Nonhomologous end-joining of ionizing radiation-induced DNA double-stranded breaks in human tumor cells deficient in BRCA1 or BRCA2

Abstract

Mutations in the BRCA1 or BRCA2 genes predispose to a wide spectrum of familial cancers. The functions of the proteins encoded by BRCA1 and BRCA2 remain to be elucidated, but their interaction and colocalization with hRAD51 suggest a role in homologous recombination and DNA double-strand break (DSB) repair. The role of BRCA1 and BRCA2 in the rejoining of ionizing radiation (IR)-induced DNA DSBs, which may represent a step in the overall process of repair, remains uncertain because recent reports provide conflicting results. Because elucidation of the role of these proteins in DNA DSB rejoining is important for their functional characterization, we reexamined this end point in cells with mutations in either BRCA1 or BRCA2. We show that two pancreatic carcinoma cell lines known to have either wild-type (BxPC3) or mutant forms (Capan-1) of BRCA2 rejoin IR-induced DNA DSBs to a similar extent following biphasic kinetics characterized by a fast and a slow component. Importantly, inactivation of DNA-dependent protein kinase (DNA-PK) by wortmannin generates similar shifts from the fast to the slow component of rejoining in BRCA2-proficient and BRCA2-deficient cells. This suggests that the functioning of either the fast, DNA-PK-dependent component or the slow, DNA-PK-independent component of rejoining is not affected by mutations in BRCA2. Also, a human breast cancer cell line with mutated BRCA1 shows normal rejoining of IR-induced DNA DSBs and levels of inhibition by wortmannin commensurate with the degree of DNA-PK inhibition. These observations fail to confirm a direct role for BRCA1 or BRCA2 in the rejoining of IR-induced DSBs in the genome of human tumor cells and, as a result, an involvement in nonhomologous end-joining. They are in line with similar observations with mutants deficient in genes implicated in homologous recombination and support the view that the radiosensitivity to killing of cells deficient in BRCA1 or BRCA2 derives from defects in this repair pathway.