Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining.
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 gamma1. In addition, the repair kinetics of the DSBs induced by calicheamicin gamma1 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.
Audebert
Marc
M
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, F-31077 Toulouse Cedex, France.
Salles
Bernard
B
Calsou
Patrick
P
D000617:Aminoglycosides / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D053281:Enediynes; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D008957:Models, Genetic; D009153:Mutagens; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D000076105:X-ray Repair Cross Complementing Protein 1
D000617:Aminoglycosides; D000903:Antibiotics, Antineoplastic; D004247:DNA; D000072481:DNA Ligase ATP; D011088:DNA Ligases; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D053281:Enediynes; D006657:Histones; D009153:Mutagens; D009841:Oligonucleotides; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000607893:LIG4 protein, human; C000619371:XRCC1 protein, human; C000619352:Xrcc1 protein, mouse; C055955:calicheamicin gamma(1)I; C475961:gamma-H2AX protein, mouse