The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons

• Main Author: Davies, O. R.
• Published: University of Cambridge 2007
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598348

I have established a method of expressing and purifying a 1,127 amino acid fragment of human BRCA2 (BRCA2_BRC1-8) in which all eight BRC repeats are housed, providing a novel tool for research into mammalian recombination. Structural and functional studies of BRCA_BRC1-8have provided important insights into the nature of the BRC repeat-containing region of BRCA2 as a whole. Furthermore, through collaborative research, BRCA2_BRC1-8was found to promote RAD51-mediated strand exchange <i>in vitro. </i>This establishes a collective function for the multiple BRC repeat region of BRCA2, providing the first evidence that the BRC repeats of BRCA2 function as recombination mediators. An additional unrelated RAD51-binding site of currently unknown function is located at the C-terminal exon 27-encoded region of BRCA2 (BRCA2_Exon27). I have established that, in contrast to the inhibitory action of BRC repeats, BRCA2_Exon27binds RAD51 through a non-inhibitory mechanism and further associates with RAD51-DNA nucleoprotein filaments. Furthermore, I have demonstrated a function of BRCA2_Exon27 in protecting RAD51-ssDNA nucleoprotein filaments from disruption by BRC repeats. This function of BRCA2_Exon27is not extended to oligomeric RAD51 in isolation or RAD51-dsDNA complexes, highlighting that BRCA2_Exon27specifically protects RAD51-ssDNA nucleoprotein filaments, the key structures required for recombination-mediated repair. These findings suggest a model in which active recombination requires an interplay between the disruptive role of BRC repeats and the protective role of BRCA2_Exon27. Within the context of BRCA2_BRC1-8, the disruptive function of BRC repeats may load RAD51 onto ssDNA to form the nucleoprotein filament. This filament is then protected from subsequent BRC repeat-dependent disruption by BRCA2_Exon27for the duration of active recombination. It has previously been reported that BRCA2_Exon27 is phosphorylated at the G₂-M phase cell cycle transition, and upon phosphorylation can no longer interact with RAD51. I have demonstrated that the protection function of BRCA2_Exon27is abrogated by its phosphorylation. This further suggests a model for the known termination of recombination during mitosis; once phosphorylated, the protective function of BRCA2_Exon27 is lost and the disruptive function of BRC repeats may then dominate in disrupting all remaining RAD51-DNA nucleoprotein filaments, thus terminating recombination.