognition of DNA lesions (4). For TCR
the potency of blocking elongation of transcription by a DNA lesion is
most likely the critical factor.
Understanding the mechanisms and identification of the factors that
determine DNA damage recognition requires lesion-specific information
on repair kinetics, transcription blockage capacity, and structural
distortion of the DNA helix. Studies with UV-irradiated mammalian cells
have provided valuable insights in the repair of structural different
lesions, i.e. CPD and 6-4 PP, although there are serious
limitations as both types of lesions are induced simultaneously by UV
light. Exposure of cells to NA-AAF provides the possibility of studying
structurally different DNA lesions, for which detailed information
exists on DNA conformation and transcription/replication blockage (for
review, see Ref. 5). NA-AAF reacts with the guanine base of DNA
inducing two major types of lesions, i.e. the deacetylated
dG-C8-AF and the acetylated dG-C8-AAF. Formation of dG-C8-AAF lesions
leads to a conformation change of the DNA which results in a local
denaturation of about 5 base pairs (6, 7). This denaturation is caused
by stacking of the aminofluorene group inside the DNA-helix, a
conformation that is stabilized by the acetyl group. Such a distortion
of the helix either does not occur or does so only at a low frequency when dG-C8-AF lesions are induced (8, 9). Moreover, convincing evidence
shows that a dG-C8-AAF lesion is an efficient block for transcription
(10-12), whereas the dG-C8-AF lesion is a poor inhibitor of
transcription (12, 13). The relative induction level of both lesions is
dependent on organism, tissue, or even animal strain. In mice and
cultured mammalian cells, including human fibroblasts, the main lesion
induced is the dG-C8-AF lesion (14-19), whereas in some rat strains
the dG-C8-AAF lesion is induced to a substantial extent (20). The
preferential induction of dG-C8-AF in most cellular systems has been
related to efficient deacetylation of NA-AAF. In rat the high frequency
of formation of dG-C8-AAF is caused by the presence of high levels of
sulfotransferase activity, which prevents the deacetylation process by
forming a stable sulfonated and acetylated compound (5).
In recent studies we have focused on the repair of NA-AAF-induced
dG-C8-AF lesions in normal and NA-AAF-sensitive human fibroblasts (18,
19). The repair of this lesion proceeds biphasically and at a much
slower rate than repair of UV-induced CPD or 6-4 PP in the same cell
lines (21-24). In normal cells repair of dG-C8-AF is dominated by GGR,
although dG-C8-AF is target for TCR as shown in cells expressing only
TCR (19). Compared with UV-induced photolesions, the effect of the
dG-C8-AF lesion on transcription and cell survival is much less
severe.
If localized unwinding of the DNA helix and transcription blockage
capacity would be critical determinants for efficient processing of DNA
lesions by GGR and TCR, respectively, then one would expect dG-C8-AAF
to be an extremely good target for both NER subpathways. Indeed,
plasmids containing dG-C8-AAF are good substrates for NER in cell-free
systems (25). However, because dG-C8-AAF is not formed in cultured
mammalian cells, no data exist for repair of dG-C8-AAF in
vivo. In the current study we established the conditions to induce
dG-C8-AAF in primary human fibroblasts, and we subsequently studied
repair of dG-C8-AAF in primary human fibroblast cell strains used
previously to study removal of CPD, 6-4 PP, and dG-C8-AF. To induce
acetylated lesions in human fibroblasts one needs to block the cellular
deacetylation activities while still allowing the formation of lesions.
Vu et al. (20) concluded that treatment of mouse primary
hepatocytes with paraoxon, a deacetylation inhibitor, before incubation
with NA-AAF not only reduced the formation of dG-C8-AF but also
increased the formation of dG-C8-AAF. In this study we show that
treatment of primary human fibroblasts with a selected concentration of
paraoxon before incubation with NA-AAF leads predominantly to the
formation of dG-C8-AAF. To gain insights in the repair of dG-C8-AAF by
GGR and TCR pathways we used proficient human fibroblasts and human
fibroblasts characterized by a deficiency in one of the two NER
subpathways. Cell lines belonging to xeroderma pigmentosum
complementation group C (XP-C) are deficient in the global genome
repair pathway but fully capable of performing transcription-coupled
repair (21, 26). On the contrary, Cockayne's syndrome (CS) cells
exhibit a deficiency in transcription-coupled repair but are proficient
in global genome repair (22, 24).
We show that repair of a major fraction of dG-C8-AAF lesions is very
efficient (much more efficient than the repair of dG-C8-AF) and occurs
without strand specificity, probably because the TCR is overruled by
GGR. CS cells remove the dG-C8-AAF lesions with kinetics similar to
that of normal cells, but they show clearly enhanced sensitivity to the
cytotoxic effects of paraoxon/NA-AAF treatment. Based on lesion
frequencies, inhibition of transcription by dG-C8-AAF is more efficient
than by UV-induced photolesions and is mediated by blockage of
initiation rather than elongation.
 |
MATERIALS AND METHODS |
Cell Lines and Culture Conditions--
Primary normal human
(VH25D), Cockayne's syndrome complementation group B (CS1AN), and
xeroderma pigmentosum complementation group C (XP21RO) fibroblasts were
cultured in Ham's F-10 medium (without hypoxanthine and thymidine)
supplemented with 15% fetal calf serum and antibiotics at 37 °C,
using a 2.5% CO2 atmosphere. In experiments aimed at
determining the frequency of adducts in defined genomic sequences,
exponentially growing cells were prelabeled for 2 days with
[3H]thymidine (0.06 µCi/ml, 82 Ci/mmol). For repair
replication and for RNA synthesis experiments, exponentially growing
cells were prelabeled with 32Pi (0.3 µCi/ml)
and [14C]uridine (0.03 µCi/ml, 60 mCi/mmol),
respectively. In all cases the medium was replaced after the labeling
period by label-free medium, and cells were allowed to grow to
confluence.
Clonal Survival Studies--
Cell survival after treatment with
NA-AAF was determined by measuring the colony forming ability of the
treated cells relative to the untreated control. 500-2,000 cells were
seeded in 94-mm Petri dishes, allowed to attach for 16 h, and
incubated with 10
8 M paraoxon (3.2 mM stock solution dissolved in 100% dimethyl sulfoxide)
for 15 min before treatment with 0, 50, 100, and 150 µM
NA-AAF for 30 min at 37 °C in complete medium. After incubation, the
cells were washed twice with PBS, and fresh medium was added to the
cells. After 7 days the medium was replaced by fresh medium; 10-14
days after plating the cells were rinsed twice with 0.9% NaCl, and
colonies were stained with methylene blue.
HPLC/ECD Analysis--
The nature and frequency of DNA lesions
induced by the paraoxon/NA-AAF treatment were measured by HPLC analysis
coupled to ECD of dG-C8-AF and dG-C8-AAF lesions. A detailed
description of the methodology is published elsewhere (27). Briefly,
DNA samples from the various cell lines treated with paraoxon/NA-AAF were dried and subsequently hydrolyzed in 50 µl of 98%
trifluoroacetic acid at 70 °C for 45 min. After hydrolysis,
H2O and ethyl acetate were added to the tubes, the DNA was
extracted with ethyl acetate, and the combined ethyl acetate layers
were evaporated. DNA samples were dissolved in water/methanol and
injected on a Chromopack Hypersil-ODS glass cartridge column. Elution
was done isocratically with 0.02 M
K2PO4 and 50% methanol, pH 6.0. Detection of
dG-C8-AF and dG-C8-AAF was performed by ECD detection employing a
glassy carbon electrode (5000 series ANTEC).
Measurement of RNA
Synthesis--
[14C]Uridine-prelabeled confluent cells
were incubated with 10
8 M paraoxon for 15 min
before treatment with 150 µM NA-AAF for 30 min at
37 °C. After washing the cells twice with PBS, fresh medium was
added to the cells. At different time intervals after NA-AAF treatment
the cells were pulse labeled with [3H]uridine (10 µCi/ml, 39.0 Ci/mmol) for 30 min at 37 °C and processed for liquid
scintillation counting as described previously (18).
Measurement of DNA Repair Replication--
DNA repair
replication was measured by the radioisotope and density labeling
technique described by Van Zeeland et al. (28). 32Pi-Prelabeled confluent cells were treated
with 10
8 M paraoxon for 15 min before
treatment with 300 µM NA-AAF for 30 min at 37 °C.
After washing twice with PBS, fresh medium was added containing 10 µM BrdUrd and 1 µM FrdUrd, with
[3H]thymidine (5 µCi/ml, 82 Ci/mmol), and the cells
were allowed to perform DNA repair at 37 °C for 24 h. Cell
lysis, DNA isolation, and cesium chloride density gradient
centrifugation were performed as described previously (18). Repair
replication was expressed as 3H cpm/µg of DNA.
DNA Probes--
Double-stranded DNA probes were radioactively
labeled with [32P]dATP by random primer extension (29).
Strand-specific single-stranded probes were radioactively labeled with
[32P]dATP by a linear polymerase chain reaction, using a
single primer recognizing specifically one strand (30).
Gene-specific Analysis of NA-AAF-induced DNA Adducts--
To
measure DNA adduct frequencies in defined genomic sequences as a
function of dose, 3H-labeled confluent cells were incubated
with 10
8 M paraoxon for 15 min before
treatment with 0, 100, 200, 300, and 400 µM NA-AAF, in
complete medium, for 30 min at 37 °C. After washing the cells twice
with PBS, they were lysed. In repair experiments 3H-labeled
confluent cells were incubated with 10
8 M
paraoxon for 15 min before treatment with 300 µM NA-AAF,
in complete medium, for 30 min at 37 °C. After washing the cells twice with PBS, they were either lysed immediately or incubated for
various time intervals in complete medium supplemented with 10 µM BrdUrd and 1 µM FrdUrd. DNA was isolated
and purified as described previously (21), resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and
digested overnight either with EcoRI at 37 °C or
BclI at 50 °C. To separate the parental DNA from the replicated DNA, neutral cesium chloride gradient centrifugation was
performed. After fractionation, the fractions containing the parental
DNA were pooled, dialyzed against 1 × TE and 0.05 × TE, and
dried by vacuum centrifugation. Finally the samples were resolved in
sterile water.
The frequency of NA-AAF-induced DNA adducts was determined by in
vitro incision at the sites of adducts employing the UvrABC excinuclease complex of E. coli as described previously
(24). Pierce et al. (25) showed that the UvrABC excinuclease
incises efficiently at sites of all dG-C8-AAF lesions in DNA fragments. Because the UvrABC complex exhibited a low but variable activity on DNA
from cells not exposed to NA-AAF, a DNA sample isolated from untreated
cells was also incubated with UvrABC (23). After incubation, the DNA
samples were purified by phenol and chloroform extraction and subjected
to 0.8% alkaline agarose gel electrophoresis followed by Southern
blotting and hybridization. The 32P intensities of full
size restriction fragments were quantified by performing electronic
autoradiography (InstantImager, Packard). The number of
UvrABC-sensitive sites/fragment was calculated from the relative band
intensities of full size restriction fragments in the lanes containing
the treated and mock-treated samples, assuming a Poisson distribution
of lesions.
 |
RESULTS |
Formation of DNA Adducts--
Because incubation of cells with
NA-AAF leads primarily to the induction of dG-C8-AF, we had to develop
a system of inducing dG-C8-AAF. Vu et al. (20) showed that
pretreatment of mice hepatocytes with paraoxon, a deacetylase
inhibitor, led to the preferential induction of dG-C8-AAF in
NA-AAF-treated cells. However, the concentration of paraoxon appeared
to be very critical because doses that are too high abolish the
formation of lesions, whereas doses that are too low lead to induction
of mainly dG-C8-AF. Consistent with their results we found that because
of the paraoxon treatment relatively high doses of NA-AAF had to be
applied to get sufficient induction of dG-C8-AAF. To determine the
frequency and relative induction level of various types of DNA lesions
after paraoxon/NA-AAF treatment we employed HPLC analysis combined with
ECD of adducted nucleosides. With this approach the principal DNA
lesions induced by NA-AAF, i.e. dG-C8-AF and dG-C8-AAF, can
be analyzed quantitatively (27). As shown in Table
I, the main lesion induced by the
combined paraoxon/NA-AAF treatment is the acetylated dG-C8-AAF (85%);
this in contrast to exposure of these cells to NA-AAF alone, which induces the deacetylated dG-C8-AF exclusively (18, 19). There are no
major differences in the relative frequency and the types of
paraoxon/NA-AAF-induced adducts among normal, XP-C, and CS-B cells.
Employing a dose of 10
8 M paraoxon alone we
did not find any negative or positive effect on transcription
inhibition, repair replication, or colony forming ability.
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Table I
Induction of dG-C8-AF and dG-C8-AAF after treatment with 10 8
M paraoxon and 300 µM NA-AAF
Lesion frequencies were measured employing HPLC/ECD analysis of DNA
obtained from normal (VH25D), Cockayne's syndrome (CS1AN), and
xeroderma pigmentosum (XP21RO) cells.
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Clonal Survival Studies--
The cytotoxic effect of the
paraoxon/NA-AAF treatment was measured by determining the colony
forming ability of the cells after treatment. Fig.
1 shows that the CS-B and the XP-C cells were much more sensitive to paraoxon/NA-AAF treatment than the normal
human cells. Paraoxon alone had no significant effect on colony forming
ability of any of the three cell lines.

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Fig. 1.
Cell killing effects of paraoxon/NA-AAF on
primary VH25D ( ), CS1AN ( ), and XP21RO complementation group C
cells ( ). Bars represent the S.E.
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Effect of Paraoxon/NA-AAF Treatment on RNA Synthesis--
A
hallmark of CS cells is the lack of recovery of inhibited RNA synthesis
after treatment with DNA-damaging agents such as NA-AAF and UV light.
On the contrary XP-C cells with a very reduced repair capacity are able
to recover RNA synthesis after NA-AAF or UV treatment. Fig.
2A shows that RNA synthesis
during the first 30 min after paraoxon/NA-AAF treatment is inhibited in
a dose-dependent manner and that the level of inhibition
was similar for VH25D and XP-C cells.

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Fig. 2.
Panel A, inhibition of RNA synthesis
after treatment with 10 8 M paraoxon and
different doses of NA-AAF. , VH25D; , XP21RO cells. Panel
B, recovery of RNA synthesis after treatment with
10 8 M paraoxon and 150 µM
NA-AAF. , VH25D; , CS1AN; , XP21RO cells. In both panels RNA
synthesis after treatment with NA-AAF is measured by 30'-pulse labeling
and expressed relative to corresponding untreated cells.
Bars represent the S.E.
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Next we addressed the question of whether the three cell lines differ
in their abilities to recover paraoxon/NA-AAF-inhibited RNA synthesis.
Fig. 2B shows that after a dose of 150 µM
NA-AAF the CS-B cells are incapable of recovering RNA synthesis,
whereas the normal and the XP-C cells exhibit recovery of RNA synthesis up to 85% of the level in untreated cells 8 h after
treatment.
Repair Replication Studies--
The genome overall capacity of the
three cell lines to repair paraoxon/NA-AAF-induced DNA lesions was
assessed by measurement of the extent of repair replication.
32P-Prelabeled cells were treated with paraoxon and 300 µM NA-AAF and were allowed to repair for 24 h in the
presence of [3H]thymidine, BrdUrd, and FrdUrd. An
untreated sample was included as a control to determine the specific
activity of the DNA (32P cpm/µg of DNA) from each cell
strain in order to quantify the level of repair replication, allowing
direct comparison of the extent of repair replication in different cell
lines. The results (Fig. 3) demonstrate
that the capacity of the CS cells to repair paraoxon/NA-AAF-induced DNA
lesions is not substantially different from that of normal cells. In
contrast, the XP-C cell line shows a reduced level of repair
replication, amounting to approximately 25% of that in normal
cells.

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Fig. 3.
Repair replication in (VH25D fibroblasts,
open bars), CS1AN fibroblasts, closed bars),
and XP-C fibroblasts (XP21RO, striped bars) after treatment
with 10 8 M paraoxon and 300 µM
NA-AAF and postincubation for 24 h. Bars represent the
S.E.
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Induction and Repair of dG-C8-AAF Adducts in Active and Inactive
Genes--
The frequency of paraoxon/NA-AAF-induced DNA adducts in
restriction fragments of (in)active genes as a function of the dose was
determined employing the UvrABC excinuclease assay as described previously (24). The UvrABC excinuclease introduces single-stranded DNA
breaks in the DNA at the site of a lesion. The frequency of these DNA
breaks can be quantified at the gene level by alkaline agarose-gel
electrophoresis, Southern blotting, and hybridization with
(strand-specific) radiolabeled probes. The presence of DNA lesions is
seen as the reduction of full size restriction fragments in lanes
containing UvrABC-digested DNA compared with undigested DNA.
To determine the formation of paraoxon/NA-AAF-induced DNA lesions in
active and inactive genes, lesion frequencies were measured in the
18.5-kb EcoRI fragment of the transcriptionally active ADA
gene and the 14-kb EcoRI fragment of the X-chromosomal
inactive 754 gene by sequential hybridization of the same membrane. A
linear relationship between the dose of NA-AAF and induction of
UvrABC-sensitive sites up to 400 µM was observed with an
average induction frequency of 0.0017 DNA lesions/µM/10
kb for both the ADA and 754 gene (data not shown). The lesion frequency
was similar in restriction fragments of active and inactive genes, and
no significant differences in induction levels among the three cell
lines were observed. To achieve the desirable induction of one
adduct/restriction fragment for repair experiments a dose of 300 µM was chosen, inducing about 0.5 adducts/10 kb.
To detect possible differences in the kinetics of removal of dG-C8-AAF
lesions between the transcribed and the nontranscribed strand of the
ADA gene, we studied strand-specific repair in the 5'-located 19.9-kb
BclI fragment covering a region of the ADA gene with only
transcription of the ADA template strand (31). Fig.
4 is an autoradiogram representing
strand-specific repair in the three cell lines. Fig.
5A shows that VH25D cells
exhibit a very rapid repair of approximately 40-60% of the adducts
during the first 4 h after treatment followed by repair of
additional 10-20% up to 24 h. No differences in kinetics and
extent of repair were observed between the transcribed and
nontranscribed strands of the ADA gene in normal cells. The extent of
repair of the ADA gene in CS1AN cells was virtually the same as in
normal cells (Fig. 5B). Repair of the ADA gene in the XP21RO
cells proceeded at a slower rate and to a lesser extent than in normal
and CS cells; but in contrast to the latter cell lines, repair in
XP21RO showed a clear strand specificity, i.e. repair in the
transcribed strand was more efficient. After longer postincubation
times repair in the transcribed strand of the ADA gene in the XP21RO
cell was almost complete, e.g. 85% of the lesions were
removed after 48 h. Repair of DNA adducts in the nontranscribed
strand proceeded slowly but to substantial level (34%) after 48 h. Basically, the same results were obtained when repair of
paraoxon/NA-AAF-induced DNA lesions was measured in the 3'-located
18.5-kb EcoRI fragment of the ADA gene (data not shown).
When repair was assayed in the transcriptionally inactive 754 gene, a
less rapid removal of lesions was observed in normal and CS cells, in
particular during the first 8 h after treatment, whereas repair of
754 was virtually absent in XP-C cells.

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Fig. 4.
Autoradiograms showing removal of dG-C8-AAF
from the 19.9-kb BclI ADA fragment of VH25D cells
(panel A), CS1AN cells (panel B), and XP21RO
cells (panel C). The repair in the ADA gene is
analyzed with strand-specific probes recognizing either the transcribed
(TS) or the nontranscribed strand (NTS).
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Fig. 5.
Removal of paraoxon/NA-AAF induced lesions
from the 19.9-kb BclI fragment of the ADA gene and the
14-kb EcoRI fragment of the 754 gene. Panel A,
VH25D fibroblasts; panel B, CS1AN fibroblasts; and
panel C, XP21RO fibroblasts. , ADA transcribed strand;
, ADA nontranscribed strand; , 754 both strands. Lesion
frequencies were measured by the UvrABC assay. Bars
represent the S.E.
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 |
DISCUSSION |
NA-AAF is known to induce two types of DNA adducts with different
DNA-distorting properties, i.e. dG-C8-AF and dG-C8-AAF. In
the case of the dG-C8-AAF adduct, addition of the bulky
acetylaminofluorene moiety to guanine is known to cause severe
distortion of the DNA helix, manifested most clearly by the local
denaturation of several bases at the site of the lesion resulting from
stacking of the acetylaminofluorene group inside the helix (6, 7). In
contrast, dG-C8-AF affects DNA conformation only marginally because the aminofluorene group resides mainly outside the helix, although no full
consensus exists on this point (8, 9). Tang and co-workers (16, 25)
have shown that in vitro the E. coli UvrABC excinuclease recognized the dG-C8-AAF adduct 3-fold more efficiently than dG-C8-AF.
In this study, we used NA-AAF and paraoxon to induce dG-C8-AAF in human
cells and studied the repair kinetics of this lesion by GGR and TCR. In
accordance with Vu et al. (20) the major lesion induced in
the presence of paraoxon was dG-C8-AAF with small (15%) amounts of
dG-C8-AF. No major differences were detected among normal, XP-C, and CS
fibroblasts with respect to the types and frequencies of
paraoxon/NA-AAF-induced DNA lesions, demonstrating that the enhanced
sensitivity of XP-C and CS to paraoxon/NA-AAF cannot be attributed to
higher levels of DNA adduct formation nor to different types of DNA
lesions. Based on equal induction frequency, dG-C8-AAF is a much more
cytotoxic lesion than dG-C8-AF (this study and Refs. 18 and 19).
Interestingly, the sensitivity of XP-C cells treated with
paraoxon/NA-AAF compared with normal cells is much more pronounced than
after treatment with NA-AAF alone (19). This enhanced sensitivity of
XP-C cells to the cytotoxic effects of paraoxon/NA-AAF is most likely
caused by persistent dG-C8-AAF lesions in nontranscribed regions,
blocking DNA replication. In contrast, dG-C8-AF is a poor inhibitor of
replication (32, 33).
In vitro studies revealed that dG-C8-AAF lesions have
profound transcription-blocking properties (12). This study shows that
also in intact cells, dG-C8-AAF induces efficient inhibition of
transcription with a target size of inhibition of approximately 45 kb,
suggesting that less than one dG-C8-AAF lesion per transcription unit
inactivates transcription and that transcription inhibition might be
mediated by blocking initiation rather than elongation. Based on lesion
frequencies, the inhibition of transcription by dG-C8-AAF is about
3-fold more effective than by UV-C-induced photolesions
(i.e. CPD and 6-4 PP) and about 15-fold more than by
dG-C8-AF. However, Petit-Frere et al. (34) showed that the immediate inhibition of RNA synthesis by UV irradiation correlates with
6-4 PP rather than the overall photolesion formation, indicating that
6-4 PP is the mediator of the inhibitory effect. Our results provide
indirect evidence for the correctness of this hypothesis as the 3-fold
lower effectiveness of RNA synthesis inhibition by UV-C (overall
photolesion formation) mimics the 3-fold higher induction of CPD over
6-4 PP (24). Indeed, a plot of UV-induced RNA synthesis inhibition
versus 6-4 PP frequency coincides with the dG-C8-AAF
inhibition curve (Fig. 6).

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Fig. 6.
Inhibition of RNA synthesis after induction
of different types of DNA lesions, based on equal lesion frequency, in
normal human fibroblasts. The data on inhibition by the 6-4 PP
are based on the assumption that 6-4 PP are the RNA synthesis-blocking
lesions. The data on inhibition by UV are based on the assumption that
both CPD and 6-4 PP are the RNA synthesis-blocking lesions. ,
dG-C8-AAF; , dG-C8-AF; , UV; , 6-4 PP.
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CS cells appeared to be incapable of recovering inhibited RNA synthesis
after paraoxon/NA-AAF treatment analogous to their response after UV
exposure or NA-AAF treatment only (18, 35), whereas normal and XP-C
cells do recover RNA synthesis. To correlate RNA synthesis recovery to
efficiency and strand specificity of repair in transcriptionally active
genes, we quantified dG-C8-AAF lesion frequencies in the ADA
housekeeping gene. The repair of dG-C8-AAF in the ADA gene in normal
and CS cells displays two characteristics: (i) repair proceeds in a
biphasic manner with approximately 50% of the lesions removed within
2-4 h followed by additional repair at a slower rate, and (ii) there
is no significant difference in repair between the transcribed and
nontranscribed strands. Part of the slow repair at late times might be
because of the presence of dG-C8-AF, which is formed as a minor
fraction after the paraoxon/NA-AAF treatment. Moreover, although the
majority of the dG-C8-AAF lesions resides in the syn conformation, a
substantial fraction might adapt the anti formation dependent on the
sequence context (36, 37); the latter is known to be less
helix-distorting and therefore presumably repaired at a slower rate. In
addition, sequence-dependent differences in the repair
efficiency of dG-C8-AAF adducts may contribute to heterogeneity of
repair since in vitro studies revealed that incision
efficiency of dG-C8-AAF by UvrABC excinuclease varied substantially
depending on the local sequence (38).
The absence of a strand specificity of repair in normal cells does not
favor a significant contribution of TCR to repair of dG-C8-AAF in
active genes. Moreover, because the repair kinetics of dG-C8-AAF in CS
cells is the same as observed in normal cells, the enhanced sensitivity
of CS cells to dG-C8-AAF adducts and the lack of RNA synthesis
restoration in those cells cannot be attributed to defective repair of
transcriptionally active genes either. Although this repair phenotype
for dG-C8-AAF contrasts the defective TCR in UV-irradiated CS cells, it
closely resembles the absence of strand-specific repair of the
nonacetylated dG-C8-AF adduct in normal cells and normal repair
kinetics of this lesion in CS cells. The enhanced sensitivity and lack
of RNA synthesis recovery after induction of dG-C8-AAF fit a previous
reported model (based on the repair kinetics of the dG-C8-AF lesion)
proposing that CS proteins act as repair-transcription uncoupling
factors in concert with the basal transcription factor TFIIH (18).
TFIIH is essential for initiation of RNA polymerase II-driven
transcription as well as NER. We propose that upon treatment of cells
with NA-AAF (and paraoxon) TFIIH will be recruited for association with
additional NER proteins to do repair and that CS proteins are essential
for its conversion back to transcription function. In summary, the enhanced sensitivity of CS cells to NA-AAF (with or without paraoxon) and UV is not caused by a defect in TCR but rather the consequence of a
defect in transcription initiation in the presence of DNA damage.
The absence of any observable strand specificity for repair of
dG-C8-AAF in normal cells does not relate to a poor substrate property
of this lesion for TCR; the results obtained with XP-C cells show that
dG-C8-AAF is a substrate for TCR. However, repair in XP-C cells
proceeds at slower rate than in normal and CS cells and is virtually
absent during the first 4 h after treatment when approximately
50% of the lesions in normal cells are repaired. The delay in repair
in XP-C cells has been observed for UV-induced photolesions as well
(24) and is likely to be caused by delayed transcription initiation.
The results described in this and previous studies (18, 19, 23, 24) are
consistent with a model in which DNA lesion conformation dictates the
inhibition of transcription initiation and the efficiencies of GGR. In
contrast, repair of DNA lesions by TCR appears to be independent of the
structure of the lesions, i.e. UV-induced CPD and 6-4 PP
are repaired with same kinetics by TCR (24). However, gross differences
exist between the repair rates of the various bulky adducts by GGR. Both 6-4PP and dG-C8-AAF are repaired extremely rapid (approximately 50% repair in 2 h at equal induction frequency) and much more rapid than CPD and dG-C8-AF lesions. It is tempting to speculate that
the degree of damage-induced DNA helix distortion is a main determinant
in the recognition of DNA damage by DNA repair proteins, i.e. XPA, XPC, and XPE. If DNA damage recognition is the
critical signal for formation of incision complexes, the rate of TFIIH depletion from transcription complexes will depend largely on the
efficiency by which different types of DNA damage are recognized by the
relevant XP factors. In such a model the depletion of TFIIH from
promoter sites will be the predominant process that causes transcription to be inhibited. We hypothesize that the conversion of
TFIIH from repair to transcription is an active process for which CS
proteins are required. However, efficient GGR as such cannot be the
signal for this process because XP-C cells show recovery of
transcription without GGR.
 |
ACKNOWLEDGEMENTS |
The HPLC/ECD measurements were performed by
A. de Groot, Department of Radiation Genetics and Chemical
Mutagenesis, Medical Genetics Center, Leiden University. The UvrABC
proteins used in this study were obtained from Dr. P. van de Putte,
Department of Molecular Genetics, Leiden Institute of Chemical
Research, Leiden University, The Netherlands.
 |
FOOTNOTES |
*
This study was supported by a grant from the Medical
Genetics Center South-West Netherlands and by European Commission
Contract EV5V-CT94-0397).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: Dept. of Radiation
Genetics and Chemical Mutagenesis, MGC, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
Tel.:31-71-527-6126; Fax: 31-71-522-1615.
1
The abbreviations used are: NER, nucleotide
excision repair; TCR, transcription-coupled repair; GGR, global genome
repair; CPD, cyclobutane pyrimidine dimer; 6-4 PP, pyrimidine 6-4
pyrimidone photoproduct; NA-AAF,
N-acetoxy-2-acetylaminofluorene; dG-C8-AF, N-(deoxyguanosine-8-yl)-2-aminofluorene;
dG-C8-AAF, N-(deoxyguanosine-8-yl)-2-acetylaminofluorene; XP, xeroderma pigmentosum; XP-C, xeroderma pigmentosum
complementation group C; CS, Cockayne's syndrome; CS-B, Cockayne's
syndrome complementation group B; PBS, phosphate-buffered saline;
HPLC, high performance liquid chromatography; ECD, electrochemical
detection; kb, kilobase(s); ADA, adenosine deaminase; BrdUrd,
5'-bromodeoxyuridine; FrdUrd, fluorodeoxyuridine.
 |
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