Abstract P3-04-06: Damage-induced BRCA1 phosphorylation contributes to the timing of end resection

Abstract

Abstract Germline mutations of BRCA1 predispose women to breast and ovarian cancers. BRCA1 functions as a tumor suppressor. A wealth of evidence has established that BRCA1 plays an important role in DNA repair and cell cycle checkpoint control. Efficient repair of double-strand-breaks (DSBs) by BRCA1 is essential to its tumor suppression function. DSBs can be repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). BRCA1 is hyperphosphorylated upon DSBs. Although phosphorylation of BRCA1 is among the earliest events documented upon DNA damage, the role of BRCA1 phosphorylation in DNA repair remains unclear after more than a decade. It has been proposed that BRCA1 serves as a scaffolding protein that recruits multiple repair proteins to the break sites. One of the BRCA1-containing complexes, MRN (Mre11-Rad50-Nbs1)-BRCA1-CtIP, promotes HR repair by initiating DNA end-resection, the first step in the HR pathway. Mre11 possesses intrinsic nuclease activity, which likely facilitates initial processing of DNA ends for subsequent extensive end-resection. Believed to stimulate the nuclease activity of the MRN complex, CtIP is recruited to the complex by BRCA1. In spite of these useful molecular models, the precise function of BRCA1 in DSB repair, especially the role of its phosphorylation, remains elusive. BRCA1 is phosphorylated by Chk2 kinase upon g-irradiation at serine 988 (human) or serine 971 (mouse). Our data shows that a point mutation in mouse BRCA1 (S971A) that abolishes the Chk2 phosphorylation site impairs end-resection after DSBs. The S971A mutation does not appear to affect the assembly of MRN and CtIP at DSBs, but specifically delays dispersion of BRCA1 from DSBs. Interestingly, BRCA1 is ubiquitinated and degraded by SCFSkp2, and loss of Chk2 phosphorylation at S988 of human BRCA1 impairs its ubiquitination. Previous work has revealed BRCA1 as a scaffold protein for the assembly of HR proteins at DSBs. Paradoxically, BRCA1 also inhibits Mre11 nuclease activity. Our study suggests that BRCA1 plays a dual role in HR. Although BRCA1 is required for initial assembly of the MRN-BRCA1-CtIP complex at DSBs, the exit of BRCA1 from the MRN-BRCA1-CtIP complex is required to relieve its inhibitory effect on Mre11 nuclease activity. Mechanistically, this critical “exit” of BRCA1 from the MRN-BRCA1-CtIP complex is regulated through phosphorylation catalyzed by Chk2 and then degraded by SCFSkp2, which is expressed at S/G2 phases, thus ensuring the initiation of end resection/HR to S/G2 phases of the cell cycle. Our study explains two seemingly paradoxical observations: BRCA1 is required for assembly of the MRN-BRCA1-CtIP complex, but it inhibits Mre11 nuclease activity required for end-resection. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-06.