Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404524200 on October 21, 2004

J. Biol. Chem., Vol. 279, Issue 53, 55117-55126, December 31, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/53/55117    most recent
M404524200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Audebert, M.
Right arrow Articles by Calsou, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Audebert, M.
Right arrow Articles by Calsou, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Involvement of Poly(ADP-ribose) Polymerase-1 and XRCC1/DNA Ligase III in an Alternative Route for DNA Double-strand Breaks Rejoining*

Marc Audebert{ddagger}, Bernard Salles§, and Patrick Calsou

From the Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, F-31077 Toulouse Cedex, France

Received for publication, April 23, 2004 , and in revised form, October 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin {gamma}1. In addition, the repair kinetics of the DSBs induced by calicheamicin {gamma}1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA double-strand breaks (DSBs)1 represent normal intermediates during physiological processes like meiosis or V(D)J recombination but also toxic lesions produced by collapsed DNA replication forks and by DNA-damaging agents such as ionizing radiation (IR) or reactive oxygen species. Repair of DSBs is critical for the maintenance of genomic integrity because unproperly repaired breaks can lead to cancer via chromosomal aberrations (1, 2).

In eukaryotic cells, DSBs are repaired through two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ) (for reviews see Refs. 3–6). It is largely admitted that DSBs are repaired by NHEJ at least in the G1 phase of the cell cycle, whereas HR operates in late S/G2 (7). The NHEJ process requires several factors that recognize and bind the double-strand break, catalyze the synapsis of the broken ends, and then process and reseal the break (8, 9). The NHEJ pathway relies on a set of proteins comprising at least (i) a DNA end binding activity, the DNA-dependent protein kinase (DNA-PK) that consists of the catalytic subunit DNA-PKcs and the Ku70/Ku80 heterodimer (10, 11), and (ii) a DNA break resealing activity, the XRCC4-DNA ligase IV complex (12, 13).

In order to gain insight into the NHEJ mechanism, both in vitro and in vivo approaches have been undertaken. The end-joining reaction has been studied in vitro by various assays, many of them using incubation with cell extracts of plasmid DNA linearized by enzymatic restriction as a model for DSBs containing substrates (17). Results of these in vitro repair experiments have brought substantial evidence for an alternative DNA-PK-independent end-joining pathway (18–24). In vivo studies also support the hypothesis that several DNA end-joining pathways exist either in yeast (25, 26) or in mammalian cells (27–31). For example, end-joining of restricted DNA remained efficient after transfection of cells deficient in various components of the DNA-PK-dependent pathway and relied mostly on an end homology-dependent mechanism (29). Also, it was shown recently that Ku was dispensable for the microhomology-directed rejoining of an intrachromosomal DSB (32). Taken together, these in vitro and in vivo studies strongly suggest that end-joining of DSBs relies on more than the DNA-PK-dependent pathway.

Any candidate for an alternative end-joining pathway of DSBs would recognize DNA ends, and its defect would have an impact on cell survival to double-strand breaking agents. Based on its high binding affinity for DNA DSBs (33, 34), the involvement of poly(ADP-ribose) polymerase-1 protein (PARP-1) in sensing and/or repairing DSBs can be hypothesized. PARP-1 is involved in different cellular processes, including the DNA base excision repair pathway (BER), responsible for the removal of alkylated bases and abasic sites (for reviews see Refs. 35–38). PARP-1 catalyzes the covalent transfer of successive units of ADP-ribose moiety from NAD to itself and other nuclear protein acceptors in a manner dependent upon the presence of DNA single-strand breaks (SSBs). A possible role of PARP-1 in the repair of DSBs is supported by the following points. (i) Ku heterodimer does not represent the sole DSBs recognition complex in cell extracts because it was originally pointed out that PARP-1 is activated in vitro not only by SSBs but also by DSBs (39) and that purified PARP-1 binds to DSBs with an efficacy higher than to SSBs (33, 34) and with an affinity even greater than that of DNA-PK (34). (ii) PARP-1 has been shown to interact with both subunits of DNA-PK (40, 41) and to catalyze their poly(ADP-ribosyl)ation (42, 43). (iii) In cells, nuclear areas of poly(ADP-ribosyl)ation were induced concomitantly to the formation of direct DSBs via V(D)J recombination in the absence of functional DNA-PK (44). (iv) DSB repair was delayed under conditions of overexpression of the catalytically inactive DNA-binding domain of PARP-1 (45) or when cells were pretreated with a PARP-1 inhibitor (46–50). Despite all these data, no biochemical experiments were yet specifically devoted to substantiate the potential role of PARP-1 in the repair of DSBs.

Classical DSB-inducing agents such as IR mostly induce SSBs and base damage but only a few percent of DSBs. Therefore, we have used calicheamicin {gamma}1 that is a more potent producer of DSBs (51). We initially found that both chemical PARP inhibition or genetic loss of PARP-1 function impaired DSBs repair. Then we developed a DNA pull-down assay using nuclear extracts (NE) from mammalian cells. By using this approach, we have identified a new synapsis and end-joining activity independent of the classical NHEJ proteins. We have reconstituted this DSB end-joining activity with recombinant PARP-1, XRCC1, and DNA ligase III, proteins otherwise involved in BER (52). Taken together, these results suggest that a PARP-1-dependent DSB end-joining mechanism operates in cells as an alternative route that complements the DNA-PK/XRCC4/ligase IV-dependent NHEJ.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells Lines and Culture—EM9 (XRCC1 mutant) and AA8 (parental Chinese hamster) cell lines were kindly provided by Dr. L. Thompson (Livermore, CA). PARP-1–/– and control mouse embryonic fibroblasts were a gift from Dr. G. de Murcia (Illkirch, France). The Ku80-deficient xrs6 cell line and the control xrs6/haKu80 cell line transfected with the hamster Ku80 cDNA were obtained from the European Collection of Animal Cell Culture (Salisbury, UK). Dr. G. Whitmore (Toronto, Ontario) generously provided us with the DNA-PKcs mutant V3 cell line. Cell lines were grown in {alpha}-minimum Eagle's medium (CHO cells) or Dulbecco's modified Eagle's medium (MEF cells) (Invitrogen) supplemented with 10% fetal calf serum, 2 mM glutamine, 125 units/ml penicillin, and 125 µg/ml streptomycin.

Clonogenic Survival Assays—Cells growing in exponential phase in tissue culture flasks were trypsinized, and 500 cells per 35-mm Petri dish were plated and left to attach for 24 h. Calicheamicin {gamma}1, a generous gift from P. R. Hamann (Wyeth Research, Pearl River, NY), was dissolved in ethanol and stored at –70 °C. The drug was diluted in uncomplemented culture medium immediately before use and added directly to the cell culture. When indicated, 1,5-dihydroxyisoquinolin (DIQ, Sigma) dissolved in Me2SO was added to the cell culture medium at 10 µM 1 h before calicheamicin {gamma}1. The dishes were then incubated at 37 °C and 5% CO2 for 7 days. Dishes were washed with phosphate-buffered saline, and colonies were stained with crystal violet (2 mg/ml). Triplicate wells were seeded per point. Colonies containing over 50 cells were scored under an inverted microscope.

Antibodies—Anti-actin, anti-Ku70 (N3H10), anti-Ku80 (clone 111), anti-Ku70/80 (clone 162), and anti-XRCC1 (33-2-5) monoclonal antibodies were from Neomarkers (Fremont, CA). The monoclonal anti-PARP-1 antibody was from Zymed Laboratories Inc., and anti-PAR monoclonal antibody was from Trevigen (Gaithersburg, MD). Polyclonal rabbit antibodies anti-XRCC4 and anti-DNA ligase IV were from Serotec Ltd. (Oxford, UK) and from Abcam Ltd. (Cambridge, UK), respectively. Monoclonal antibodies anti-phosphorylated H2AX (JBW301) and anti-proliferating cell nuclear antigen (PC10) were from Upstate Cell Signaling Solutions (Milton Keynes, UK) and DAKO (Denmark), respectively.

Immunoblotting—Protein samples were heated with SDS sample buffer at 95 °C for 5 min, resolved by SDS-PAGE, and electrotransferred onto polyvinylidene difluoride membrane (Amersham Biosciences). Membranes were blocked with 5% milk in PBS for 1 h at room temperature, and proteins were immunoblotted with primary antibodies overnight at 4 °C and then probed with secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch). Specific proteins were visualized as immunoreactive bands by the enhanced chemiluminescence detection system (SuperSignal West Pico, Pierce).

{gamma}-H2AX Dephosphorylation Assay—Cells growing in exponential phase in tissue culture flasks were trypsinized, and 6 x 105 cells per well were plated in 6-well tissue culture dishes and left to attach for 24 h. Cells were pretreated for 1 h with or without 100 µM DIQ and then treated in serum-free media with 3 pM calicheamicin {gamma}1 in the presence or absence of DIQ. The cells were then rinsed twice and post-incubated in fresh culture medium still in the presence of DIQ when necessary, for the time indicated. At each time point, cells were washed with phosphate-buffered saline and scraped, spun down for 4 min at 1000 rpm, and resuspended in lysis buffer (PBS-SDS 1%). Extracts were boiled for 10 min and sonicated for 20 s. Protein samples were resolved on 15% SDS-PAGE, electrotransferred onto polyvinylidene difluoride membrane (Amersham Biosciences), and immunoblotted as described above.

Nuclear Protein Extracts Preparation—HeLa nuclear extracts were purchased from Computer Cell Culture Center (Seneffe, Belgium). Nuclear extracts from EM9, AA8, and PARP-1–/– cells were prepared as described previously (53). For actin or Ku immunodepletions, anti-actin and anti-Ku70/80 (clone 162) were coupled to magnetic anti-mouse IgG beads (Dynabeads M-450, Dynal). For XRCC4 immunodepletion, anti-XRCC4 antibodies were coupled to magnetic anti-rabbit IgG beads (Dynabeads M-280, Dynal), according to the manufacturer's recommendations. Two successive depletions with each antibody were performed as described (54).

Oligonucleotides Substrates—Oligodeoxyribonucleotides were purchased from Sigma Genosys. The blunt-end YC0 DNA fragment was constructed by annealing the 30-nucleotide oligomer 5'-TAAAGGGAACAAAAGCTGGGTACCGGTGTT-3' biotinylated on the 5'-end with the complementary C0 nonbiotinylated oligonucleotide. The 5' YC protruding oligonucleotides were constructed by annealing the 30-mer biotinylated as above with nonbiotinylated oligonucleotides bearing various protruding 5'-ends (5'-CG-3' for C2, 5'-GGCC-3' for C4, 5'-CGATCG-3' for C6, 5'-CGTTAACG-3' for C8, and 5'-AATT-3' for NC, respectively). The 4-nucleotide 3'-protruding DNA fragment YC4 –3' was constructed by annealing the C0 oligonucleotide with the oligonucleotide 5'-TAAAGGGAACAAAAGCTGGGTACCGGTG-TTCCGG-3', biotinylated on the 5'-end. All YC fragments contained an AluI restriction site at position +15. In each case, the nonbiotinylated oligomer was phosphorylated at the 5'-end by T4 polynucleotide kinase (New England Biolabs), and unincorporated ATP was removed by gel filtration (Sepharose G-50, Amersham Biosciences). In order to construct radiolabeled oligomers, the T4 polynucleotide kinase reaction was performed in the presence of [{gamma}-32P]ATP (4500 Ci mmol–1, ICN Pharmaceuticals), and unincorporated [{gamma}-32P]ATP was removed by gel filtration as above.

DNA Pull-down Assay—40 pmol of YC oligonucleotide were immobilized on 100 µl of streptavidin paramagnetic beads (Dynabeads M280 streptavidin, Dynal) as recommended by the manufacturer. 5 µl of mock- or DNA-treated beads were washed in reaction buffer A (40 mM HEPES, pH 7.8, 10 mM MgCl2, 60 mM KOAc, 0.5 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.05% Nonidet P-40) and then incubated under gentle agitation in a final 10-µl reaction at 30 °C for 1 h with 30 µg of NE, with or without 1 mM NAD (Sigma) as indicated. The supernatant was removed, and the beads were washed twice in an excess volume of reaction buffer, and 0.2 pmol of radioactive YC DNA fragment preincubated with 5 pmol of soluble streptavidin (Sigma) was added in a final 10-µl reaction with or without 1 mM ATP for 1 h at 30°C. Beads were washed twice in an excess volume of reaction buffer. For analysis of the pulled down DNA, beads were heat-inactivated at 65 °C for 10 min and incubated in a final 20-µl reaction with 10 units of AluI, plus 10 units of HaeIII, 10 units of MseI, or 10 units of MboI when necessary (all from New England Biolabs) for 2 h at 37 °C. Reaction products were analyzed by electrophoresis on 20% polyacrylamide denaturing gels, and autoradiography of the gel was processed with a PhosphorImager (Storm SystemTM, Amersham Biosciences). Quantitative analysis of the gel was performed with the ImageQuant software (5.2 version).

Adenylation Assay—After incubation with nuclear extracts as above, DNA beads were reacted for 1 h at 30 °C under gentle agitation in reaction buffer containing 5 µCi of [{alpha}-32P]ATP (3000 Ci/mmol, ICN Pharmaceuticals, Inc.). After five washes in reaction buffer, DNA beads were heated in SDS sample buffer and resolved by 8% SDS-PAGE followed by electrotransfer onto polyvinylidene difluoride membrane (Amersham Biosciences). Adenylated proteins were detected by autoradiography of the membrane processed with a PhosphorImager (Storm SystemTM, Amersham Biosciences).

DNA Rejoining Assay with Purified and Recombinant Proteins—The vector coding for the histidine-tagged recombinant human DNA ligase III was a kind gift from Dr. T. Lindahl and Dr. D. Barnes, and the protein was produced as described (55). Recombinant human XRCC1 was provided by J. P. Radicella (CEA, Fontenay-aux-Roses, France) and was produced as described (56). Human recombinant PARP-1 protein (95% purity) was from Trevigen (Gaithersburg, MD). For the reaction with purified recombinant proteins PARP-1, XRCC1 and DNA ligase III were preincubated in reaction buffer B (20 mM Tris, pH 7.5, 10 mM MgCl2, 60 mM KOAc, 0.5 mM dithiothreitol, 0.3 mg/ml bovine serum albumin) at 30 °C for 10 min prior to 30 min of incubation with DNA beads at 30 °C, and the reaction was then conducted in reaction buffer B as in the DNA pull-down assay with nuclear extracts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PARP-1 Inhibition on the Cytotoxicity of Calicheamicin {gamma}1—If PARP-1 is involved in an end-joining reaction in vivo, a specific inhibitor should potentiate the cytotoxic effect of a DSB inducer. Indeed, previous reports (48–50) have shown such potentiation effect when IR was used in combination with a PARP-1 inhibitor. However, IR produce mainly SSBs, despite the fact that DSBs are believed to be responsible for cell toxicity (57). Consequently, we used calicheamicin {gamma}1 as a DSB generator. This natural hydrophobic enediyne antibiotic has been shown to produce DSBs with selectivity and efficiency higher than IR, yielding a 1:3 ratio of DNA DSBs to SSBs in vivo, compared with a 1:20 ratio for IR (51). In addition, we anticipated that the sensitization effect by a PARP inhibitor might rely on an end-joining reaction independent of DNA-PK. Therefore, we investigated by clonogenic assay the sensitivity of DNA-PK-proficient and -deficient rodent cells to the radiomimetic compound calicheamicin {gamma}1, in the presence or absence of a nontoxic concentration of the potent PARP-1 inhibitor DIQ (58). We treated in parallel two paired CHO cell lines, the control AA8 and DNA-PKcs-deficient V3 cell lines (59), and the hamster control Ku80 cDNA-complemented xrs6 (xrs6/haKu80) and the xrs6 Ku-deficient cell lines (60).

First, we confirmed that DIQ was a potent inhibitor of PARP-1 activity in vivo (61, 62) since we observed a dose-dependent inhibition by DIQ of the H2O2-induced overall cellular protein poly(ADP-ribosyl)ation by Western blotting with an anti-PAR antibody (data not shown). By performing clonogenic assay on the four rodent cell lines, we measured the cytotoxicity of DIQ and chose 10 µM as the highest drug concentration that still allowed 100% cell survival (data not shown). Then we assessed the clonogenic survival to calicheamicin {gamma}1 of rodent cells pretreated or not with 10 µM DIQ. Fig. 1, A and B, showed that DNA-PK deficiency sensitizes cells to calicheamicin {gamma}1 as compared with the respective control, 5.5-fold for xrs6 and 3.5-fold for V3 cells at LD50 (concentration of drug that inhibited cell survival by 50%), as already reported (63). To assess the effect of PARP-1 inhibition on cell sensitivity, we calculated for each cell line the potentiating factor as the mean ratio of LD50 for calicheamicin {gamma}1 alone divided by LD50 in the presence of DIQ. As shown in Table I, all the ratios were significantly superior to 1, clearly indicating that DIQ potentiates the cytotoxicity of calicheamicin {gamma}1 in both DNA-PK-proficient and -deficient cells.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1.
Effect of the PARP-1 inhibitor DIQ on cell survival after calicheamicin {gamma}1 treatment of DNA-PK-proficient and -deficient cell lines. Exponentially growing cells were preincubated or not for 1 h in the presence of 10 µM DIQ in the culture medium and then exposed to the indicated concentration of calicheamicin {gamma}1 in the same medium (three wells/dose). Stained colonies were counted after 7 days. Each point represent the mean of at least four experiments ± S.E. A, xrs6/haKu80 and xrs6 cells; B, AA8 and V3 cells. Open symbols, calicheamicin {gamma}1 alone; filled symbols, calicheamicin {gamma}1 + DIQ.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Comparison of PF50 values derived from survival curves

 
Kinetics of DSB Repair Determined by H2AX Dephosphorylation—Although calicheamicin {gamma}1 induces DSBs at a rate higher than IR, it also produces SSBs. Thus, it was necessary to focus on the repair of DBSs. It has been shown recently that a PARP inhibitor decreased DNA DSB repair in both DNA-PK-proficient and -deficient CHO cells exposed to IR (48, 50). Because DSB repair was quantified by neutral filter elution in these reports, we thought to use an alternative method in order to assess DSBs repair under similar conditions. Recent studies have demonstrated that the intracellular content of the phosphorylated histone variant H2AX (named {gamma}-H2AX) can be used to measure the repair of DSBs in CHO cells at low yield of DSBs (1 gray (7)). Under experimental conditions similar to ours, it has been calculated that calicheamicin {gamma}1 produced about 400 DSBs/nM (51). Accordingly, we chose a drug dose (3 pM) corresponding to the LD10 for xrs6/haKu80 cells as measured by clonogenic assay (Fig. 1). Then we followed the kinetics of loss of {gamma}-H2AX phosphorylation after a 1-h treatment of both DNA-PK-proficient and -deficient cells with this concentration of calicheamicin {gamma}1, in the presence or absence of DIQ. Proliferating cell nuclear antigen was used as a control of protein loading. As shown in Fig. 2A, {gamma}-H2AX was detected in whole cells extracts only after treatment of both DNA-PK-proficient and -deficient cells with the same dose of calicheamicin {gamma}1 (Fig. 2A, compare lanes 1 and 3). In the absence of DIQ, only DNA-PK-proficient cells showed a kinetics of {gamma}-H2AX dephosphorylation over the 1-h post-treatment period examined (Fig. 2A, compare lanes 3 and 9 for xrs6/haKu80 and xrs6 cell lines), indicating that it is directly correlated to the cellular DSB repair activity. In addition, the half-time of {gamma}-H2AX loss is generally longer than the DSB rejoining half-time (64). This could explain that phosphorylated H2AX was still detected 1-h post-treatment even in DNA-PK-proficient cells (Fig. 2A, lane 9 for xrs6/haKu80). In the presence of DIQ, there was an obvious potentiation of {gamma}-H2AX phosphorylation already detected at time point 0 post-treatment, in both cell lines (Fig. 2A, compare lanes 3 and 4). This observation is compatible with the possibility that a PARP-1-dependent DSBs repair pathway already takes place within the 1-h time interval of the drug treatment. Additionally, H2AX phosphorylation was maintained over the 1-h post-treatment period in both cell lines in the presence of DIQ (Fig. 2A, compare lanes 4 and 10).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 2.
Effect of DIQ on the kinetics of {gamma}-H2AX dephosphorylation after cell exposure to calicheamicin {gamma}1. Either CHO xrs6/haKu80 and xrs6 (A) or PARP-1–/– and wild type proficient cells (B) were pretreated for 1 h with or without 100 µM DIQ as indicated and then treated or not with 3 pM calicheamicin {gamma}1 in the same medium for 1 h. Then the cells were rinsed and post-incubated in fresh culture medium, still in the presence of DIQ when necessary, for the time indicated. At each time point, cells were scraped and resuspended in lysis buffer. Proteins were analyzed on 15% SDS-PAGE followed by Western blotting with antibodies as indicated.

 
These results support the possibility that PARP-1 is involved in the early repair of a significant fraction of DNA DSBs that escape DNA-PK-dependent end-joining. However, DIQ could have an effect on {gamma}-H2AX dephosphorylation independently of DSB repair. In addition, PARP-1 inhibition could block the enzyme on DNA breaks and impair their repair by conventional mechanisms, rather than preventing PARP-1 to participate in a PARP-dependent repair route. In order to check these possibilities, we followed the kinetics of loss of {gamma}-H2AX phosphorylation in both PARP-1-proficient and -deficient MEF cells after a short treatment with calicheamicin {gamma}1, in the presence or absence of DIQ. As shown in Fig. 2B, the kinetics of {gamma}-H2AX dephosphorylation in PARP-1-proficient cells was similar to that observed in xrs6/haKu80 (compare lanes 3 and 9 in Fig. 2B, upper panel, and in Fig. 2A, upper panel). In PARP-1–/– cells, there is an obvious potentiation of {gamma}-H2AX phosphorylation already detected at time point 0 post-treatment as compared with PARP-1-proficient cells (Fig. 2B, upper panel, compare lanes 3 and 4), and this difference was maintained all over the post-treatment period examined (Fig. 2B, upper panel, compare lanes 9 and 10). In the presence of DIQ, there was no observable effect on the kinetics of loss of {gamma}-H2AX phosphorylation in PARP-1–/– cells (Fig. 2B, compare lanes 6, 8 or 10 in the lower panel with the same lanes in the upper panel); in contrast, DIQ slowed down the kinetics of {gamma}-H2AX dephosphorylation in the PARP-1-proficient cells (Fig. 2B, compare lanes 7 or 9 in the lower panel with the same lanes in the upper panel) that then became indistinguishable with the PARP-1–/– cells (Fig. 2B, lower panel, compare lanes 9 and 10).

These results confirm recent reports (48) that indicate a role of PARP-1 activity in cell survival to DSBs. Moreover, cell sensitization to calicheamicin {gamma}1 treatment using PARP-1 inhibitor paralleled the effect on DSBs repair, as determined by the kinetics of {gamma}-H2AX dephosphorylation. These results suggest that the inhibition of a PARP-dependent route for DSBs repair by DIQ could play a role in its sensitization effect toward DSBs generators.

Characterization of a Mammalian End-joining Activity via a Two-stage DNA Pull-down Assay—Because a DSB repair mechanism independent of DNA-PK but relying on PARP-1 activity might contribute to cell survival, we decided to set up an in vitro assay in order to test the involvement of PARP-1 in an end-joining activity. A biochemical assay dependent on the presence of DNA-PK and XRCC4/ligase IV has already been reported (65). Because the end-joining process dependent on DNA-PK most probably requires a synapsis reaction between proteins present at each DNA end (66), we deliberately unfavored such reaction by using a two-step in vitro DNA pull-down assay with NEs from mammalian cells (Fig. 3). We used a 30-bp double-stranded DNA fragment (YC) biotinylated on one 5'-end and bearing a 5'-phosphate group on the opposite end allowing ligation between two YC fragments. In a first step, YC bound to streptavidin-conjugated magnetic beads (YC beads) was incubated with HeLa NEs, allowing recruitment of protein complexes recognizing DNA ends, namely at least DNA-PK and PARP-1. After a wash, the same oligonucleotide radiolabeled on the 5'-end (YC*), and bound to soluble streptavidin in order to orientate synapsis of DNA ends, was then added to YC beads and incubated with or without ATP. Consequently, only proteins bound to the DNA on beads in the first step were able to perform synapsis of DNA ends and the subsequent ligation reactions (66). Two washes were then performed to limit non-specific association. YC fragments with a free blunt end (YC0) or with 2-, 4-, 6-, or 8-base complementary ends (YC2, YC4, YC6, and YC8, respectively) were used in parallel experiments. After the two-step pull-down reaction, soluble DNA was recovered by incubation of the DNA beads with AluI restriction enzyme, which allows us to differentiate the ligated YC duplex from the native unligated YC monomers. As shown in a representative experiment (Fig. 4A) and after quantification of the data (Fig. 4B), radiolabeled DNA was marginally retained on beads if NEs were omitted in the reaction. In contrast, with NE and in absence of ATP, a significant amount of YC* was trapped on beads whatever the extent of the 5'-overhang (Fig. 4A), leading to approximately the same quantity of radioactivity pulled down for all the oligonucleotides (Fig. 4B). Only unligated YC* monomers accounted for the pulled down radioactivity in the absence of ATP (Fig. 4A). With 1 mM ATP, a ligation product with the expected length could be observed with all the oligonucleotides tested, including the blunt-ended YC0 oligonucleotide (Fig. 4A). Moreover, the yield of oligonucleotide retained in the presence of ATP varied with the 5'-overhang when compared with the conditions without ATP (3.7, 16.1, 16.5, 7., and 2.6-fold increase for YC0, YC2, YC4, YC6, and YC8, respectively). These results indicate that proteins on beads exhibit an ATP-independent synapsis between YC on beads and YC* DNA fragments without discrimination of DNA ends, followed by an ATP-dependent ligation that shows a marked preference for a 4-nucleotide overhang.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3.
Scheme of the DNA pull-down assay. In a first step, oligonucleotide bound to streptavidin-conjugated paramagnetic beads (YC beads) was incubated with HeLa nuclear protein extracts and then washed. The same oligonucleotide radiolabeled on the 5'-end (YC*) and bound to soluble streptavidin in order to orientate synapsis of DNA ends was then mixed with YC beads and incubated with or without ATP. Then two washes were performed to limit nonspecific association. For analysis of the pulled down DNA, beads were incubated with AluI endonuclease, and the digestion products were analyzed by electrophoresis on 20% polyacrylamide denaturing gels, and autoradiography of the gel was processed with a PhosphorImager (Storm SystemTM, Amersham Biosciences).

 



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 4.
Synapsis and DSBs DNA end-joining activities by HeLa nuclear extracts. YC fragments with a free blunt end (YC0) or with a 2-, 4-, 6-, or 8-base 5'-protruding end (YC2, YC4, YC6, and YC8, respectively) were used in parallel experiments as indicated. A, analysis on a 20% polyacrylamide denaturing gel of the pulled down DNA after AluI digestion. B, quantification of the radioactive DNA pulled down with a PhosphorImager. Shown is the mean of three experiments with S.D. nt, nucleotide.

 
To test the fidelity of this end-joining mechanism, the AluI digestion products of the ligated duplex were incubated with specific restriction enzymes that could digest the duplex form only in case of an error-free rejoining reaction. We found that the observed end-joining reaction was essentially error-free because most of the YC ligated products could be digested by the corresponding endonucleases (YC0 and YC8 ligation products were fully digested with the MseI endonuclease, YC4 duplex was digested with HaeIII, and YC6 duplex was digested with MboI, data not shown).

Effect of the Polarity of the Protruding End on the Endjoining Activity—We ran in parallel time course experiments with the 4-nucleotide protruding oligonucleotides either at the 5'- or the 3'-end (YC4 –5' and YC4 –3', respectively). An end-joining activity was obtained with DSBs bearing either 5'- or 3'-protruding extremities (Fig. 5A). Although the global amount of pulled down radioactivity was similar for both oligonucleotides (Fig. 5B), quantification of the time course of ligation indicated that nuclear extracts catalyzed rejoining for 3'-protruding ends at a slower rate when compared with 5'-protruding ends (Fig. 5C). In addition, the yield of total radioactive oligonucleotide trapped on the beads already reached a plateau after 3 min (Fig. 5B), whereas the duplex form still accumulated up to 30 min (Fig. 5C). These data suggest that the synapsis step of the end-joining reaction is uncoupled from the ligation step.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 5.
Time course of pull-down and end-joining reactions of 3'- or 5'-protruding DNA. A, YC fragments with a 4-base 5'- or 3'-protruding end (YC4 and YC4 –3', respectively) were used in parallel experiments. The reaction was stopped at the indicated time, and the pulled down DNA was analyzed by electrophoresis on 20% polyacrylamide denaturing gels after AluI digestion. nt, nucleotide. B, quantification of total DNA pulled down as in A. Shown is the mean of three experiments with S.D. C, quantification of the ligation efficiency, expressed as the percentage of ligated product out of total pulled down DNA. Squares, 5'-protruding termini; circles, 3'-protruding termini. Shown is the mean of three experiments with S.D.

 
End-joining Activity Is Independent of Classical NHEJ—The major DNA end-joining process in mammalian cells relies on the XRCC4/DNA ligase IV, Ku, and DNA-PKcs proteins (4, 66). Because we designed the pull-down assay in order to observe an end-joining activity independent of DNA-PK, we performed two lines of control experiments to verify this point. First, nuclear extracts were immunodepleted either for the Ku heterodimer or XRCC4 proteins by two rounds of incubation with the corresponding immunobeads (53, 54). Ku- and XRCC4-immunodepleted extracts retained less than 5% of the respective protein (Fig. 6A). As a control protein, PARP-1 was not immunodepleted along with Ku or XRCC4. Conversely, DNA-ligase IV was co-immunodepleted with XRCC4, because both proteins form a stable complex (67). We then performed a YC4* pull-down experiment with HeLa NE immunodepleted for Ku or XRCC4 proteins, as compared with the control actin-immunodepleted extract and an HeLa undepleted extract. Under standard conditions, YC4 joining was identically catalyzed by all the immunodepleted extracts or by the control extract (Fig. 6B). The same result was observed with the YC0* DNA fragment (data not shown). This clearly indicates that the mechanism involved in the present DSBs-joining activity is essentially not dependent on DNA-PK or XRCC4/DNA ligase IV. Second, we assessed the effect of wortmannin, a DNA-PK inhibitor that covalently binds to the catalytic DNA-PKcs subunit. As expected, the reaction was resistant to wortmannin (data not shown), contrary to DNA-PK-dependent end-joining in vitro (65).



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 6.
End-joining activity by Ku- or XRCC4-immunodepleted extracts. A, Western blot analysis of HeLa protein extracts immunodepleted for actin, Ku, or XRCC4. nt, nucleotide. B, gel electrophoresis analysis of a YC4 oligonucleotide pull-down experiment with immunodepleted and control nuclear extracts.

 
The fact that we did not observe a DNA-PK-dependent NHEJ reaction under our conditions could be explained by the following: (i) the specificity of our two-step pull-down assay that would not allow synapsis in trans of two NHEJ proteins-DNA end complexes (66), and (ii) the preparation of NE used here, which differed from that of NHEJ-competent extracts (65).

The End-joining Activity Is Dependent on PARP-1—We then investigated whether PARP-1 was involved in the end-joining activity detected in our assay. Under high concentration of {beta}-NAD, poly(ADP-ribose) synthesis induces a loss of PARP-1 affinity for the DNA breaks (68). When 1 mM {beta}-NAD was added to reaction buffer, only a marginal amount of radioactive YC4* DNA was pulled down and ligated (Fig. 7A, compare lanes 2 and 4), whereas {alpha}-NAD, which is not the natural PARP-1 substrate, had no effect on the reaction (Fig. 7A, compare lanes 2 and 3). Most interestingly, {beta}-NAD also abolished the retention of radioactive YC4* DNA in the absence of ATP (Fig. 7A, compare lanes 5 and 6). These results indicate either that PARP-1 activity could modulate the synapsis and end-joining reactions that we observed or that its binding was necessary for these reactions to occur.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 7.
Implication of PARP-1 and the XRCC1-DNA ligase III complex in a DSBs end-joining activity. A, analysis on denaturing gel of YC4 DNA pulled down by HeLa nuclear extracts in the presence or absence of 1 mM ATP and 1 mM {alpha}- or {beta}-NAD as indicated. B, analysis on denaturing gel of YC4 DNA pulled down by nuclear extracts of XRCC1-deficient (EM9), control (AA8), and PARP-1–/– cells, in the presence or absence of 1 mM ATP. PARP-1–/– extracts were complemented with recombinant PARP-1 (100 ng) as indicated. C, comparative Western blot analysis of the protein fractions on beads and in the supernatant under the same conditions as in A and B.

 
To distinguish between these two possibilities, we have tested NE from PARP-1–/– mouse embryonic fibroblasts. A very faint retention of radioactive YC4* DNA was detected with PARP-1–/– NE, and no end-joining occurred (Fig. 7B, lanes 5 and 6). However, addition of purified human recombinant PARP-1 protein to the reaction with PARP-1–/– NE restores both synapsis and formation of the YC4 joined product (Fig. 7B, lanes 7 and 8). Thus, PARP-1 was necessary for both synapsis and end-joining but could not account for the ligation activity. Therefore, we hypothesized that a ligase other than DNA ligase IV was involved in the reaction.

End-joining Activity Is Dependent on the XRCC1-DNA Ligase III Complex—PARP-1 interacts with XRCC1 (69), a protein also involved in BER (70), and XRCC1 is tightly associated with DNA ligase III (71). Because this protein complex could conceivably be responsible for the ligation step of the end-joining reaction observed here, we compared the activity of NE from XRCC1-deficient cells (EM9) (72) with NE from the parental line (AA8) in our YC4 DNA pull-down assay. As compared with AA8 extracts, we observed essentially no ligated product with EM9 NE (Fig. 7B, compare lanes 2 and 4) strongly suggesting that the XRCC1-associated ligase III was responsible for the ligation activity.

To assess the presence of PARP-1 and XRCC1 on the DNA beads, we performed Western blotting experiments on both the protein fractions retained on DNA beads and remaining in the supernatant (Fig. 7C). Without DNA on the beads, PARP-1 and XRCC1 remained in the supernatant (Fig. 7C, lane 1). In the presence of DNA, these two proteins associated with the DNA beads (Fig. 7C, lanes 2 and 3), but if {beta}-NAD was added, less PARP-1 and XRCC1 remained in the DNA-bound protein fraction (Fig. 7C, lane 4). Note the mobility shift of PARP-1 associated with the beads, which corresponds to the poly(ADP)-ribosylated form of the protein (69), as assessed by reblotting the membrane with anti-PAR antibodies (data not shown). With EM9 NE devoid of XRCC1, PARP-1 was still detected on the beads (Fig. 7C, lane 5), like with extracts from the AA8 parental line (Fig. 7C, lane 6). In contrast, no XRCC1 protein was retained on the DNA beads with NE extracts from PARP-1–/– cells (Fig. 7C, lane 7), showing that PARP-1 is necessary for XRCC1 recruitment on DNA, as already suggested (69). Indeed, addition of recombinant PARP-1 in NE extracts from PARP-1–/– cells promoted the recruitment of XRCC1 to the protein complex associated with the DNA beads (Fig. 7C, lane 8).

Because DNA ligase III is associated with XRCC1 (71), we assessed its presence by an adenylation assay on DNA beads preincubated with various NE (Fig. 8). A major adenylated protein was detected with HeLa NE with an apparent molecular mass of 105 kDa (Fig. 8, lane 1). The band intensity decreased when 5 pmol of YC4 was added after the reaction of ligase adenylation (Fig. 8, lane 2). Moreover, adenylation was almost abolished when {beta}-NAD was added to the NE (Fig. 8, lane 3), most likely due to dissociation of XRCC1 and PARP-1 from the DNA beads (see Fig. 7C, lane 4). When HeLa extracts immunodepleted for XRCC4, which contained only residual amounts of DNA ligase IV (Fig. 6A) (54), were used, an adenylation signal similar to the control undepleted HeLa NE was observed (Fig. 8, lane 4), indicating that DNA ligase IV did not correspond to the adenylated protein. We next tested NE from EM9 containing about 25% of DNA ligase III compared with extracts from the AA8 parental line (70). In contrast to AA8 NE that exhibited the same adenylated band as HeLa NE (Fig. 8, compare lanes 1 and 6), essentially no adenylated protein was detected with EM9 NE (Fig. 8, lane 5). These data allow to identify the major adenylated protein present on DNA beads as DNA ligase III and to establish that it is part of the synaptic complex.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 8.
Adenylation reaction on the DNA-binding protein fraction. Adenylated proteins on the DNA beads resolved by SDS-PAGE followed by electro-transfer and autoradiography of the membrane.

 
Reconstitution of the End-joining Reaction with Recombinant Proteins—To confirm the results obtained with NE, we performed the YC4 end-joining reaction on DNA beads with only recombinant PARP-1 and the XRCC1-DNA ligase III complex (XL) (Fig. 9).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 9.
Reconstitution of the DNA DSBs rejoining reaction with purified proteins. A, pull-down assay with YC4 DNA, XRCC1-DNA ligase III complex (XL), and recombinant PARP-1 protein without ATP. Upper panel, gel electrophoresis analysis of the pulled down DNA; lower panel, quantification of the radioactivity pulled down as the ratio to the radioactivity pulled down without protein. Shown is the mean of three experiments with S.D. B, pull-down assay with YC4 DNA, XL complex, and recombinant PARP-1 protein in the presence of 1 mM ATP. Upper panel, gel electrophoresis analysis of the pulled down DNA; lower panel, quantification of the amount of ligated product. The ratios of the yield of ligated product with PARP-1 + XL to the yield with XL alone are shown. Shown is the mean of four experiments with S.D. C, gel electrophoresis analysis of pull-down assays with HeLa extracts and YC fragments with either a 4-base complementary 5'- or 3'-protruding end (YC4–5' and YC4–3', respectively), a blunt end (YCO), or a 4-base 5'-noncomplementary end (YCN). Reactions were carried out with 400 fmol of XL complex and 400 fmol of PARP-1 in the presence or absence of 1 mM ATP.

 
First, we tested the synapsis activity of XL or PARP-1. As shown in Fig. 9A, the presence of increasing amounts of XL did not promote retention of radiolabeled DNA above the amount that was marginally retained on beads in the absence of proteins in the reaction. In contrast, PARP-1 alone could promote DNA pull-down in a dose-dependent manner (Fig. 9A), and the presence of XL did not change significantly the synapsis activity of PARP-1. In the presence of ATP, this allowing ligation, XL alone promoted some end-joining in a concentration-dependent manner (Fig. 9B). However, addition of an optimal amount of PARP-1 for synapsis, as determined in Fig. 9A, strongly increased the yield of joined product (Fig. 9B, 7.5-, 6.2-, and 9.4-fold for 0.5, 1, and 2 molar ratios of XL to PARP-1, respectively). Addition of {beta}-NAD strongly decreased both the amount of pulled down oligonucleotide and of ligated product. Taken together, these data confirm that both PARP-1 protein and the XL complex are necessary and sufficient in vitro for both the synapsis and ligation steps of the DSBs rejoining reaction under the experimental conditions reported here.

Finally, by using the reconstituted system with purified proteins, we tested the effect of the type of DNA ends on the end-joining reaction. As shown in Fig. 9C, the synapsis activity of PARP-1/XL was similar with 5'-, 3'-protruding or blunt DNA ends. Most interestingly, a noncomplementary 5'-protruding oligonucleotide was pulled down with a similar efficiency (Fig. 9C, lane 4). In contrast, the ligation efficiency was affected by the kind of DNA ends with a similar efficiency for 5'- or 3'-protruding ends, a lower efficiency for blunt ends, and no ligation with noncomplementary ends.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PARP-1 is an abundant nuclear protein that binds not only to SSBs but also to DSBs. Through its biochemical properties, PARP-1 is involved in DNA repair, recombination, and genomic stability (35–38). Although the affinity of PARP-1 for nicks is lower than for some types of DNA ends (34), most of the studies have concentrated on PARP-1 function in DNA repair via nick recognition (68).

We report here the involvement of PARP-1 in an end-joining reaction independent of the main DNA-PK reaction. Under cell-free reaction conditions, our results emphasize a new function of PARP-1 for synapsis of DNA ends that is uncoupled from the subsequent ligation step dependent on XRCC1/DNA ligase III (XL). We propose a model for a PARP-1-dependent DSBs-rejoining pathway (Fig. 10), the main features of which are the following. (i) Based on its high abundance and binding affinity for DSBs, it is likely that PARP-1 binds first to one end of the break, most probably as a PARP-1 catalytic homodimer (73); at this step and as shown here with the purified protein, PARP-1 is sufficient to bring DNA ends together. Then PARP-1 recruits XL to DNA ends. Although in our two-step pull-down assay, XL was recruited before the synapsis step, we cannot exclude that it could be recruited after this step. Anyhow, XL does not affect the synapsis activity of PARP-1. (ii) Finally, DNA ligase III reseals the break. Further characterization of the DNA-protein complexes would be necessary in order to know if two XL complexes associate with a PARP-1 homodimer.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 10.
Model for a DNA DSBs end-joining reaction dependent on PARP-1 and the XRCC1-DNA ligase III complex.

 
This model is reminiscent of the reaction steps in the DNA-PK-dependent NHEJ mechanism in which DNA-PKcs recruited by Ku catalyzes DNA-ends synapsis (66), and then both end-positioned Ku and DNA-PKcs mediate the recruitment of the XRCC4-DNA ligase IV complex responsible for the ligation step (54).

By analogy with the current model for SSBs repair (52), XL recruitment may be favored by a low level of self-ADP-ribosylation of the enzyme (69). In vivo also, PARP-1 is necessary for H2O2-induced foci formation of XRCC1 that colocalize with sites of PAR synthesis (61, 74). Notably, both PARP-1 in nuclear extracts and as recombinant protein exhibited a reactivity on Western blots with anti-PAR antibodies specific for short PAR oligomers (data not shown). However, poly(ADP-ribosyl)ated PARP-1 has been shown to dissociate from DNA (68), and under these conditions, XL retained a high affinity for heavily automodified PARP-1 molecules (62). Indeed, we found that extensive modification impairs synapsis activity and promotes dissociation of both PARP-1 and XL from DNA. Thus, a small amount of PARP-1 self-ADP-ribosylation may allow XL recruitment onto DNA ends although subsequent extensive modification may promote dissociation of the protein-DNA complex. So, efficient end-joining is likely to require a tight coordination between the PARP-1 and DNA ligase activities at DNA ends, probably resulting from a competition between the kinetics of both reactions.

Is this PARP-1-dependent DSBs rejoining activity in vitro relevant to a function in vivo? Here we have demonstrated a potentiation by a PARP-1 inhibitor of the cell sensitivity to the double-strand breaking agent calicheamicin {gamma}1. In addition, we have reported a reduced kinetics of DSBs rejoining both in PARP-1-proficient cells pretreated with a PARP-1 inhibitor and in PARP-1-deficient cells, as assessed by loss of {gamma}-H2AX phosphorylation. Because there was a clear potentiation of {gamma}-H2AX phosphorylation in PARP-1–/– cells already detected after 1 h of drug treatment (Fig. 2), this observation is compatible with the possibility that a PARP-1-dependent DSB repair pathway operates early after DSB generation.

Altogether, these data support the possibility of the involvement of PARP-1 in vivo in an end-joining mechanism for DSBs, independent of DNA-PK. Accordingly, an impaired rejoining of DSBs repair after ionizing radiation has been demonstrated recently in PARP-1–/– cells, with only ~50% DNA DSBs rejoined within 60 min after irradiation (48). In addition, it was shown that a potentiation of ionizing radiation cytotoxicity by PARP-1 inhibition in both DNA-PK-proficient or -deficient rodent cells, which correlated with a severe and early inhibition of DNA DSBs rejoining as assessed by neutral filter elution (48, 50), is in full agreement with the sensitization effect by a PARP inhibitor both in DNA-PK-proficient or -deficient cells that we obtained here with calicheamicin {gamma}1.

Although PARP-1 and XRCC1 have been implicated mostly in BER and single-strand breaks rejoining (75), several reports have already substantiated an involvement of these proteins in an end-joining pathway for DSBs repair. XRCC1-deficient cell lines displayed a significant defect in rejoining of radiation-induced DNA DSBs (76, 77). In addition, XRCC1 is a clue determinant of cell resistance to camptothecin, an indirect DSBs-generating agent via collision of the DNA replication fork with topoisomerase I/DNA intermediates (70, 78). Notably, part of the hypersensitivity of XRCC1 mutant cells to camptothecin is S-phase-dependent and may be linked to a true defect in DSBs repair (79). Also, it has been shown that inhibition of PARP-1 catalytic activity retards DBSs rejoining (49). Moreover, overexpression of the catalytically inactive DNA-binding domain of PARP-1 inhibits rejoining of radiation-induced DSBs (45).

Regarding DSBs repair, PARP-1 was until now assigned to a sole anti-recombinogenic function. Its has been postulated that its binding to DNA might transiently prevent inappropriate recombination initiation (80). Accordingly, PARP-1–/– cells exhibited a hyper-recombination phenotype (81). In cells lacking DNA-PKcs, inactivation of PARP-1 by genetic knock-out was required for the occurrence of a low levels of V(D)J recombination rescue (82), also arguing for a PARP-1 anti-recombinogenic function under these conditions. Actually, PARP-1 anti-recombinogenic activity could also rely in part on the synapsis function described here that might help to avoid the formation of free DSBs intermediates. Indeed, inactivation of both PARP-1 and Ku80 cellular activities by genetic knock-out in mice causes early embryonic lethality after probable improper attempts to repair extensive chromosome breaks due to endogenous DNA damage (83, 84).

The potential DSBs joining pathway involving enzymes of the BER pathway that we report here might be important if NHEJ becomes saturated or inefficient at a subset of DSBs. Indeed, substantial evidence in the literature argues for an alternative DNA-PK-independent end-joining pathway, based on both in vitro (18–23) and in vivo data (25–31). The fact that we found the same extent of potentiating effect of PARP-1 inhibition toward DSBs cytotoxicity in both DNA-PK-proficient and -deficient cells could be explained by a subclass of DNA DSBs that could solely be repaired by a PARP-1-dependent end-joining. If not repaired, these DNA breaks would impact on cell survival, despite an efficient DNA-PK end-joining. The additive potentiating effects of PARP-1 and DNA-PK inhibitors on both ionizing radiation cytotoxicity and inhibition of DNA DSBs rejoining (48) also support the hypothesis of independent end-joining mechanisms. Since it was reported recently that PARP-1 was not essential in vivo for repair of extrachromosomal I-SceI breaks by HR or NHEJ (85), we favor the involvement of PARP-1 in an alternative route of NHEJ dealing with damage that is a poor substrate of the DNA-PK machinery. For example, PARP-1 and XL-dependent DSBs repair might realize a functional coupling between BER and end-joining; this pathway could operate on a subclass of DSBs arising from repair attempts at localized clusters of multiple base damage which are significantly induced by ionizing radiation (86, 87). In addition to genotoxic agents, stalled replication forks could also generate breaks that could be handled by a PARP-1-dependent repair pathway as suggested (85). This end-joining pathway could be responsible for the formation of chromosome aberrations observed in cells under NHEJ-deficiency conditions (30, 31, 86, 87). Because Ku-independent end-joining generally relies on microhomology-directed ligation (29, 32), our observation that PARP-1 can promote synapsis of noncomplementary DNA ends may suggest that it could be implicated in this pathway.

In conclusion, the data presented here help to elucidate the mammalian mechanisms responsible for the repair of DNA DSBs. They emphasized a potential role of PARP-1 as a key factor in an end-joining mechanism involved partly in cellular resistance to DSBs cytotoxicity. In addition, they suggest that PARP-1 could represent an important alternative cellular target in tumor radiosensitization strategy.


   FOOTNOTES
 
* This work was supported in part by grants from the Association pour la Recherche Contre le Cancer, Commissariat à l'Energie Atomique, the Ligue Nationale Contre le Cancer, and Electricité de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a post-doctoral fellowship from Association pour la Recherche Contre le Cancer and Fondation pour la Recherche Médicale. Back

§ To whom correspondence should be addressed: Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 Route de Narbonne, F-31077 Toulouse Cedex, France. Tel.: 33-5-61-17-59-36; Fax: 33-5-61-17-59-33; E-mail: bernard.salles{at}ipbs.fr.

1 The abbreviations used are: DSBs, DNA double-strand breaks; NHEJ, nonhomologous end-joining; HR, homologous recombination; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, catalytic subunit of DNA-PK; IR, ionizing radiations; SSBs, single-strand breaks; PARP-1, poly(ADP-ribose) polymerase-1; XL, XRCC1/DNA ligase III; BER, base excision repair; DIQ, 1,5-dihydroxyisoquinolin; CHO, Chinese hamster ovary; NE, nuclear extracts; XL, XRCC1-DNA ligase III. Back


   ACKNOWLEDGMENTS
 
We thank D. Barnes and T. Lindahl for providing the DNA ligase III construct. We are grateful to J.P. Radicella and to S. Marsin for the gift of XRCC1 protein, to Dr. G. De Murcia for the gift of PARP-1–/– cells, to G. Villani for critical reading of the manuscript, and to Dr. C. Muller for helpful suggestions on the experiments. We thank P. R. Hamann (Wyeth Research) for the generous gift of calicheamicin {gamma}1.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1998) Nature 396, 643–649[CrossRef][Medline] [Order article via Infotrieve]
  2. Mills, K. D., Ferguson, D. O., and Alt, F. W. (2003) Immunol. Rev. 194, 77–95[CrossRef][Medline] [Order article via Infotrieve]
  3. Jackson, S. P. (2002) Carcinogenesis 23, 687–696[Abstract/Free Full Text]
  4. van Gent, D. C., Hoeijmakers, J. H., and Kanaar, R. (2001) Nat. Rev. Genet. 2, 196–206[CrossRef][Medline] [Order article via Infotrieve]
  5. Valerie, K., and Povirk, L. F. (2003) Oncogene 22, 5792–5812[CrossRef][Medline] [Order article via Infotrieve]
  6. Krejci, L., Chen, L., Van Komen, S., Sung, P., and Tomkinson, A. (2003) Prog. Nucleic Acids Res. Mol. Biol. 74, 159–201[Medline] [Order article via Infotrieve]
  7. Rothkamm, K., Kruger, I., Thompson, L. H., and Lobrich, M. (2003) Mol. Cell. Biol. 23, 5706–5715, and references therein[Abstract/Free Full Text]
  8. Lieber, M. R., Ma, Y., Pannicke, U., and Schwarz, K. (2003) Nat. Rev. Mol. Cell. Biol. 4, 712–720[CrossRef][Medline] [Order article via Infotrieve]
  9. Lees-Miller, S. P., and Meek, K. (2003) Biochimie (Paris) 85, 1161–1173
  10. Gottlieb, T. M., and Jackson, S. P. (1993) Cell 72, 131–142[CrossRef][Medline] [Order article via Infotrieve]
  11. Lees-Miller, S. P., Chen, Y. R., and Anderson, C. W. (1990) Mol. Cell. Biol. 10, 6472–6481[Abstract/Free Full Text]
  12. Li, Z., Otevrel, T., Gao, Y., Cheng, H. L., Seed, B., Stamato, T. D., Taccioli, G. E., and Alt, F. W. (1995) Cell 83, 1079–1089[CrossRef][Medline] [Order article via Infotrieve]
  13. Grawunder, U., Zimmer, D., Fugmann, S., Schwarz, K., and Lieber, M. R. (1998) Mol. Cell 2, 477–484[CrossRef][Medline] [Order article via Infotrieve]
  14. Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M. R. (2002) Cell 108, 781–794[CrossRef][Medline] [Order article via Infotrieve]
  15. Moshous, D., Callebaut, I., de Chasseval, R., Corneo, B., Cavazzana-Calvo, M., Le Deist, F., Tezcan, I., Sanal, O., Bertrand, Y., Philippe, N., Fischer, A., and de Villartay, J. P. (2001) Cell 105, 177–186[CrossRef][Medline] [Order article via Infotrieve]
  16. Dai, Y., Kysela, B., Hanakahi, L. A., Manolis, K., Riballo, E., Stumm, M., Harville, T. O., West, S. C., Oettinger, M. A., and Jeggo, P. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2462–2467[Abstract/Free Full Text]
  17. Labhart, P. (1999) Eur. J. Biochem. 265, 849–861[Medline] [Order article via Infotrieve]
  18. Cheong, N., Perrault, A. R., Wang, H., Wachsberger, P., Mammen, P., Jackson, I., and Iliakis, G. (1999) Int. J. Radiat. Biol. 75, 67–81[CrossRef][Medline] [Order article via Infotrieve]
  19. Feldmann, E., Schmiemann, V., Goedecke, W., Reichenberger, S., and Pfeiffer, P. (2000) Nucleic Acids Res. 28, 2585–2596[Abstract/Free Full Text]
  20. Johnson, A. P., and Fairman, M. P. (1997) Mutat. Res. 383, 205–212[Medline] [Order article via Infotrieve]
  21. North, P., Ganesh, A., and Thacker, J. (1990) Nucleic Acids Res. 18, 6205–6210[Abstract/Free Full Text]
  22. Zhong, Q., Boyer, T. G., Chen, P. L., and Lee, W. H. (2002) Cancer Res. 62, 3966–3970[Abstract/Free Full Text]
  23. Wang, H., Perrault, A. R., Takeda, Y., Qin, W., Wang, H., and Iliakis, G. (2003) Nucleic Acids Res. 31, 5377–5388[Abstract/Free Full Text]
  24. Perrault, R., Wang, H., Wang, M., Rosidi, B., and Iliakis, G. (2004) J. Cell. Biochem. 92, 781–794[CrossRef][Medline] [Order article via Infotrieve]
  25. Wilson, T. E., Grawunder, U., and Lieber, M. R. (1997) Nature 388, 495–498[CrossRef][Medline] [Order article via Infotrieve]
  26. Boulton, S. J., and Jackson, S. P. (1996) EMBO J. 15, 5093–50103[Medline] [Order article via Infotrieve]
  27. DiBiase, S. J., Zeng, Z. C., Chen, R., Hyslop, T., Curran, W. J., Jr., and Iliakis, G. (2000) Cancer Res. 60, 1245–1253[Abstract/Free Full Text]
  28. Liang, F., and Jasin, M. (1996) J. Biol. Chem. 271, 14405–14411[Abstract/Free Full Text]
  29. Verkaik, N. S., Esveldt-van Lange, R. E., van Heemst, D., Bruggenwirth, H. T., Hoeijmakers, J. H., Zdzienicka, M. Z., and van Gent, D. C. (2002) Eur. J. Immunol. 32, 701–709[CrossRef][Medline] [Order article via Infotrieve]
  30. Virsik-Kopp, P., Rave-Frank, M., Hofman-Huther, H., and Schmidberger, H. (2003) Int. J. Radiat. Biol. 79, 61–68[Medline] [Order article via Infotrieve]
  31. Zhu, C., Mills, K. D., Ferguson, D. O., Lee, C., Manis, J., Fleming, J., Gao, Y., Morton, C. C., and Alt, F. W. (2002) Cell 109, 811–821[CrossRef][Medline] [Order article via Infotrieve]
  32. Guirouilh-Barbat, J., Huck, S., Bertrand, P., Pirzio, L., Desmaze, C., Sabatier, L., and Lopez, B. S. (2004) Mol. Cell 14, 611–623[CrossRef][Medline] [Order article via Infotrieve]
  33. Weinfeld, M., Chaudhry, M. A., D'Amours, D., Pelletier, J. D., Poirier, G. G., Povirk, L. F., and Lees-Miller, S. P. (1997) Radiat. Res. 148, 22–28[CrossRef][Medline] [Order article via Infotrieve]
  34. D'Silva, I., Pelletier, J. D., Lagueux, J., D'Amours, D., Chaudhry, M. A., Weinfeld, M., Lees-Miller, S. P., and Poirier, G. G. (1999) Biochim. Biophys. Acta 1430, 119–126[CrossRef][Medline] [Order article via Infotrieve]
  35. Bouchard, V. J., Rouleau, M., and Poirier, G. G. (2003) Exp. Hematol. 31, 446–454[CrossRef][Medline] [Order article via Infotrieve]
  36. Burkle, A. (2001) BioEssays 23, 795–806[CrossRef][Medline] [Order article via Infotrieve]
  37. Tong, W. M., Cortes, U., and Wang, Z. Q. (2001) Biochim. Biophys. Acta 1552, 27–37[Medline] [Order article via Infotrieve]
  38. Shall, S., and de Murcia, G. (2000) Mutat. Res. 460, 1–15[Medline] [Order article via Infotrieve]
  39. Benjamin, R. C., and Gill, D. M. (1980) J. Biol. Chem. 255, 10502–10508[Abstract/Free Full Text]
  40. Galande, S., and Kohwi-Shigematsu, T. (1999) J. Biol. Chem. 274, 20521–20528[Abstract/Free Full Text]
  41. Ariumi, Y., Masutani, M., Copeland, T. D., Mimori, T., Sugimura, T., Shimotohno, K., Ueda, K., Hatanaka, M., and Noda, M. (1999) Oncogene 18, 4616–4625[CrossRef][Medline] [Order article via Infotrieve]
  42. Li, B., Navarro, S., Kasahara, N., and Comai, L. (2004) J. Biol. Chem. 279, 13659–13667[Abstract/Free Full Text]
  43. Ruscetti, T., Lehnert, B. E., Halbrook, J., LeTrong, H., Hoekstra, M. F., Chen, D. J., and Peterson, S. R. (1998) J. Biol. Chem. 273, 14461–14467[Abstract/Free Full Text]
  44. Brown, M. L., Franco, D., Burkle, A., and Chang, Y. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 4532–4537[Abstract/Free Full Text]
  45. Rudat, V., Bachmann, N., Kupper, J. H., and Weber, K. J. (2001) Int. J. Radiat. Biol. 77, 303–307[CrossRef][Medline] [Order article via Infotrieve]
  46. Bryant, P. E., and Johnston, P. J. (1993) Mutat. Res. 299, 289–296[CrossRef][Medline] [Order article via Infotrieve]
  47. Chung, H. W., Phillips, J. W., Winegar, R. A., Preston, R. J., and Morgan, W. F. (1991) Radiat. Res. 125, 107–113[CrossRef][Medline] [Order article via Infotrieve]
  48. Veuger, S. J., Curtin, N. J., Richardson, C. J., Smith, G. C., and Durkacz, B. W. (2003) Cancer Res. 63, 6008–6015[Abstract/Free Full Text]
  49. Boulton, S., Kyle, S., and Durkacz, B. W. (1999) Carcinogenesis 20, 199–203[Abstract/Free Full Text]
  50. Veuger, S. J., Curtin, N. J., Smith, G. C., and Durkacz, B. W. (2004) Oncogene 23, 7322–7329[CrossRef][Medline] [Order article via Infotrieve]
  51. Elmroth, K., Nygren, J., Martensson, S., Ismail, I. H., and Hammarsten, O. (2003) DNA Repair 2, 363–374[Medline] [Order article via Infotrieve]
  52. Caldecott, K. W. (2003) DNA Repair 2, 955–969[Medline] [Order article via Infotrieve]
  53. Calsou, P., Frit, P., and Salles, B. (1996) J. Biol. Chem. 271, 27601–27607[Abstract/Free Full Text]
  54. Calsou, P., Delteil, C., Frit, P., Drouet, J., and Salles, B. (2003) J. Mol. Biol. 326, 93–103[CrossRef][Medline] [Order article via Infotrieve]
  55. Nash, R. A., Caldecott, K. W., Barnes, D. E., and Lindahl, T. (1997) Biochemistry 36, 5207–5211[CrossRef][Medline] [Order article via Infotrieve]
  56. Vidal, A. E., Boiteux, S., Hickson, I. D., and Radicella, J. P. (2001) EMBO J. 20, 6530–6539[CrossRef][Medline] [Order article via Infotrieve]
  57. Iliakis, G. (1991) BioEssays 13, 641–648[CrossRef][Medline] [Order article via Infotrieve]
  58. Banasik, M., Komura, H., Shimoyama, M., and Ueda, K. (1992) J. Biol. Chem. 267, 1569–1575[Abstract/Free Full Text]
  59. Blunt, T., Gell, D., Fox, M., Taccioli, G. E., Lehmann, A. R., Jackson, S. P., and Jeggo, P. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10285–10290[Abstract/Free Full Text]
  60. Singleton, B. K., Priestley, A., Steingrimsdottir, H., Gell, D., Blunt, T., Jackson, S. P., Lehmann, A. R., and Jeggo, P. A. (1997) Mol. Cell. Biol. 17, 1264–1273[Abstract]
  61. Okano, S., Lan, L., Caldecott, K. W., Mori, T., and Yasui, A. (2003) Mol. Cell. Biol. 23, 3974–3981[Abstract/Free Full Text]
  62. Leppard, J. B., Dong, Z., Mackey, Z. B., and Tomkinson, A. E. (2003) Mol. Cell. Biol. 23, 5919–5927[Abstract/Free Full Text]
  63. van Duijn-Goedhart, A., Zdzienicka, M. Z., Sankaranarayanan, K., and van Buul, P. P. (2000) Mutat. Res. 471, 95–105[Medline] [Order article via Infotrieve]
  64. Olive, P. L., and Banath, J. P. (2004) Int. J. Radiat. Oncol. Biol. Phys. 58, 331–335[CrossRef][Medline] [Order article via Infotrieve]
  65. Baumann, P., and West, S. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14066–14070[Abstract/Free Full Text]
  66. DeFazio, L. G., Stansel, R. M., Griffith, J. D., and Chu, G. (2002) EMBO J. 21, 3192–3200[CrossRef][Medline] [Order article via Infotrieve]
  67. Lee, K. J., Huang, J., Takeda, Y., and Dynan, W. S. (2000) J. Biol. Chem. 275, 34787–34796[Abstract/Free Full Text]
  68. Ferro, A. M., and Olivera, B. M. (1982) J. Biol. Chem. 257, 7808–7813[Free Full Text]
  69. Masson, M., Niedergang, C., Schreiber, V., Muller, S., Menissier-de Murcia, J., and de Murcia, G. (1998) Mol. Cell. Biol. 18, 3563–3571[Abstract/Free Full Text]
  70. Thompson, L. H., and West, M. G. (2000) Mutat. Res. 459, 1–18[Medline] [Order article via Infotrieve]
  71. Caldecott, K. W., McKeown, C. K., Tucker, J. D., Ljungquist, S., and Thompson, L. H. (1994) Mol. Cell. Biol. 14, 68–76[Abstract/Free Full Text]
  72. Thompson, L. H., Brookman, K. W., Jones, N. J., Allen, S. A., and Carrano, A. V. (1990) Mol. Cell. Biol. 10, 6160–6171[Abstract/Free Full Text]
  73. Pion, E., Bombarda, E., Stiegler, P., Ullmann, G. M., Mely, Y., de Murcia, G., and Gerard, D. (2003) Biochemistry 42, 12409–12417[CrossRef][Medline] [Order article via Infotrieve]
  74. El-Khamisy, S. F., Masutani, M., Suzuki, H., and Caldecott, K. W. (2003) Nucleic Acids Res. 31, 5526–5533[Abstract/Free Full Text]
  75. Caldecott, K. W. (2001) BioEssays 23, 447–455[CrossRef][Medline] [Order article via Infotrieve]
  76. Nocentini, S. (1999) Radiat. Res. 151, 423–432[Medline] [Order article via Infotrieve]
  77. Taverna, P., Hwang, H. S., Schupp, J. E., Radivoyevitch, T., Session, N. N., Reddy, G., Zarling, D. A., and Kinsella, T. J. (2003) Cancer Res. 63, 838–846[Abstract/Free Full Text]
  78. Park, S. Y., Lam, W., and Cheng, Y. C. (2002) Cancer Res. 62, 459–465[Abstract/Free Full Text]
  79. Barrows, L. R., Holden, J. A., Anderson, M., and D'Arpa, P. (1998) Mutat. Res. 408, 103–110[Medline] [Order article via Infotrieve]
  80. Lindahl, T., Satoh, M. S., Poirier, G. G., and Klungland, A. (1995) Trends Biochem. Sci. 20, 405–411[CrossRef][Medline] [Order article via Infotrieve]
  81. Schultz, N., Lopez, E., Saleh-Gohari, N., and Helleday, T. (2003) Nucleic Acids Res. 31, 4959–4964[Abstract/Free Full Text]
  82. Morrison, C., Smith, G. C., Stingl, L., Jackson, S. P., Wagner, E. F., and Wang, Z. Q. (1997) Nat. Genet. 17, 479–482[CrossRef][Medline] [Order article via Infotrieve]
  83. Henrie, M. S., Kurimasa, A., Burma, S., Menissier-de Murcia, J., de Murcia, G., Li, G. C., and Chen, D. J. (2003) DNA Repair 2, 151–158[Medline] [Order article via Infotrieve]
  84. Tong, W. M., Cortes, U., Hande, M. P., Ohgaki, H., Cavalli, L. R., Lansdorp, P. M., Haddad, B. R., and Wang, Z. Q. (2002) Cancer Res. 62, 6990–6996[Abstract/Free Full Text]
  85. Yang, Y. G., Cortes, U., Patnaik, S., Jasin, M., and Wang, Z. Q. (2004) Oncogene 23, 3872–3882[CrossRef][Medline] [Order article via Infotrieve]
  86. Stenerlow, B., Karlsson, K. H., Cooper, B., and Rydberg, B. (2003) Radiat. Res. 159, 502–510[CrossRef][Medline] [Order article via Infotrieve]
  87. Sutherland, B. M., Bennett, P. V., Sidorkina, O., and Laval, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 103–108[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
E. A. Ahmed, A. D. B.-v. Rijbroek, H. B. Kal, H. Sadri-Ardekani, S. C. Mizrak, A. M.M. v. Pelt, and D. G. d. Rooij
Proliferative Activity In Vitro and DNA Repair Indicate that Adult Mouse and Human Sertoli Cells Are Not Terminally Differentiated, Quiescent Cells
Biol Reprod, June 1, 2009; 80(6): 1084 - 1091.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
I. Robert, F. Dantzer, and B. Reina-San-Martin
Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination
J. Exp. Med., May 11, 2009; 206(5): 1047 - 1056.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
J. B. Kirkland
Niacin status and treatment-related leukemogenesis
Mol. Cancer Ther., April 1, 2009; 8(4): 725 - 732.
[Abstract] [Full Text] [PDF]

Home page
 Phil Trans R Soc BHome page
A. Kotnis, L. Du, C. Liu, S. W Popov, and Q. Pan-Hammarstrom
Non-homologous end joining in class switch recombination: the beginning of the end
Phil Trans R Soc B, March 12, 2009; 364(1517): 653 - 665.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
L. Han and K. Yu
Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells
J. Exp. Med., November 24, 2008; 205(12): 2745 - 2753.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
S. Iiizumi, A. Kurosawa, S. So, Y. Ishii, Y. Chikaraishi, A. Ishii, H. Koyama, and N. Adachi
Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting
Nucleic Acids Res., November 1, 2008; 36(19): 6333 - 6342.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
A. Kinner, W. Wu, C. Staudt, and G. Iliakis
{gamma}-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin
Nucleic Acids Res., October 1, 2008; 36(17): 5678 - 5694.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
C. Godon, F. P. Cordelieres, D. Biard, N. Giocanti, F. Megnin-Chanet, J. Hall, and V. Favaudon
PARP inhibition versus PARP-1 silencing: different outcomes in terms of single-strand break repair and radiation susceptibility
Nucleic Acids Res., August 1, 2008; 36(13): 4454 - 4464.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
W. Y. Mansour, S. Schumacher, R. Rosskopf, T. Rhein, F. Schmidt-Petersen, F. Gatzemeier, F. Haag, K. Borgmann, H. Willers, and J. Dahm-Daphi
Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks
Nucleic Acids Res., July 1, 2008; 36(12): 4088 - 4098.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
L. Liang, L. Deng, S. C. Nguyen, X. Zhao, C. D. Maulion, C. Shao, and J. A. Tischfield
Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks
Nucleic Acids Res., June 1, 2008; 36(10): 3297 - 3310.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
L. Schulte-Uentrop, R. A. El-Awady, L. Schliecker, H. Willers, and J. Dahm-Daphi
Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks
Nucleic Acids Res., May 1, 2008; 36(8): 2561 - 2569.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Cotner-Gohara, I.-K. Kim, A. E. Tomkinson, and T. Ellenberger
Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III
J. Biol. Chem., April 18, 2008; 283(16): 10764 - 10772.
[Abstract] [Full Text] [PDF]

Home page
 aacredbookHome page
E. R. Plummer and H. Calvert
Targeting Poly(ADP-Ribose) Polymerase: A Two-Armed Strategy for Cancer Therapy
Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 15 - 22.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
B. Rosidi, M. Wang, W. Wu, A. Sharma, H. Wang, and G. Iliakis
Histone H1 functions as a stimulatory factor in backup pathways of NHEJ
Nucleic Acids Res., March 1, 2008; 36(5): 1610 - 1623.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Guirouilh-Barbat, E. Rass, I. Plo, P. Bertrand, and B. S. Lopez
Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends
PNAS, December 26, 2007; 104(52): 20902 - 20907.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P.-Y. Wu, P. Frit, L. Malivert, P. Revy, D. Biard, B. Salles, and P. Calsou
Interplay between Cernunnos-XLF and Nonhomologous End-joining Proteins at DNA Ends in the Cell
J. Biol. Chem., November 2, 2007; 282(44): 31937 - 31943.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
E. R. Plummer and H. Calvert
Targeting Poly(ADP-Ribose) Polymerase: A Two-Armed Strategy for Cancer Therapy
Clin. Cancer Res., November 1, 2007; 13(21): 6252 - 6256.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
X. Cui and K. Meek
Linking double-stranded DNA breaks to the recombination activating gene complex directs repair to the nonhomologous end-joining pathway
PNAS, October 23, 2007; 104(43): 17046 - 17051.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. L. Heacock, R. A. Idol, J. D. Friesner, A. B. Britt, and D. E. Shippen
Telomere dynamics and fusion of critically shortened telomeres in plants lacking DNA ligase IV
Nucleic Acids Res., October 8, 2007; 35(19): 6490 - 6500.
[Abstract] [Full Text] [PDF]

Home page
 Eukaryot CellHome page
P. Burton, D. J. McBride, J. M. Wilkes, J. D. Barry, and R. McCulloch
Ku Heterodimer-Independent End Joining in Trypanosoma brucei Cell Extracts Relies upon Sequence Microhomology
Eukaryot. Cell, October 1, 2007; 6(10): 1773 - 1781.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
N. Hegarat, G. M. Cardoso, F. Rusconi, J.-C. Francois, and D. Praseuth
Analytical biochemistry of DNA protein assemblies from crude cell extracts
Nucleic Acids Res., July 26, 2007; 35(13): e92 - e92.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
F. Karimi-Busheri, A. Rasouli-Nia, J. Allalunis-Turner, and M. Weinfeld
Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination
Cancer Res., July 15, 2007; 67(14): 6619 - 6625.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. M. Hinz, P. B. Nham, S. S. Urbin, I. M. Jones, and L. H. Thompson
Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis
Nucleic Acids Res., June 28, 2007; 35(11): 3733 - 3740.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
L. F. Povirk, R.-Z. Zhou, D. A. Ramsden, S. P. Lees-Miller, and K. Valerie
Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts
Nucleic Acids Res., June 9, 2007; 35(12): 3869 - 3878.
[Abstract] [Full Text] [PDF]

Home page
 MutagenesisHome page
S. Kuhfittig-Kulle, E. Feldmann, A. Odersky, A. Kuliczkowska, W. Goedecke, A. Eggert, and P. Pfeiffer
The mutagenic potential of non-homologous end joining in the absence of the NHEJ core factors Ku70/80, DNA-PKcs and XRCC4-LigIV
Mutagenesis, May 1, 2007; 22(3): 217 - 233.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
J. L. Rose, K. C. Reeves, R. I. Likhotvorik, and D. G. Hoyt
Base Excision Repair Proteins Are Required for Integrin-Mediated Suppression of Bleomycin-Induced DNA Breakage in Murine Lung Endothelial Cells
J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 318 - 326.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
E. Despras, P. Pfeiffer, B. Salles, P. Calsou, S. Kuhfittig-Kulle, J. F. Angulo, and D. S.F. Biard
Long-term XPC Silencing Reduces DNA Double-Strand Break Repair
Cancer Res., March 15, 2007; 67(6): 2526 - 2534.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
K. Ratnam and J. A. Low
Current Development of Clinical Inhibitors of Poly(ADP-Ribose) Polymerase in Oncology
Clin. Cancer Res., March 1, 2007; 13(5): 1383 - 1388.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Wang, W. Wu, W. Wu, B. Rosidi, L. Zhang, H. Wang, and G. Iliakis
PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways
Nucleic Acids Res., December 4, 2006; 34(21): 6170 - 6182.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
R. Brem, D. G. Cox, B. Chapot, N. Moullan, P. Romestaing, J.-P. Gerard, P. Pisani, and J. Hall
The XRCC1 -77T->C variant: haplotypes, breast cancer risk, response to radiotherapy and the cellular response to DNA damage
Carcinogenesis, December 1, 2006; 27(12): 2469 - 2474.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Drouet, P. Frit, C. Delteil, J.-P. de Villartay, B. Salles, and P. Calsou
Interplay between Ku, Artemis, and the DNA-dependent Protein Kinase Catalytic Subunit at DNA Ends
J. Biol. Chem., September 22, 2006; 281(38): 27784 - 27793.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Yin and J. Glass
In Prostate Cancer Cells the Interaction of C/EBP{alpha} with Ku70, Ku80, and Poly(ADP-ribose) Polymerase-1 Increases Sensitivity to DNA Damage
J. Biol. Chem., April 28, 2006; 281(17): 11496 - 11505.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
D. Li, M. Frazier, D. B. Evans, K. R. Hess, C. H. Crane, L. Jiao, and J. L. Abbruzzese
Single Nucleotide Polymorphisms of RecQ1, RAD54L, and ATM Genes Are Associated With Reduced Survival of Pancreatic Cancer
J. Clin. Oncol., April 10, 2006; 24(11): 1720 - 1728.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
H. E. Bryant and T. Helleday
Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair
Nucleic Acids Res., March 23, 2006; 34(6): 1685 - 1691.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
C. Baumann, G. S. Boehden, A. Burkle, and L. Wiesmuller
Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53
Nucleic Acids Res., February 9, 2006; 34(3): 1036 - 1049.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
N. Levy, A. Martz, A. Bresson, C. Spenlehauer, G. de Murcia, and J. Menissier-de Murcia
XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage
Nucleic Acids Res., January 5, 2006; 34(1): 32 - 41.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Martin, A. Genesca, L. Latre, I. Jaco, G. E. Taccioli, J. Egozcue, M. A. Blasco, G. Iliakis, and L. Tusell
Postreplicative Joining of DNA Double-Strand Breaks Causes Genomic Instability in DNA-PKcs-Deficient Mouse Embryonic Fibroblasts
Cancer Res., November 15, 2005; 65(22): 10223 - 10232.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Wang, B. Rosidi, R. Perrault, M. Wang, L. Zhang, F. Windhofer, and G. Iliakis
DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining
Cancer Res., May 15, 2005; 65(10): 4020 - 4030.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/53/55117    most recent
M404524200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Audebert, M.
Right arrow Articles by Calsou, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Audebert, M.
Right arrow Articles by Calsou, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement