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Title: The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons
Author: Davies, O. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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I have established a method of expressing and purifying a 1,127 amino acid fragment of human BRCA2 (BRCA2BRC1-8) in which all eight BRC repeats are housed, providing a novel tool for research into mammalian recombination. Structural and functional studies of BRCABRC1-8 have provided important insights into the nature of the BRC repeat-containing region of BRCA2 as a whole. Furthermore, through collaborative research, BRCA2BRC1-8 was found to promote RAD51-mediated strand exchange in vitro. 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 (BRCA2Exon27). I have established that, in contrast to the inhibitory action of BRC repeats, BRCA2Exon27 binds RAD51 through a non-inhibitory mechanism and further associates with RAD51-DNA nucleoprotein filaments. Furthermore, I have demonstrated a function of BRCA2Exon27 in protecting RAD51-ssDNA nucleoprotein filaments from disruption by BRC repeats. This function of BRCA2Exon27 is not extended to oligomeric RAD51 in isolation or RAD51-dsDNA complexes, highlighting that BRCA2Exon27 specifically 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 BRCA2Exon27. Within the context of BRCA2BRC1-8, 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 BRCA2Exon27 for the duration of active recombination. It has previously been reported that BRCA2Exon27 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 BRCA2Exon27 is abrogated by its phosphorylation. This further suggests a model for the known termination of recombination during mitosis; once phosphorylated, the protective function of BRCA2Exon27 is lost and the disruptive function of BRC repeats may then dominate in disrupting all remaining RAD51-DNA nucleoprotein filaments, thus terminating recombination.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available