Open AccessKu counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks
Author(s) -
Qiao Cheng,
Nadia Barboule,
Philippe Frit,
Dennis Gómez,
Oriane Bombarde,
Bettina Couderc,
Guo Sheng Ren,
Bernard Salles,
Patrick Calsou
Publication year - 2011
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkr656
Subject(s) - ku70 , ku80 , biology , chromatin , parp1 , microbiology and biotechnology , genome instability , non homologous end joining , dna repair , dna damage , dna , poly adp ribose polymerase , genetics , transcription factor , dna binding protein , gene , polymerase
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.