From the Commissariat à l'Energie Atomique
(CEA), Direction des Sciences du Vivant, Département de
Radiobiologie et Radiopathologie, Unité Mixte de Recherche 217 CNRS-CEA Radiobiologie Moléculaire et Cellulaire, 92265 Fontenay aux Roses, France and ¶ Unité Propre de Recherche
9003, CNRS, Université Louis Pasteur, Ecole Supérieur
de Biotechnologie de Strasbourg, 67400 Illkirch, France
Received for publication, December 18, 2002, and in revised form, February 28, 2003
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ABSTRACT |
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Oxidative DNA base damage is mainly corrected by
the base excision repair (BER) pathway, which can be divided into two
subpathways depending on the length of the resynthetized patch, either
one nucleotide for short patch BER or several nucleotides for long patch BER. The role of proteins in the course of BER processes has been investigated in vitro using purified enzymes and
cell-free extracts. In this study, we have investigated the repair of
8-oxo-7,8-dihydroguanine (8-oxoG) in vivo using
wild-type, polymerase Reactive oxygen species, generated either endogenously by
cellular metabolism or by exposure to environmental oxidants, induce DNA damages that have been implicated in human pathologies such as
cancer, neurodegenerative diseases, or aging (1-4). An oxidatively damaged guanine 8-oxo-7,8-dihydroguanine
(8-oxoG),1 is an important
mutagenic DNA lesion due to its potential to mispair with adenine, thus
generating G:C Several studies pointed to Pol To investigate the role of Pol Cell Lines and Culture Conditions--
Spontaneously
immortalized 3T3 wild-type, homozygous PARP1 Western Blotting--
Cell-free protein extracts were prepared
from clonal isolates as described previously (59). The protein content
was determined according to Bradford, and 50 µg of protein were
analyzed by 8% SDS-PAGE and immunoblotting. For immunodetection, blots
were incubated with anti-PARP-1 (1/4000, Montevideo) polyclonal
antibodies. Blots were then probed with horseradish peroxidase-coupled
secondary antibodies (goat anti-rabbit 1/25000, Sigma) and
immunoreactivity was enhanced by chemiluminescence according to the
manufacturer (ECL, Amersham Biosciences).
8-OxoG:C Cleavage Assay--
Standard assay mixture (15-µl
final volume) contained 25 mM Tris-HCl, pH 7.6, 2 mM Na2EDTA, 70 mM NaCl, 25 fmol of
5'-32P-labeled 8-oxoG:C 34-mer DNA duplex and 10 µg of
cell-free protein extract (62).
In Vivo 8-OxoG Repair Kinetics--
Construction of
pS Mutagenesis Assay--
Transfection and plasmid extraction were
performed as above. Recovered plasmid DNA was used to transform
E. coli strain (BH990) fpg Primer Extension--
Recovered plasmid DNA was used as template
for an "asymmetrical PCR amplification." An 18-mer, located 80 bp
from the site of the lesion and used as a primer, was labeled in the
5'-end by [ Combination of Pol
To further investigate the role of Pol Removal of 8-OxoG in NTS Is Not Affected in
Pol Incomplete Repair of 8-OxoG in Double
Pol Poly(ADP-ribose) Polymerase Activity of PARP-1 Is Required for the
8-OxoG:C Repair on NTS in Vivo in
Pol The identification of proteins involved in the processes of BER in
mammalian cells is subject to intense investigation. Most studies are
carried out using cell extracts or purified proteins (22-24, 26, 30,
46, 70-72). The role of "nonessential factors" such as PARP-1 is
also a subject of discussion, (for reviews, see Refs. 49, 73, and 74).
In this work, we used an in vivo approach based on the
transfection of a monomodified plasmid in intact cells to study the
repair of 8-oxoG in the cell context. In the last decade, shuttle
plasmids have been used to analyze mutagenesis and repair in mammalian
cell lines and mutagenic potency of specific lesions (75, 76). In every
case, results gave a quite good preview of the process in genomic DNA.
This system also allowed us to study the repair of a 8-oxoG in either a
TS or a NTS condition (59, 77).
In this study, we investigated the repair of 8-oxoG in 3T3 cells
deficient in PARP-1 or/and Pol Our study has also shown that Pol/
(Pol
/
),
poly(ADP-ribose) polymerase-1
/
(PARP-1
/
), and
Pol
/
PARP-1
/
3T3 cell lines. We used
non replicating plasmids containing a 8-oxoG:C base pair to study the
repair of the lesion located in a transcribed sequence (TS) or in a
non-transcribed sequence (NTS). The results show that 8-oxoG repair in
TS is not significantly impaired in cells deficient in Pol
or PARP-1
or both. Whereas 8-oxoG repair in NTS is normal in Pol
-null cells,
it is delayed in PARP-1-null cells and greatly impaired in cells
deficient in both Pol
and PARP-1. The removal of 8-oxoG and
presumably the cleavage at the resulting apurinic/apyrimidinic site are
not affected in the PARP-1
/
Pol
/
cell
lines. However, 8-oxoG repair is incomplete, yielding plasmid molecules
with a nick at the site of the lesion. Therefore,
PARP-1
/
Pol
/
cell lines cannot
perform 5'-dRP removal and/or DNA repair synthesis. Furthermore, the
poly(ADP-ribosyl)ation activity of PARP-1 is essential for 8-oxoG
repair in a Pol
/
context, because expression of the
catalytically inactive PARP-1 (E988K) mutant does not restore 8-oxoG
repair, whereas an wild type PARP-1 does.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
T:A transversions. The biological significance of
8-oxoG is revealed by the spontaneous mutator phenotype of bacterial
and yeast mutants impaired in 8-oxoG repair (5-9). In all organisms,
8-oxoG is primarily repaired by the base excision repair (BER) pathway,
which is the major process for the elimination of oxidative base
damage, alkylation base damage, and apurinic/apyrimidinic (AP) sites
(10, 11). In mammalian cells, BER of 8-oxoG is initiated by the action
of the Ogg1 DNA N-glycosylase, which catalyzes the
hydrolysis of the N-glycosyl bond linking damaged bases to
the sugar phosphate backbone-generating AP sites. Ogg1 is also endowed
with an AP lyase activity that can incise the phosphodiester bond
immediately 3' of an AP site, yielding a 3'-terminal sugar phosphate
(3'-dRP) (12-19). However, in the presence of the major AP
endonuclease Ape1, AP sites resulting from the removal of 8-oxoG
residues by Ogg1 are primarily processed by Ape1, which catalyzes the
hydrolytic cleavage of the phosphodiester bond immediately 5' to the AP
site, generating a 5'-terminal sugar phosphate (5'-dRP) (20, 21). Afterward, the 5'-dRP is removed by a dRPase activity associated with
DNA polymerase
(Pol
), which simultaneously adds one nucleotide. The nick is finally sealed by DNA ligase III, which is associated with
the x-ray cross-complementing factor 1 (Xrcc1) (22-24). The entire
process results in the removal of 8-oxoG and its replacement with a
guanine and constitutes the short patch base excision repair (SP-BER)
pathway. In repair proficient cells, SP-BER is thought to be the major
repair pathway for 8-oxoG (25). Another form of BER, the long patch BER
(LP-BER), results in the removal of the DNA damage and the replacement
of 2-10 nucleotides extending 3' to the lesion (26-31). In the course
of LP-BER, the early steps of 8-oxoG repair are performed by Ogg1 and
Ape1 as described for SP-BER. Then, a DNA polymerase (Pol
,
or
) adds few nucleotides and displaces the 5'-dRP residue,
generating a 5'-flap structure with a 5'-dRP end. The 5'-flap is
removed by the Flap endonuclease 1 (Fen1), and a DNA ligase seals the
nick. The role of different DNA polymerases in LP-BER in the wild type
cellular context is unclear. Recently, DNA polymerase
- or
-dependent LP-BERs have been reconstituted with purified
human proteins (32). For efficient repair of a regular AP site, in
addition to Ape 1 and DNA polymerase
the reaction assay required
replication factor C (RF-C), proliferating cell nuclear antigen (PCNA),
Fen1, and Ligase I (30). LP-BER is a minor pathway for the repair of
8-oxoG and regular AP sites in wild type cell-free extracts (26, 33).
In contrast, LP-BER is thought to be the major pathway for the repair
of reduced or oxidized AP sites (31).
as the major repair DNA polymerase in
eukaryotes, involved in both SP- and LP-BER (24, 30, 34-36). Pol
lacks accessory functions such as 3' to 5' exonuclease, dNMP turnover,
or pyrophosphorolysis (37-40). On the other hand, Pol
possesses a
robust AP lyase activity that allows the removal of 5'-dRP in the
course of BER (41). Pol
performs an essential function in
development, because knockout mice for Pol
are not viable (42, 43).
However, Pol
-null 3T3 cells are viable but hypersensitive to
methylating agents (39, 44-46). The hypersensitivity to a methylating
agent can be suppressed by the expression of the AP lyase domain of
Pol
(44). Although non-essential, the poly(ADP-ribose) polymerase 1 (PARP-1) has been shown to intervene in the course of BER in living
cells (47, 48). PARP-1 is a nuclear protein found in proliferating
tissues of eukaryotes with the exception of yeast (49, 50). PARP-1
binds with high affinity DNA containing single-strand breaks. Upon
binding to DNA strand breaks, PARP-1 catalyzes the synthesis of
poly(ADP-ribose) from NAD+ and covalently modifies several
nuclear proteins involved in chromatin architecture (such as histones
and lamin B) and DNA metabolism (such as topoisomerases, DNA
polymerases, and BER factors). The automodification of PARP-1 induces
its dissociation from DNA breaks and inhibition of its catalytic
activity. PARP-1 and poly(ADP-ribosyl)ation are proposed to be
critical for cellular processes such as DNA repair, transcription, or
energy depletion-induced cell death during inflammatory injury (for
review see Refs. 50 and 51). Evidence for the involvement of PARP-1 in
BER was provided by the fact that PARP-1-null mice are hypersensitive
to ionizing radiation and alkylating agents (52-54). Moreover, the
physical interaction of PARP-1 with proteins such as Pol
and Xrcc1
also points to its role in BER (55, 56). Recently, PARP-1 was shown to
bind with high affinity to BER intermediates harboring a 5'-dRP (57).
Furthermore, reconstitution of BER using purified proteins shows that
PARP-1 stimulates two of the key steps of LP-BER, i.e. strand displacement synthesis by Pol
and 5'-flap cleavage by Fen1
(58). In addition repair of AP sites is impaired in cell-free extracts
of PARP-1-null mice cell lines (46). This study also shows that
PARP-1-null Pol
-null cell-free extracts present a dramatic decrease
in LP-BER when compared with PARP-1-null cells. Therefore, results with
purified proteins and cell-free extracts point to a role of PARP-1
in LP-BER.
and PARP-1 in the course of BER in
the cellular context, we measured the repair of 8-oxoG in murine cell
lines (3T3s), either wild type (WT), those deficient in Pol
(Pol
/
) or PARP-1 (PARP-1
/
), or cells
that present the two deficiencies
(PARP-1
/
Pol
/
). We measured the
repair kinetics for a single 8-oxoG:C base pair in these four cell
lines using shuttle vectors that contain 8-oxoG in a transcribed
sequence (TS) or a non-transcribed sequence (NTS) (59). Our results
show that the repair of 8-oxoG in a TS plasmid is not affected in wild
type, Pol
-null, PARP-1-null, and double PARP-1-null Pol
-null 3T3
cells. On the other hand, repair of 8-oxoG in an NTS plasmid, which is
not affected in Pol
-null cells, occurs at a 2-fold reduced rate in
PARP-1-null cells. Furthermore, 8-oxoG repair in the NTS plasmid is
nearly completely abolished in cells deficient in both Pol
and
PARP-1. The role of the poly(ADP-ribose) synthesis during BER is also
discussed in this study.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
,
homozygous Pol
/
, and double deficient
PARP-1
/
Pol
/
clones were established
in Dulbecco's modified Eagle's medium, 4.5g/liter glucose
medium supplemented with 10% fetal bovine serum, and 0.5%
gentamycin (46). To obtain
PARP1
/
Pol
/
3T3 cell lines corrected
by the expression of wild-type or mutant human PARP-1, double deficient
cell lines were transfected with an empty vector (pECV-23Xho) or a
vector containing the cDNA encoding the wild type PARP-1
(pECV-PARP-1) (60) or the E988K mutant human PARP-1 protein
(pECV-PARP-1E988K) (61). Transfectants were selected by
growth in medium containing hygromycin at increasing concentrations up
to 400 µg/ml. Single clones were isolated after 15 days, propagated
in 12-well plates, and analyzed for PARP-1 expression by Western blotting.
oriSV-[8-oxoG:C] or pS
(ori-p)SV-[8-oxoG:C] used for repair
kinetics has been described (59). pS
oriSV-[8-oxoG:C] (TS) or
pS
(ori-p)SV-[8-oxoG:C] (NTS) plasmids were transfected into
semiconfluent 3T3 cell lines (Effectene reagent, Qiagen). Cells were
incubated from 2 to 24 h and harvested, and plasmid DNA was
recovered (63). Assays for removal kinetics of 8-oxoG were carried out
using a procedure described previously (59). Briefly, recovered
extrachromosomal DNA was treated or not with 5 ng of the
Escherichia coli Fpg protein (64) and analyzed on a 0.8%
agarose gel containing ethidium bromide. Plasmid DNA was detected by
Southern blotting, and quantification was done using a PhosphorImager
(Amersham Biosciences). The repair of 8-oxoG at each time point
corresponds to the ratio between covalently closed molecules (CC) after
an Fpg treatment and the sum of CC molecules and open circles (OC).
This assay requires that the integrity of the CC molecules is preserved
during DNA extraction. Thus conversion into the relaxed form (OC) of
the plasmid DNA depends on Fpg treatment (59).
mutY
by
electroporation, and transformants were selected by ampicillin resistance. The repair of 8-oxoG:C was monitored by NgoMIV
digestion of plasmid DNA extracted from individual bacterial clones as
described previously (65).
-32P]ATP and T4 polynucleotide kinase.
Primer extensions were performed using an automatic thermocycler and an
E. coli DNA polymerase I Klenow fragment, 250 µM dNTP in a reaction mixture containing 1 mM
dithiothreitol, 6.7 mM Tris-HCl, pH 8.8, and 6.6 mM MgCl2 (Biolabs). Amplification conditions
were 29 cycles with 30 s at 94 °C, 1 min at 54 °C, and 2 min
at 72 °C. Reactions were quenched by denaturing loading buffer, and
the reaction products were resolved on a 6% polyacrylamide/7 M urea
gel. A sequence of plasmid DNA using the unlabeled 18-mer primer was
performed according to the standard protocol provided by the
manufacturer (Sequenase 2.0 DNA sequence kit, Upstate Biotechnology)
and migrated in parallel to the primer extension synthesis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and PARP-1 Deficiencies Abrogates 8-OxoG:C
Repair on NTS but Not in TS--
To investigate the role of Pol
and
PARP-1 proteins in the repair of 8-oxoG in the cellular context, we
used the 3T3 cell lines WT, PARP-1
/
,
Pol
/
, and
PARP-1
/
Pol
/
, which have been
characterized previously (46). The different 3T3 cells were transfected
with non-replicative plasmids that contain a single 8-oxoG:C base pair.
Two constructs were used to allow repair analysis of 8-oxoG located in
the same sequence context but with a different transcriptional status
(59, 66). One plasmid allows the analysis of 8-oxoG repair in a TS,
whereas the other allows the analysis of 8-oxoG repair in the same
sequence but non-transcribed because of the deletion of the SV40
promoter (59, 66). To monitor the repair of 8-oxoG, plasmid DNA was recovered after incubation in 3T3 cells and digested by the Fpg protein, which specifically nicks DNA at the 8-oxoG:C base pair. In
this assay, 8-oxoG repair is characterized by increasing amounts of CC
plasmid molecules that are resistant to cleavage by Fpg, indicating the
replacement of the 8-oxoG:C pair with a G:C pair in DNA and the sealing
of the repair-induced nick or gap (59). Fig.
1 (right panel) gives an
illustration of different Southern blots obtained with NTS constructs
in each cell line used. Fig. 1 (left panel) shows a
comparison of 8-oxoG:C repair kinetics in TS and NTS in WT and mutant
3T3 cell lines. Fig. 1B shows that repair kinetics in
Pol
-null cells are very similar to those obtained in WT cells (Fig.
1A) independently of the transcriptional status of the
lesion, TS or NTS. Therefore, DNA polymerase
is not required for the repair of 8-oxoG:C in the cellular context, indicating the
action of other DNA polymerases. Fig. 1C shows the repair kinetics of 8-oxoG:C in PARP-1-null cell lines. Again, there is no
obvious difference between PARP-1
/
and WT with 8-oxoG:C
in the TS status. In contrast, 8-oxoG:C repair on NTS is delayed in
PARP-1-null cells when compared with that observed in the WT cell line.
However, full repair of 8-oxoG:C is observed at 24 h in the
PARP-1
/
cells (Fig. 1C). These results
suggest that the absence of PARP-1 impairs an efficient replacement of
8-oxoG with a guanine, even if the protein is not absolutely required
in the cellular context.
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Fig. 1.
Kinetics of 8-oxoG:C repair in 3T3 cell
lines. The non replicating shuttle vectors pS oriSV-[8-oxoG:C]
(TS) and pS
(ori+p)SV-[8-oxoG:C] (NTS)
were transfected into WT (A), Pol
/
(B), PARP-1
/
(C), or
PARP-1
/
Pol
/
(D) 3T3 cell
lines. Plasmid DNA was recovered after 4-24-h incubations. Extracted
plasmid DNA was treated by Fpg and migrated on an agarose gel
containing ethidium bromide. DNA migration was observed after Southern
blotting, and the nicked (OC) and CC plasmids were
quantified using a PhosphorImager. Control lanes are designated by
C. Right panels show representative Southern
blots of pS
(ori+p)SV-[8-oxoG:C] in Pol
/
,
PARP-1
/
and
PARP-1
/
Pol
/
3T3 cell lines.
Left panels show the percentage of repair corresponding to the
ratio of covalently closed molecules to the total amount of recovered
plasmid DNA. Experimental values are the average of two or three blots
resulting from independent transfections with two independent
preparations of monomodified plasmid DNA. Error bars are shown.
and PARP-1, we analyzed
the processing of 8-oxoG:C in a double deficient
PARP-1
/
Pol
/
cell line. Fig.
1D shows that the 8-oxoG:C base pair is efficiently repaired
when located on a TS plasmid in both WT and
PARP-1
/
Pol
/
cell lines. In contrast,
until 18 h after transfection we did not observe detectable repair
of 8-oxoG:C in the NTS plasmid, whereas all molecules were repaired in
WT cells. Finally, less than 10% of repair may be observed in the
double knockout cells 24 h after transfection (Fig.
1D). These results show that full repair of 8-oxoG:C is
greatly impaired in the NTS plasmid in cells lacking both Pol
and
PARP-1 proteins.
/
PARP-1
/
Double Knockout
Cells--
The absence of repair of 8-oxoG:C in the NTS plasmid
12 h after transfection in the PARP-1-null Pol
-null cells could
be due to an impaired recognition and/or excision of 8-oxoG by the Ogg1 DNA N-glycosylase. Therefore, Ogg1 enzyme activity in crude
extracts was assayed using as substrate a 34-mer oligonucleotide
containing an 8-oxoG:C base pair. Fig. 2
shows that a cell-free extract of the PARP-1-null Pol
-null 3T3 cells
efficiently cleaves the 8-oxoG:C duplex. Furthermore, Western blotting
analysis using anti-human Ogg1 antibodies reveals a normal expression
of the murine Ogg1 in PARP-1-null Pol
-null cells (data not shown).
These results strongly suggest that removal of 8-oxoG is not globally
affected. If 8-oxoG is efficiently processed by Ogg1, the plasmid DNA
recovered from PARP-1-null Pol
-null cells should contain a nick at
or near the site of the lesion. Fig. 3
shows that NTS plasmid DNA recovered from PARP-1-null Pol
-null cells
12 h after transfection migrates as an OC with and without
Fpg-treatment. This result indicates that these plasmids have been
nicked in the cell, presumably after removal of 8-oxoG. Unspecific
cleavage of the NTS plasmid in PARP-1-null Pol
-null cells is
unlikely, because TS plasmid transfected in the same cells is recovered
as s covalently closed circle (Fig. 3, bottom). The kinetics
of strand cleavage of the NTS plasmid in PARP-1-null Pol
-null cells
was compared with those of 8-oxoG repair in WT cells. Fig.
4 shows that the kinetic of cleavage in
PARP-1-null Pol
-null cells is very similar to that of full 8-oxoG
repair in WT. To demonstrate the removal of 8-oxoG from the NTS plasmid
in PARP-1-null Pol
-null cells, the recovered plasmid DNA was
transformed into the
fpg
mutY
mutant strain
of E. coli. If 8-oxoG is still present in DNA, it has to
induce specific G:C
T:A transversion during DNA replication in this
bacterial strain (9, 67). Therefore, after amplification, the
8-oxoG-containing plasmids generate a population that contains one or
two NgoMIV restriction sites. In contrast, a plasmid without 8-oxoG generates a pure population containing a single
NgoMIV restriction site as described previously (68). Our
results show that only 1 of 96 clones tested contained the 8-oxoG:C
pair (data not shown). Taken together, these data strongly suggest that
the removal of the 8-oxoG lesion in NTS is not affected by the deletion of Pol
and PARP-1 in the cell.
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Fig. 2.
Cleavage of a 34 mer 8-oxoG:C duplex by crude
cell-free protein extracts of 3T3 cell lines. A 34-mer
oligodeoxyribonucleotide containing a single 8-oxoG was 5'
32P-labeled and hybridized with a complementary strand
yielding a duplex containing a cytosine opposite 8-oxoG. The 8-oxoG:C
duplex was incubated with 10 µg of crude cell-free extracts
from WT, PARP-1 /
, Pol
/
, or double
PARP-1
/
Pol
/
. The assay was performed
at 37 °C for 15 min, and the reaction products were analyzed in a
20% denaturing PAGE. Control (
) is an 8-oxoG:C duplex incubated with
buffer. The substrate corresponds to the 34-mer, and the product to the
16-mer obtained after successive cleavages by Ogg1 DNA glycosylase and
AP endonuclease- containing extract.
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Fig. 3.
Visualization of the
pS oriSV-[8-oxoG:C] (TS) and
pS
(ori+p)SV-[8-oxoG:C]
(NTS) plasmid molecules after 12 h of incubation
in WT and PARP-1
/
Pol
/
cell line. Extracted
DNA was treated or not by Fpg, migrated on an agarose gel containing
ethidium bromide and revealed after Southern blotting. As controls,
migration of OC and CC molecules are indicated.
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Fig. 4.
. Kinetics of cleavage of
pS (ori+p)SV-[8-oxoG:C] (NTS) plasmid in
PARP-1
/
Pol
/
cell line and full repair of the same plasmid in WT cell line.
pS
(ori+p)SV-[8-oxoG:C] construct was transfected in
PARP-1
/
Pol
/
, recovered after 2-12 h
of incubation, and analyzed by Southern blotting without previous Fpg
treatment. In parallel, repair kinetics of the constructs were obtained
as described in the Fig. 1 legend. Curves obtained for the double
deficient (
) and WT (
) lines correspond to the quantitative
analysis of two to six Southern blots by time point. Error
bars are shown.
/
PARP-1
/
Cells in NTS Results from
a Defect in 5'-dRPase and/or DNA Repair Synthesis
Activities--
Repair assays with purified proteins or cell-free
extracts show that AP sites generated by Ogg1, after removal of 8-oxoG, are primarily incised at the 5'-side by Ape1 yielding 5'-dRP and 3'-OH
(20, 69). The results reported in this in vivo study show
that removal of 8-oxoG in the NTS or TS plasmid occurs normally in WT,
PARP-1
/
, Pol
/
, and the
PARP-1
/
Pol
/
double mutant. However,
incomplete repair of 8-oxoG in NTS is observed in the double deficient
cell line, suggesting a defect in a late step in the course of the
repair process. As an attempt to identify the repair intermediate that
accumulates in PARP-1-null Pol
-null 3T3 cells, we performed primer
extension studies, using as template the NTS plasmid DNA recovered from
the double deficient cell lines 12 h after transfection. The
principle of this assay is described briefly in Fig.
5A. Extracted plasmid DNA is
hybridized with a 5'-end, 32P-labeled 18-mer primer
specific to the strand containing the lesion. Afterward, primer
extension is performed using the Klenow fragment of E. coli
DNA polymerase I. Fig. 5B shows a strong arrest of
polymerization with DNA recovered from
PARP-1
/
Pol
/
cells. In contrast, no
polymerization arrest is observed using DNA recovered from
WT or Pol
/
(Fig. 5B). Location of the
arrest band was determined by comparing primer extension and plasmid
sequencing using the same primer. Sequence analysis indicates that the
arrest band corresponds to an incorporation opposite to the original
position of 8-oxoG in the plasmid DNA (Fig. 5B). This
observation strongly suggest a cleavage of DNA by Ape1 at the site of
the lesion resulting in the formation of a 5'dRP residue (Fig.
5A).
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Fig. 5.
Primer extension study using NTS
plasmid recovered from deficient and WT cell lines.
pS (ori+p)SV-[8-oxoG:C] (NTS) construct was transfected in 3T3 cell
lines deficient in PARP-1
/
Pol
/
and
Pol
/
or WT and recovered after 12 h incubation.
A, rational of the study. X indicates the site of
the lesion; it may be an intact guanine if the repair is complete
or an 8-oxoG/nick/gap if it is not (arrows show the incision
site by Ogg1 and Ape1). B, analysis of primer extension
products on 6% denaturing polyacrilamide gel. A sequence obtained
using the same primer shows the complementary strand of the sequence
containing the lesion, and the location of the arrest of
polymerization. NgoMIV restriction site is indicated. |C
is the base opposite the lesion.
/
Cells--
The results indicate that complete
repair of 8-oxoG:C on NTS plasmid requires the PARP-1 in a Pol
-null
background. To investigate the role of the poly(ADP-ribose) synthesis
in the 8-oxoG:C repair process, we constructed two 3T3 cell lines,
pECV-PARP-1 and pECV-PARP-1E988K, that express either the
wild-type human PARP-1 or a catalytically inactive PARP-1 mutant
(E988K) in the PARP-1
/
Pol
/
background. The E988K mutation inhibits PARP-1 activity without affecting its DNA binding capacity (61). Selected clones express the
PARP-1 protein at a level similar to that of the wild type 3T3 cell
line (Fig. 6). These cells lines were
used to study the repair of 8-oxoG:C on NTS. As expected, the
PARP-1
/
Pol
/
cells expressing the WT
PARP-1 (pECV-PARP-1) efficiently repair 8-oxoG:C on NTS (Fig.
7). In contrast, PARP-1
/
Pol
/
cells expressing the mutant PARP-1
(pECV-PARP-1E988K), do not allow the repair of 8-oxoG:C on
NTS (Fig. 7). Therefore, the poly(ADP-ribosyl)ation reaction performed
by PARP-1 is required for the repair of 8-oxoG:C in vivo in
the absence of Pol
.
View larger version (11K):
[in a new window]
Fig. 6.
Identification of stably PARP-1-expressing
clones. PARP-1 /
Pol
/
cell lines
were transfected with a mock vector, a vector containing the cDNA
encoding a WT PARP-1 cDNA, or a vector containing the cDNA
encoding a mutated PARP-1 gene (E988K). Total cell extracts were
prepared and analyzed by Western blotting using anti-PARP-1 polyclonal
antibodies.
View larger version (17K):
[in a new window]
Fig. 7.
Kinetics of 8-oxoG:C repair in
PARP-1 /
Pol
/
3T3 cell lines expressing WT and mutated PARP-1. The
non-replicating shuttle vector pS
(ori+p)SV-[8-oxoG:C] (NTS) was
transfected into PARP-1
/
Pol
/
3T3
cell lines, recovered after 6- and 12-h incubations, and analyzed for
the repair of 8-oxoG:C base pair. A, Southern blotting after
transfection in the different clones and treatment by Fpg as described
in the Fig. 1 legend. Control lanes (C) correspond to
monomodified plasmids incubated with (+) or without (
) Fpg.
B, quantitative analysis of the Southern blots. Experimental
values are the average of two blots resulting from two transfections
from independent preparations of monomodified plasmid DNA. Errors
bars are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. Our data show that the repair of
8-oxoG in TS is not significantly affected by the inactivation of
Pol
and/or PARP-1 proteins. This last result points again to the
occurrence of a specific DNA repair pathway, associated with
transcription, acting at oxidative DNA damage such as 8-oxoG in human
and mice cells. In human cells, repair of 8-oxoG in TS requires TFIIH,
XPG, CSB, BRCA1, and BRCA2 but is independent of XPA (66, 78). In mouse
cells, this pathway is dependent on CSB but independent of Ogg1,
Pol
, and PARP-1 (Refs. 59 and 77 and our study). In contrast, 8-oxoG
repair in NTS requires BER proteins such as Ogg1 and the combination of
Pol
and PARP-1 but not nucleotide excision repair (NER) proteins
(our study and Refs. 59, 66, and 77). PARP-1 has been reported to be a negative or positive regulator of transcription by modifying and/or binding several transcription factors (see Ref. 51 for a review). The
absence of PARP-1 has no effect on TS, indicating that the function of
PARP-1 in transcription is not related to DNA repair but is more likely
to participate in the organization of the chromatin architecture
(79).
, primarily involved in SP-BER, is
not essential in vivo for 8-oxoG repair in NTS, which is in
agreement with studies using cell-free extracts (26, 80). Presumably,
in Pol
-deficient cells other DNA polymerases such as Pol
and
Pol
would achieve DNA repair resynthesis. The removal of the 5'-dRP
normally carried out by Pol
5'dRP lyase activity during SP-BER could
be achieved by Fen1 according to a LP-BER process (Fig.
8). On the other hand, PARP-1
substantially influences the repair of 8-oxoG located in NTS. Indeed,
we observed delayed repair kinetics (~2-fold) of 8-oxoG in NTS in
PARP-1
/
cells compared with WT cells. This delayed
repair, if it happens in the genomic DNA, might have dramatic
consequences in the cells. Indeed, results obtained from "Comet"
assays using PARP-1
/
cells treated with methyl methane
sulfonate (MMS) also showed a delayed DNA strand break resealing,
causing cell growth retardation, G2/M accumulation, and
chromosome instability (48). Furthermore, inactivation of both Pol
and PARP-1 dramatically impairs the repair of 8-oxoG in NTS. The lack
of 8-oxoG repair in PARP-1-null Pol
-null cells indicates that, at
the least, one step in the course of the BER processes cannot be
performed in these cells. In the present work we show that the removal
of 8-oxoG by Ogg1, which is absolutely required to initiate BER of
8-oxoG in NTS, is efficiently performed in PARP-1-null Pol
-null
cells. Furthermore, the rate of incision of an 8-oxoG:C-containing NTS
plasmid in PARP-1
/
Pol
/
cells is very
similar to the rate of full repair in WT cells. These results strongly
suggest that the removal of 8-oxoG by Ogg1 occurs normally in
PARP-1
/
Pol
/
cells. They also
indicate that removal of 8-oxoG by Ogg1 is the rate-limiting step in
the course of BER in the cellular context as well as in cell-free
extracts. Because Ape1 is a very abundant and efficient AP
endonuclease, the NTS plasmids recovered from PARP-1
/
Pol
/
cells most probably
contain a nick at the site of the lesion. This is consistent with the
primer extension analysis, which shows a strong block at the site of
the lesion. Therefore, we conclude that inactivation of both PARP-1 and
Pol
does not impair early stages but rather a late stage(s) in the
course of BER of 8-oxoG, either the excision of the 5'-dRP or/and DNA
resynthesis.
View larger version (22K):
[in a new window]
Fig. 8.
A scheme for an efficient 8-oxoG repair in WT
cell lines showing the role of Pol and PARP-1
proteins. After removal of the lesion by Ogg1 DNA
glycosylase and cleavage of the resulting AP site by Ape1 endonuclease,
two subpathways would co-exist in vivo, one SPR-Pol
dependent (8.1) and the other LPR-PARP-1 dependent
(8.2). An overlapping role of PARP-1 and pol
would be
when PARP-1 would accelerate the recruitment of the DNA polymerase
.
The 5'dRP only or a short oligonucleotide containing the 5'dRP would be
removed, depending on the subpathway used, and BER would be achieved.
GO corresponds to the lesion.
The role of PARP-1 in the DNA polymerization step of LP-BER of the AP
site in cell-free extract has been proposed (46). However, a direct
involvement of this protein remains unclear despite physical
interactions shown between PARP-1 and Xrcc1 and Pol and DNA ligase
III (46, 55, 56, 81). Recently, Lavrik et al. (57). have
demonstrated the high affinity of PARP-1 for a BER intermediate
harboring a nick with a 5'-dRP end. Theses studies were recently
extended to show that PARP-1 stimulates FEN1 and Pol
during strand
displacement synthesis in a reconstituted system (58). Therefore a
defect in PARP-1 would lead to a reduction of LP-BER efficiency. Our
in vivo study shows that 8-oxoG repair in NTS is delayed in
PARP-1-null cells and abolished in PARP-1-null Pol
-null cells. In
PARP-1-null cells, removal of the 5'-dRP and DNA repair synthesis would
be SP-BER and dependent upon Pol
(Fig. 8). However, the delay
observed in 8-oxoG repair led us to conclude that Pol
is
rate-limiting in PARP-1-deficient cells. In
PARP-1
/
Pol
/
cells we may think that
the 5'-dRP is processed at a very slow rate because of Pol
inactivation and a low level of activity or expression of Fen1 to act
in absence of PARP-1 (31, 74). Alternatively, the recruitment of
replicative DNA polymerase may be very inefficient in the absence of
PARP-1. Our results also indicate that poly(ADP-ribosyl)ation of PARP-1
or other factors is essential in that specific context. Other studies
suggested a correlation between PARP-1 automodification and improved
DNA repair (82, 83). Poly(ADP-ribosyl)ation might be important either
for the recruitment of BER proteins or its dissociation from the nick
DNA. In addition, the lack of poly(ADP-ribosyl)ation activity was shown
to be the cause of the hypersensitivity of PARP-1
/
cell lines to ionizing radiation and alkylating
agents (71, 84)
In conclusion, the efficient repair of 8-oxoG observed in WT cells may
reflect the requirement for PARP-1 and Pol in BER of 8-oxoG in
distinct but overlapping subpathways, each of which can compensate for
loss of the other (Fig. 8). Our work is the first demonstration for a
role of PARP-1 and poly(ADP-ribosylation) in late stage(s) in the
course of BER of 8-oxoG in the cellular context.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank, Dr Josiane Ménissier-de Murcia for the establishment of the 3T3 cell lines and Drs. Lionel Gellon, Stephanie Marsin, and J. Pablo Radicella for helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by CNRS and CEA, the Association pour la Recherche sur le Cancer (ARC) Grant 5432 (to S. B.) and European Commission Grant FIGH-CT-2002-00207, and additional funds from CNRS, the Association pour la Recherche Contre le Cancer, Electricité de France, Ligue Nationale Contre le Cancer, and Commissariat à l'Energie Atomique (to G. d. M.).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: 00331-46548939; Fax: 00331-46548859; E-mail: lepage@dsvidf.cea.fr.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M212905200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
8-oxoG, 8-oxo-7,8-dihydroguanine;
BER, base excision repair;
SP-BER, short
patch BER;
LP-BER, long patch BER;
AP, apurinic/apyrimidic;
dRP, 2-deoxyribose 5-phosphate;
Pol, polymerase
;
Xrcc1, x-ray
cross-complementing factor 1;
Fen1, Flap endonuclease 1;
PARP-1, poly(ADP-ribose) polymerase 1;
WT, wild type;
TS, transcribed sequence;
NTS, non-transcribed sequence;
CC, covalently closed;
OC, open
circle.
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