Analysis of The RING Domain And BRCT Repeats of BRCA1

Mutations within BRCA1 often contribute to breast cancer susceptibility. Many of these mutations cluster within two highly conserved regions that may be important for BRCA1's functions: the RING domain and the BRCT repeats. BRCA1's RING domain has E3 ubiquitin ligase activity, which is greatly enhanced when it forms a heterodimer with Bard1. This region is of particular interest because it displays the only known enzymatic activity of BRCA1. The BRCT repeats have phosphopeptide binding activity, which is necessary for BRCA1's interaction with DNA repair proteins BACH1, ABRAXAS, and CtIP. To test the importance of these two domains we generated cell lines and mouse models with point mutations that either eliminate the E3 ligase activity of Brca1 while maintaining its interaction with Bard1 (I26A) or eliminate its phosphopeptide binding activity (M1717R). We found that the E3 ubiquitin ligase activity of Brca1 is dispensable for its role in cell viability, embryonic development, double strand break repair, and tumor suppression. Interestingly, we were unsuccessful at generating homozygous BRCT mutant ES cells and homozygous M1717R Brca1 MEFs displayed a proliferation defect, spontaneous chromosomal aberrations, and centrosomal amplification. Our data shows that the BRCT repeats are crucial for BRCA1's role in DNA repair because BRCT mutant MEFs do not recruit Brca1 or Rad51 to IR induced DNA damage sites, they have a defect in homology directed repair, and M1717R Brca1 is not hyperphosphorylated in response to DNA damage in these cells. We were able to generate homozygous M1717R Brca1 mice, but with a very low frequency (<1%). All Brca1M17171R/M1717R mice produced thus far have been males and they are sterile. Our analysis indicates that the fertility defect is not due to a defect in meiosis. Introduction of the M1717R mutation in a conditional mammary or pancreatic tumor model also reduces the tumor latency to the same degree as the introduction of a Brca1 null mutation. Therefore our data shows that BRCA1 carries out the majority of its functions through its BRCT repeats.