The BRCA 2 gene product functionally interacts with p 53 and RAD 51

Germ-line mutations in the human BRCA2 gene confer susceptibility to breast cancer. Efforts to elucidate its function have revealed a putative transcriptional activation domain and in vitro interaction with the DNA repair protein RAD51. Other studies have indicated that RAD51 physically associates with the p53 tumor suppressor protein. Here we show that the BRCA2 gene product is a 460-kDa nuclear phosphoprotein, which forms in vivo complexes with both p53 and RAD51. Moreover, exogenous BRCA2 expression in cancer cells inhibits p53’s transcriptional activity, and RAD51 coexpression enhances BRCA2’s inhibitory effects. These findings demonstrate that BRCA2 physically and functionally interacts with two key components of cell cycle control and DNA repair pathways. Thus, BRCA2 likely participates with p53 and RAD51 in maintaining genome integrity. Germ-line mutations in the human BRCA1 and BRCA2 breast cancer suppressor genes confer susceptibility to breast and ovarian cancers (1–4). Mutations in BRCA1 and BRCA2 are believed to be responsible for most hereditary breast cancers (3–10), which account for 5–10% of all breast cancer cases (11). About 52% of the families with four or more breast cancer cases have inherited mutations in BRCA1, and 32% possess BRCA2 mutations (12). In contrast, somatic mutations in BRCA1 and BRCA2 are rare in sporadic cases of breast cancer (5, 13–15). Both BRCA1 and BRCA2 are up-regulated in proliferating and differentiating cells, expressed in a cell cycle-dependent manner peaking at the G1yS boundary (19– 21), widely expressed during development (22–24), and essential for early embryonic development (25–29). Recent evidence indicates that BRCA1 is involved in transcriptional regulation (30, 31). The BRCA2 gene is composed of 27 exons and encodes a predicted 384-kDa protein possessing no obvious homology to other sequences in publicly available databases (4). There are eight repetitive units in the BRCA2 protein sequence (BRC motifs; refs. 16 and 17) and a potential nuclear localization signal within its C terminus (18). BRCA2 also possesses a putative transcriptional activation domain in exon 3, suggesting a role of BRCA2 in the regulation of gene expression (32). Evidence for a possible function of BRCA2 in DNA repair has been suggested by recent findings that Brca2 mutant mouse embryos are defective in DNA repair (27, 33, 34). Moreover, yeast two-hybrid and glutathione S-transferase (GST) pulldown analysis revealed that BRCA2 interacts in vitro with RAD51, a protein involved in DNA double-strand break repair and homologous recombination (27, 35, 36). Binding sites for RAD51 have been mapped to each of the eight BRC motifs in human BRCA2 (36) and to a C-terminal region of mouse Brca2 (27, 35). Structural and functional characterization of the endogenous BRCA2 protein have been hampered by the large size of the protein and the lack of suitable immunological reagents for its detection. In the present studies, we generated polyclonal and monoclonal antibodies to characterize endogenous BRCA2 and identify proteins that form complexes with this protein. We demonstrate that BRCA2 is a nuclear phosphoprotein that associates in vivo with a significant portion of the endogenous pool of RAD51. Because an immediate cellular reponse to DNA damage is p53-mediated cell cycle arrest (37), and RAD51 has been reported to physically associate with p53 (38, 39), we also investigated whether a physical and functional relationship could be detected between BRCA2 and p53. MATERIALS AND METHODS Generation of BRCA2 Antibodies. Polyclonal antibodies were raised in rabbits, and mAbs were generated in mice. The peptides were conjugated to keyhole limpet hemacyanin. The three GST-fusion regions were generated by reverse transcription–PCR amplification using the following primers: 59GGTCAAGTTCTTTAGCTACAGGATCCACCC-39 and 59CTCCATCTGGGCTCCATGTCGACCTGAAAG-39 for GSTB1, 59-CCTGCAACTTGTTACAGAATTCAGTCCCC-39 and 59-AGGGTGAAGAGCTAGTCTCGAGTTCCTCAA-39 for GSTB2, and 59-GTCAGTGAATCCACTAGAATTCCTCCCACC-39 and 59-TGGTCTTGAACTCCTGGCCTCGAGCACT-39 for GSTB3. These regions then were subcloned into the GST-fusion protein expression vector pGEX-5X-2 or pGEX-4T-2 (Pharmacia) and verified by sequencing. All antibodies were affinity-purified. Plasmid Constructs. pGFPB2 containing human full-length BRCA2 cDNA fused in-frame with green fluorescent protein (GFP) was constructed as follows. A fragment at the N terminus of BRCA2 was generated from pUCBRCA2 by PCR amplification using a high-fidelity PCR system (Boehringer Mennheim) and the following primers: 59-GGCCAGATCTATGCCTATTGGATCCAAAGAGAGG-39 and 59-GGCCGTCGACTGCTTGTTTATCACCTGTGTCTCC-39. This fragment was subcloned into the pEGFPC1 vector (CLONTECH) at BglII and SalI sites to generate pGFPB2N. A Sse8387I–SalI BRCA2 fragment released from pUCBRCA2 then was ligated with pGFPB2N digested with BglII and Sse8387I to generate pGFPB2. Sequencing confirmed the identity of the fragment generated by PCR. A human fulllength RAD51 cDNA with BamHI and SalI f lanking was amplified from a human testis cDNA library (CLONTECH) by using Pfu DNA polymerase (Stratagene) and the following primers: 59-GGCCGGATCCATGGCAATGCAGATGCAGCTTG-39 and 59-GGCCGTCGACTCTTTGGCATCTCCCACTCCAT-39. Cloning of this cDNA fragment into pcDNA3HA generated an N-terminal hemagglutinin (HA)The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9513869-6$2.00y0 PNAS is available online at www.pnas. org. Abbreviations: GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; RB, retinoblastoma protein. *To whom reprint requests should be addressed. e-mail: Aaronson@ smtplink.mssm. edu.