Poly(ADP-ribose) Polymerase-1 (PARP-1) Is Required in Murine Cell Lines for Base Excision Repair of Oxidative DNA Damage in the Absence of DNA Polymerase beta *

Florence Le PageDagger §, Valérie Schreiber, Claudine DhérinDagger , Gilbert de Murcia, and Serge BoiteuxDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -/- (Polbeta -/-), poly(ADP-ribose) polymerase-1-/- (PARP-1-/-), and Polbeta -/-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 Polbeta or PARP-1 or both. Whereas 8-oxoG repair in NTS is normal in Polbeta -null cells, it is delayed in PARP-1-null cells and greatly impaired in cells deficient in both Polbeta 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-/-Polbeta -/- cell lines. However, 8-oxoG repair is incomplete, yielding plasmid molecules with a nick at the site of the lesion. Therefore, PARP-1-/-Polbeta -/- 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 Polbeta -/- 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 right-arrow 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 beta  (Polbeta ), 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 (Polbeta , delta  or epsilon ) 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 delta - or epsilon -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 delta  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).

Several studies pointed to Polbeta as the major repair DNA polymerase in eukaryotes, involved in both SP- and LP-BER (24, 30, 34-36). Polbeta lacks accessory functions such as 3' to 5' exonuclease, dNMP turnover, or pyrophosphorolysis (37-40). On the other hand, Polbeta possesses a robust AP lyase activity that allows the removal of 5'-dRP in the course of BER (41). Polbeta performs an essential function in development, because knockout mice for Polbeta are not viable (42, 43). However, Polbeta -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 Polbeta (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 Polbeta 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 Polbeta 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 Polbeta -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.

To investigate the role of Polbeta 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 Polbeta (Polbeta -/-) or PARP-1 (PARP-1-/-), or cells that present the two deficiencies (PARP-1-/-Polbeta -/-). 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, Polbeta -null, PARP-1-null, and double PARP-1-null Polbeta -null 3T3 cells. On the other hand, repair of 8-oxoG in an NTS plasmid, which is not affected in Polbeta -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 Polbeta and PARP-1. The role of the poly(ADP-ribose) synthesis during BER is also discussed in this study.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell Lines and Culture Conditions-- Spontaneously immortalized 3T3 wild-type, homozygous PARP1-/-, homozygous Polbeta -/-, and double deficient PARP-1-/-Polbeta -/- 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-/-Polbeta -/- 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.

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 pSDelta oriSV-[8-oxoG:C] or pSDelta (ori-p)SV-[8-oxoG:C] used for repair kinetics has been described (59). pSDelta oriSV-[8-oxoG:C] (TS) or pSDelta (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).

Mutagenesis Assay-- Transfection and plasmid extraction were performed as above. Recovered plasmid DNA was used to transform E. coli strain (BH990) fpg-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).

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 [gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Combination of Polbeta and PARP-1 Deficiencies Abrogates 8-OxoG:C Repair on NTS but Not in TS-- To investigate the role of Polbeta and PARP-1 proteins in the repair of 8-oxoG in the cellular context, we used the 3T3 cell lines WT, PARP-1-/-, Polbeta -/-, and PARP-1-/-Polbeta -/-, 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 Polbeta -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 beta  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 pSDelta oriSV-[8-oxoG:C] (TS) and pSDelta (ori+p)SV-[8-oxoG:C] (NTS) were transfected into WT (A), Polbeta -/- (B), PARP-1-/- (C), or PARP-1-/-Polbeta -/- (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 pSDelta (ori+p)SV-[8-oxoG:C] in Polbeta -/-, PARP-1-/- and PARP-1-/-Polbeta -/- 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.

To further investigate the role of Polbeta and PARP-1, we analyzed the processing of 8-oxoG:C in a double deficient PARP-1-/-Polbeta -/- 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-/-Polbeta -/- 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 Polbeta and PARP-1 proteins.

Removal of 8-OxoG in NTS Is Not Affected in Polbeta -/- 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 Polbeta -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 Polbeta -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 Polbeta -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 Polbeta -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 Polbeta -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 Polbeta -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 Polbeta -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 Polbeta -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 Polbeta -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 right-arrow 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 Polbeta 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-/-, Polbeta -/-, or double PARP-1-/-Polbeta -/-. 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 pSDelta oriSV-[8-oxoG:C] (TS) and pSDelta (ori+p)SV-[8-oxoG:C] (NTS) plasmid molecules after 12 h of incubation in WT and PARP-1-/- Polbeta -/- 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 pSDelta (ori+p)SV-[8-oxoG:C] (NTS) plasmid in PARP-1-/- Polbeta -/- cell line and full repair of the same plasmid in WT cell line. pSDelta (ori+p)SV-[8-oxoG:C] construct was transfected in PARP-1-/-Polbeta -/-, 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 (triangle ) and WT (black-square) lines correspond to the quantitative analysis of two to six Southern blots by time point. Error bars are shown.

Incomplete Repair of 8-OxoG in Double Polbeta -/-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-/-, Polbeta -/-, and the PARP-1-/-Polbeta -/- 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 Polbeta -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-/-Polbeta -/- cells. In contrast, no polymerization arrest is observed using DNA recovered from WT or Polbeta -/- (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. pSDelta (ori+p)SV-[8-oxoG:C] (NTS) construct was transfected in 3T3 cell lines deficient in PARP-1-/-Polbeta -/- and Polbeta -/- 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.

Poly(ADP-ribose) Polymerase Activity of PARP-1 Is Required for the 8-OxoG:C Repair on NTS in Vivo in Polbeta -/-Cells-- The results indicate that complete repair of 8-oxoG:C on NTS plasmid requires the PARP-1 in a Polbeta -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-/-Polbeta -/- 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-/-Polbeta -/- cells expressing the WT PARP-1 (pECV-PARP-1) efficiently repair 8-oxoG:C on NTS (Fig. 7). In contrast, PARP-1-/- Polbeta -/- 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 Polbeta .


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Fig. 6.   Identification of stably PARP-1-expressing clones. PARP-1-/-Polbeta -/- 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.


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Fig. 7.   Kinetics of 8-oxoG:C repair in PARP-1-/- Polbeta -/- 3T3 cell lines expressing WT and mutated PARP-1. The non-replicating shuttle vector pSDelta (ori+p)SV-[8-oxoG:C] (NTS) was transfected into PARP-1-/-Polbeta -/- 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Polbeta . Our data show that the repair of 8-oxoG in TS is not significantly affected by the inactivation of Polbeta 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, Polbeta , 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 Polbeta 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).

Our study has also shown that Polbeta , 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 Polbeta -deficient cells other DNA polymerases such as Poldelta and Polepsilon would achieve DNA repair resynthesis. The removal of the 5'-dRP normally carried out by Polbeta 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 Polbeta and PARP-1 dramatically impairs the repair of 8-oxoG in NTS. The lack of 8-oxoG repair in PARP-1-null Polbeta -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 Polbeta -null cells. Furthermore, the rate of incision of an 8-oxoG:C-containing NTS plasmid in PARP-1-/-Polbeta -/- 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-/-Polbeta -/- 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-/-Polbeta -/-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 Polbeta 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.


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Fig. 8.   A scheme for an efficient 8-oxoG repair in WT cell lines showing the role of Polbeta 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-Polbeta dependent (8.1) and the other LPR-PARP-1 dependent (8.2). An overlapping role of PARP-1 and polbeta would be when PARP-1 would accelerate the recruitment of the DNA polymerase beta . 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 Polbeta 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 Polbeta 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 Polbeta -null cells. In PARP-1-null cells, removal of the 5'-dRP and DNA repair synthesis would be SP-BER and dependent upon Polbeta (Fig. 8). However, the delay observed in 8-oxoG repair led us to conclude that Polbeta is rate-limiting in PARP-1-deficient cells. In PARP-1-/-Polbeta -/- cells we may think that the 5'-dRP is processed at a very slow rate because of Polbeta 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 Polbeta 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; Polbeta , polymerase beta ; 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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