Homozygous germ line mutation in exon 27 of murine Brca2 disrupts the Fancd2-Brca2 pathway in the homologous recombination-mediated DNA interstrand cross-links' repair but does not affect meiosis

The role of the region encoded by exon 27 of the Brca2 gene in DNA repair was studied in cells and tissues from Brca2(Delta 27/27) mice. The COOH-terminal truncated Brca2 localized to the nucleus in primary mouse embryo fibroblasts from Brca2(Delta 27/Delta 27) mice. Fluorescence-activated cell sorting (FACS) analysis demonstrated that these fibroblasts were hypersensitive to mitomycin C-induced cross-links, but not to double-strand breaks (DSBs) induced by irradiation. The gamma H2AX appearance kinetics and comet assay showed that DSBs were repaired through non-homologous end joining pathways, while interstrand cross-links were not repaired due to deficient homologous recombination pathways. Immunoprecipitation experiments showed that Fancd2 did not coprecipitate with the mutated Brca2. There were also no detectable Rad51-positive foci formed in these cells after damage. On the other hand, we did not find any difference during gametogenesis in mice harboring exon 27 truncating mutation of the Brca2 gene and control mice, and in both cases, Rad51 localized to the recombination foci. Our results suggest that exon 27 of murine Brca2 is crucial for the interaction of Brca2 and Fancd2 in Rad51-mediated recombination in response to DNA damage, but that this interaction is not taking place in the homologous recombination during meiosis. Published 2005 Wiley-Liss, Inc.(dagger)