A novel model to characterize structure and function of BRCA1.

BRCA1 plays a central role in DNA repair. Although N-terminal RING and C-terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B-lymphocyte cell line DT40. The combination of "homologous recombineering" and "RT-cassette" enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock-in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2-9 BRCA1Δ/Δ181-1415 (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1Δ/Δ126-136 decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1Δ/Δ525-881 within DNA-binding region, even the conserved 7-amino acid deletion BRCA1Δ/Δ872-878 within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage-induced Rad 51 foci formation in human BRCA1 knock-in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.