BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.

Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.

Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2.

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.