Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

Abstract

BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)-and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.Germ line mutations in the breast cancer susceptibility gene BRCA1 predispose women to breast and ovarian cancers (26, 37). The human BRCA1 protein is composed of 1,863 amino acid (aa) residues and contains a RING domain in the N terminus. Two tandem BRCT domains, which are frequently found in DNA repair proteins and function as a binding module for phospho-serine peptides (34), are present in the C terminus. The N-terminal region of BRCA1 directly interacts with BARD1 (51), and the association with BARD1 enhances the ubiquitin polymerase activity of BRCA1 (2,11,33).BRCA1 is involved in many cellular processes, including DNA repair, transcription, cell cycle regulation, chromatin remodeling, and apoptosis. Several lines of evidences suggest that the involvement of BRCA1 in the DNA repair pathway is associated with the tumor suppressor activity of BRCA1. For example, mouse and human cells deficient in BRCA1 are more sensitive to DNA damage, including ionizing irradiation and drugs that produce double-strand breaks (DSBs) or interstrand cross-linking of DNA. Clinically observed missense mutations often result in a nonfunctional BRCA1 protein that has lost the ability to repair DNA damage (43). BRCA1 interacts with a number of DNA repair factors such as Rad51, the Mre11-Rad50-Nbs1 (MRN) complex, BLM, and the DNA helicase BACH1 (also called BRIP1or FANCJ) (8,42,50,57). BRCA1 localizes to nuclear foci during S phase of the cell cycle. Various types of DNA damage result in hyperphosphorylation of BRCA1 and alterations in BRCA1 localization to nuclear foci (21, 41). Normally, BRCA1 redistributes to nuclear foci where multiple DNA repair factors accumulate. BRCA1 colocalizes with phosphor...

Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

“…Previously, we showed that BRCA1 promotes precise rejoining of DSBs in human breast carcinoma cells while suppressing microhomology-mediated error-prone end joining and restricting sequence deletion at the break junction during repair (34). In addition, BRCA1 interacts with Ku80, which may be required for BRCA1-Ku80 foci formation in response to laser-induced DSBs (35). BRCA1 also interacts with the Mre11⅐Rad50⅐Nbs1⅐CtIP supercomplex and is critical for the function of CtIP in HR.…”

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

“…40 Finally, Ku80 was shown to recruit BRCA1 to sites of DSB. 41 As BRCA1 is a mediator of the HR function of Rad51, 42 lack of Ku80 may have impact also on HR on blocked replication forks. Thus, the data suggests that Ku80 has multiple functions related to the recognition and recruitment of repair factors, including HR proteins, to the site of DSB induced upon ICL processing.…”

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

“…It is possible that BRCA1 associates only with Ku in G 1 but forms a distinct complex with MRN and CtIP upon entry into S phase in order to activate the MRN nuclease activity to initiate resection. The N terminus of BRCA1 interacts with both Ku and Nbs1, but mutants differentially affected for either interaction are not yet available (82,100,101). Whether the MRN nuclease activity is required for Ku removal in mammalian cells remains an open question, but recent advances in assessing Ku localization should allow this important experiment to be done (102).…”