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Title: Control of the RAD51 recombinase by the BRC repeat motifs in the breast cancer susceptibility protein BRCA2
Author: Bates, D. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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The interaction between the breast cancer susceptibility protein BRCA2 and the RAD51 DNA recombinase is essential for DNA repair via homologous recombination. Its disruption in human cells causes genomic instability and cancer predisposition. Studies to define the biochemical and biological features of the BRCA2:RAD51 interaction are described. Human BRCA2 features eight BRC repeat motifs encoded within exon 11 through which it can bind RAD51. The BRC repeat motifs are believed to mimic and disrupt RAD51 oligomerisation at its self-association interface. I defined the minimal structural determinants required for RAD51 binding by ‘humanising’ a primitive RAD51 from an Archaeon species lacking BRCA2. Surface Plasmon Resonance (SPR) technology supported by cell biology was employed to study the characteristics of RAD51 binding to each of the BRC repeat motifs, both independently and collectively within the context of the BRCA2 protein. A single point mutation within an individual BRC repeat motif impaired RAD51 binding. Further, RAD51 was unable to interact effectively with a BRC repeat motif in which the proposed interaction interface had been disrupted by mutagenesis. Kinetic data for the interaction of an individual BRC repeat motif with RAD51 were obtained. An SPR competition assay was developed, revealing that the binding affinity of each BRC repeat motif for RAD51 differs significantly, and that their organisation within the scaffold of BRCA2 contributes to efficient interaction. Thus, I propose that both the differential binding affinities of the individual BRC repeat motifs for RAD51, and their observed cooperativity, contribute to the control of RAD51 by BRCA2. The implications of this proposal for DNA repair via homologous recombination, and for the role of BRCA2 mutations in human carcinogenesis, are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available