Alexander K. C. Wong

RAD51 Interacts with the Evolutionarily Conserved BRC Motifs in the Human Breast Cancer Susceptibility Gene brca2

Recent work has shown that the murine BRCA2 tumor suppressor protein interacts with the murine RAD51 protein. This interaction suggests that BRCA2 participates in DNA repair. Residues 3196–3232 of the murine BRCA2 protein were shown to be involved in this interaction. Here, we report the detailed mapping of additional domains that are involved in interactions between the human homologs of these two proteins. Through yeast two-hybrid and biochemical assays, we demonstrate that the RAD51 protein interacts specifically with the eight evolutionarily conserved BRC motifs encoded in exon 11 ofbrca2 and with a similar motif found in aCaenorhabditis elegans hypothetical protein. Deletion analysis demonstrates that residues 98–339 of human RAD51 interact with the 59-residue minimal region that is conserved in all BRC motifs. These data suggest that the BRC repeats function to bind RAD51.

Characterization of a carboxy-terminal BRCA1 interacting protein.

There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain. Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602–1863. A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP. The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription. A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants. In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele. Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1.