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Structural and biochemical characterization of the BRCA2 breast cancer tumour suppressor

Final Report Summary - BRCA2COMPLEXES (Structural and biochemical characterization of the BRCA2 breast cancer tumour suppressor)

The tumor suppressor protein BRCA2 is essential for DNA repair by homologous recombination (HR). Mutations in BRCA2 gene confer increased risks of breast, ovarian and possibly, other cancer types, and biallelic BRCA2 mutations have been associated with cancer-predisposition syndrome Fanconi anemia (FA) sub-type FA-D1. Therefore understanding the precise function of this tumor suppressor and its interplay with partner proteins is important to uncover the key molecular steps in cancer development. BRCA2 is implicated in the assembly of nucleoprotein filaments formed by the human recombinase RAD51 and delivery of RAD51 to sites of DNA damage. Until recently the exact mechanism how BRCA2 coordinates RAD51-mediated steps of HR remained elusive.
The results obtained in this study provide first structural and mechanistic glimpse into the role that BRCA2 plays in promoting RAD51 nucleoprotein filament formation. In collaboration with Prof. Xiaodong Zhang at Imperial College London, we visualized full-length BRCA2 protein and BRCA2-RAD51 complex using negative stain electron microscopy combined with single particle techniques to generate their three-dimensional (3D) reconstructions. We found that BRCA2 forms dimers and that single-stranded DNA (ssDNA) binds across long axis of the dimeric protein. Our structural analysis of BRCA2-RAD51 complex revealed that BRCA2 remains in a dimeric form upon binding of RAD51 and associates with two sets of RAD51 molecules oriented in opposite directions. Thus, due to polarity of ssDNA, BRCA2 will always deliver at least one set of RAD51 arranged in an optimal manner for filament formation. Our mechanistic studies demonstrated that BRCA2 increases the number of RAD51 filaments, but shows no significant effect on average RAD51 filament length highlighting an important role of this tumor suppressor in the nucleation of RAD51 filaments. Analysis of BRCA2/RAD51/ssDNA complexes further revealed that BRCA2 is present at one end of the RAD51 filament and directs unidirectional 3’-5’ growth of RAD51 filaments along the ssDNA acting as a molecular chaperone.
Together, our data uncover novel molecular insight into mechanistic aspects of BRCA2 action in HR and have potential implications for designing more efficient therapeutic intervention of cancer.