| The BRCA1 gene encodes a multifunctional protein involved in tumor formation in hereditary breast cancer. To establish a model system that facilitates comparison of BRCA1 alleles in an isogenic context, I have cloned the chicken orthologue of the human BRCA1 gene. Although the sequence homology with the mammalian orthologues is limited, phylogentic analysis indicates that the chicken gene (ggBRCA1) is a bona fide BRCA1 orthologue. Alignment of the sequences allows for the identification of nine highly-conserved motifs in the otherwise poorly-conserved central domain of the protein. Conservation of these regions is likely to reflect particular importance for BRCA1 function. Cloning of ggBRCA1 allowed for the creation of homozygous BRCA1 deletion derivatives of the chicken bursal B cell line, DT40. DT40 cells have the advantage that they are highly proficient at recombination, greatly facilitating manipulation of the genome through homologous targeting. Two deletion derivatives were constructed: one removed the ATG and additional 5′ sequences and the second removed all coding sequences for ggBRCA1. The perfect null mutation and the 5′ deletion mutation confer identical phenotypes, indicating that the 5′ deletion is also a null. Consistent with mutation analysis in other organisms, DT40 BRCA1−/− cells display several of the canonical phenotypes associated with defects in homologous recombination: they are sensitive to DNA damaging agents, have decreased targeted integration frequency, and fail to make RAD51 foci after DNA damage. While the last defect can be rescued by overexpression of RAD51, such overexpression fails to rescue the BRCA1−/− sensitivity to cisplatin. Assuming that RAD51 foci mark sites of damage in the overexpression line, as is known to be the case in normal cells, the results suggest that BRCA1 function in recombinational repair is not limited to promotion of recombinase assembly. Epistasis analysis with null mutants of BRCA1 and the gene for the end joining protein, KU70, indicates that BRCA1 and KU70 act independently to promote resistance to X-ray induced damage in DT40 cells indicating that BRCA1 is largely if not completely, dispensable for DNA end joining. |
