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.

Alternative end-joining pathway(s): Bricolage at DNA breaks

“…Finally, we have used a cellular fractionation protocol to study A-EJ in native chromatin. After treatment with a strong DSB inducer followed by western-blotting or immunostaining, recruitment of A-EJ proteins to damaged chromatin can be studied in cells engineered for Ku depletion [56].…”

Prednisolone and vitamin D(3) modulate oxidative metabolism and cell death pathways in blood and bone marrow mononuclear cells

“…Whole bone marrow cell extracts were prepared as described previously [8]. Bone marrow cells isolated from rats were lysed in RIPA buffer containing protease inhibitor cocktail (PIC, Sigma, USA).…”

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice

“…Thus, this pathway has been designated as microhomology-mediated end joining (MMEJ). Many proteins involved in the altNHEJ pathway, such as poly (ADP-ribose) polymerase1 (PARP1), meiotic recombination 11 (MRE11)-radiation sensitive 50 (Rad50)-Nijmegen Breakage Syndrome 1 (NBS1), histone H, DNA Ligase III, and XRCC1, have been identified in mammalian cells (Audebert et al, 2004;Rosidi et al, 2008;Cheng et al, 2011). Recent studies have shown that Arabidopsis XRCC1, XPF, Lig1, and PARP1/PARP2 also function in the altNHEJ pathway (Waterworth et al, 2009;Charbonnel et al, 2010Charbonnel et al, , 2011Jia et al, 2013).…”