Molecular and Cellular Pathobiology Compensatory Functions and Interdependency of the DNA- Binding Domain of BRCA2 with the BRCA1–PALB2–BRCA2 Complex

BRCA1, BRCA2, and PALB2 are key players in cellular tolerance to chemotherapeutic agents, including camptothecin, cisplatin, and PARP inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1–PALB2–BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2 mutant cells. Although previous studies have suggested that BRCA1–PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2 mutant cells displayed a considerably milder phenotype than did BRCA2 / null-deficient cells. We hypothesized that the DNA-binding domain (DBD) of BRCA2 might compensate for a defect in BRCA2 that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DBD of BRCA2 in wild-type and BRCA2 cells. Remarkably, although the resulting BRCA2 cells displayed a moderate phenotype, the BRCA2DNþDDBD cells displayed a very severe phenotype, as did the BRCA2 / cells, suggesting that the N-terminal segment and the DBD play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1–PALB2–BRCA2 complex and the DBD is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1–PALB2–BRCA2 complex and the DBD in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of antimalignant therapies for patients carrying mutations in the BRCA2 gene. Cancer Res; 74(3); 1–11. 2013 AACR. Introduction Homologous recombination plays a central role in maintaining genomic DNA as well as cellular tolerance to various DNA-damaging agents (1, 2). Specifically, homologous recombination restores stalled replication at damaged template strands by using the intact sister chromatid as a template for DNA synthesis (3–6) and repairs any double-strand breaks (DSB) that may occur in one of the sister chromatids during replication or as a result of chemotherapeutic treatments with camptothecin, cisplatin, and PARP inhibitor. Individual homologous recombination factors contribute in different ways to various types of homologous recombination (7, 8). In homologous recombination–dependent DSB repair, nucleases initiate homologous recombination by processing DSBs to generate 30 overhangs, followed by the polymerization of RAD51 recombinase on the overhang (9–11). The resulting RAD51 polymer is responsible for homology search and subsequent strand exchange with intact homologous duplex DNA. RAD51 is required for every type of homologous recombination reaction, and inactivation of RAD51 in the chicken DT40 cell line causes numerous spontaneous chromosomal breaks (12). The polymerization of RAD51 is strictly regulated by a number of RAD51 mediator proteins, including BRCA1, BRCA2, Gemin2 (13), the 5 RAD51 paralogs, RAD52, SFR1, and SWS1 (14, 15). The breast-cancer-susceptibility gene 2 (BRCA2) encodes a tumor-suppressor protein carrying 3,418 amino acids. Although the overall amino-acid identity between the human and the chicken BRCA2 protein is only 40%, several regions are conserved, including the N-terminal segment (approximately 100 amino acids), 8 BRC motifs, a carboxy-proximal region containing aDNA/DSS1-binding (DDB) domain, and the C-terminal end (16). The BRCmotifs, the DDB domain, and the C-terminal end may involve RAD51 loading onto DNA, an association with damaged DNA sites, and an interaction with Authors' Affiliations: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Sakyo-ku; and Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for D. Dejsuphong: Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. CorrespondingAuthor:Shunichi Takeda,Department ofRadiationGenetics, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan. Phone: 81-75-753-4410; Fax: 81-75-753-4419; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-1443 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 14, 2017. © 2013 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1443