title: Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks.
abstract: In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs.
authors: CNRS, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France.
authors: Qiao
authors: Q
authors: Cheng
authors: Nadia
authors: N
authors: Barboule
authors: Philippe
authors: P
authors: Frit
authors: Dennis
authors: D
authors: Gomez
authors: Oriane
authors: O
authors: Bombarde
authors: Bettina
authors: B
authors: Couderc
authors: Guo-Sheng
authors: GS
authors: Ren
authors: Bernard
authors: B
authors: Salles
authors: Patrick
authors: P
authors: Calsou
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D000072200:Ku Autoantigen; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
chemical_list: D034961:Antigens, Nuclear; D002843:Chromatin; D004268:DNA-Binding Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen