BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation, BRCA1 was detected in discrete foci in the nucleus, which colocalize with hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50, hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells carrying a homozygous mutation in BRCA1 but was restored by transfection of wild-type BRCA1. Ectopic expression of wild-type, but not mutated, BRCA1 in these cells rendered them less sensitive to the DNA damage agent, methyl methanesulfonate. These data suggest that BRCA1 is important for the cellular responses to DNA damage that are mediated by the hRad50-hMre11-p95 complex.

The BRCA2 gene was identified based on its involvement in familial breast cancer. The analysis of its sequence predicts that the gene encodes a protein with 3,418 amino acids but provides very few clues pointing to its biological function. In an attempt to address this question, specific antibodies were prepared that identified the gene product of BRCA2 as a 390-kDa nuclear protein. BRCA2 was identified (1, 2) based on its initial mapping to chromosome 13q12-13 by linkage analysis of families with inherited breast cancer not attributed to mutations in BRCA1 (3). Germ-line mutations in BRCA2 account for the same percentage of familial breast cancers as BRCA1 (1). Together, these two breast cancer susceptibility genes are responsible for a large percentage of familial cases. In addition to breast cancer, BRCA2 mutations are also linked to other cancers including ovarian (4, 5), hepatocellular (6), pancreatic (5, 7), and prostate (4-6) tumors. However, mutations in BRCA2, like BRCA1, are mainly found in familial breast cancer but seldom occur in sporadic cases (8, 9). There have been over 100 distinct mutations spanning the sequence of this large gene (Breast Cancer Information Core). The majority of these mutations lead to truncation of the gene product.BRCA2 has 27 exons and expresses an mRNA 11 kb in size (1). The expression pattern of BRCA2 mRNA is similar to that of BRCA1, with highest levels in the testis, thymus, and ovaries (10). During mouse development, Brca2 mRNA is first detected on embryonic day 7.5, a time of rapid proliferation (11). At the cellular level, expression is regulated in a cell cycledependent manner with peak expression of BRCA2 mRNA in S phase (12). These results suggest BRCA2 may have a role in proliferating cells. Using a gene knockout method to create mice with BRCA2 mutations, homozygous mutant mice with BRCA2 truncated from the 5Ј half of exon 11 cannot survive embryogenesis (refs. 11, 13, and 14, and our unpublished results), suggesting that Brca2, like Brca1, plays an essential role in early embryonic development. Similar to Brca1, Brca2 heterozygotes are phenotypically normal and fertile. Although they are predicted to be genetically predisposed to cancer, they show no evidence to date of increased tumor formation.The identification of the BRCA2 gene was accomplished, quickly giving rise to the hope that the function(s) of the gene product would soon become clear. However, BRCA2 presents dilemmas similar to BRCA1, as very little insight in its function has been determined. To address this question systematically, we have identified the cellular BRCA2 protein as a nuclear protein and determined the domain responsible for interactions with human RAD51. Furthermore, BRCA2 apparently has a critical role in response to DNA damage. These results provide a molecular basis that begins to explain how mutations of BRCA2 contribute to carcinogenesis.

The BRCA1 gene product was identified as a 220-kilodalton nuclear phosphoprotein in normal cells, including breast ductal epithelial cells, and in 18 of 20 tumor cell lines derived from tissues other than breast and ovary. In 16 of 17 breast and ovarian cancer lines and 17 of 17 samples of cells obtained from malignant effusions, however, BRCA1 localized mainly in cytoplasm. Absence of BRCA1 or aberrant subcellular location was also observed to a variable extent in histological sections of many breast cancer biopsies. These findings suggest that BRCA1 abnormalities may be involved in the pathogenesis of many breast cancers, sporadic as well as familial.

BRCA2 is a breast tumor suppressor with a potential function in the cellular response to DNA damage. BRCA2 binds to Rad51 through its BRC repeats. In support of the biological significance of this interaction, we found that the complex of BRCA2 and Rad51 in breast cancer MCF-7 cells was diminished upon conditional expression of a wild-type, but not a mutated, BRC4 repeat using the tetracycline-inducible system. Cells expressing a wild-type BRC4 repeat showed hypersensitivity to ␥-irradiation, an inability to form Rad51 radiationinduced foci, and a failure of radiation-induced G 2 /M, but not G 1 /S, checkpoint control. These results strongly suggest that the interaction between BRCA2 and Rad51 mediated by BRC repeats is critical for the cellular response to DNA damage. BRCA2 was cloned based on an analysis of mutations in families predisposed to breast cancer showing that a large percentage of the kindred had alterations within this locus (1, 2). The expression pattern of BRCA2 is remarkably similar to that of BRCA1 (3-5), with highest levels in the testis, thymus, and ovaries (5). At the cellular level, expression is regulated in a cell-cycle dependent manner and peak expression of BRCA2 mRNA is found in S phase (6). These results suggest BRCA2 may participate in regulating cell proliferation.Recent studies indicate that BRCA2 is important for the cellular response to DNA damage. Brca2-null mouse embryos are nonviable at a very early stage of development and blastocysts derived from these embryos are very sensitive to ␥-irradiation (7). Mouse embryonic fibroblasts predicted to express BRCA2 that is C-terminally truncated at amino acid 1492 also demonstrated sensitivity to DNA damaging agents, particularly methyl methanesulfonate and UV light (8). Furthermore, Capan-1, a human pancreatic cancer line, that expresses a 220-kDa C-terminally truncated BRCA2 protein, is hypersensitive to a panel of DNA damaging agents (9). Importantly, ectopic expression of wild-type, but not mutated, BRCA2 in Capan-1 cells restores resistance to treatment with MMS (10). These results provided convincing evidence that BRCA2 plays a critical role in the DNA repair process.Interestingly, BRCA2 was shown to interact with Rad51 (7, 10 -12), a key protein in DNA recombinational repair. Human Rad51 encodes a 40-kDa protein with a structure related to the Escherichia coli recombination protein RecA (13) and mediates homologous DNA pairing and strand exchange (14, 15). Similar to mBrca2, inactivation of mouse Rad51 results in an embryonic lethal phenotype, indicating that Rad51 protein is essential for development (16,17). Beyond serving as a DNA repair protein through its interactions with other Rad proteins including Rad52 and Rad54 (18), how Rad51 may participate in cell growth and development remains unclear.While an association between BRCA2 and Rad51 is well documented, there is, nonetheless, some discrepancy concerning the regions of BRCA2 that bind to Rad51. It was reported that the C-terminal region of mouse Brca2 binds to mouse Rad...

The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.