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Ku counteracts mobilization of PARP1 and MRN inchromatin damaged with DNA double-strand breaks
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Edité par CCSD ; Oxford University Press -
International audience. In mammalian cells, the main pathway for DNAdouble-strand breaks (DSBs) repair is classicalnon-homologous end joining (C-NHEJ). An alternativeor back-up NHEJ (B-NHEJ) pathway hasemerged which operates preferentially underC-NHEJ defective conditions. Although B-NHEJappears particularly relevant to genomic instabilityassociated with cancer, its components and regulationare still largely unknown. To get insights intothis pathway, we have knocked-down Ku, the maincontributor to C-NHEJ. Thus, models of human celllines have been engineered in which the expressionof Ku70/80 heterodimer can be significantly loweredby the conditional induction of a shRNA againstKu70. On Ku reduction in cells, resulting NHEJ competentprotein extracts showed a shift from CtoB-NHEJ that could be reversed by additionof purified Ku protein. Using a cellular fractionationprotocol after treatment with a strong DSBs inducerfollowed by western blotting or immunostaining, weestablished that, among C-NHEJ factors, Ku is themain counteracting factor against mobilization ofPARP1 and the MRN complex to damaged chromatin.In addition, Ku limits PAR synthesis andsingle-stranded DNA production in response toDSBs. These data support the involvement ofPARP1 and the MRN proteins in the B-NHEJ routefor the repair of DNA DSBs.
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