Mutations in the human BRCA2 gene are a leading cause of susceptibility to breast, ovarian and prostate cancers. Its protein equivalent BRCA2, is the key mediator in repair of double-stranded DNA breaks and interstrand crosslinks - the most dangerous forms of DNA damage, via RAD51 effected homologous recombination. Thereby, preferably the sister chromatid, or homologous chromosome is utilised to drive the repair. Described here is the first structural and related mechanistic characterisation of full-length (384 kDa) BRCA2, and its complex with RAD51 and DNA. Electron microscopy reconstruction reveals that BRCA2 exists as a dimer in head-to-tail conformation, which binds two oppositely directed sets of RAD51 molecules. Single-stranded DNA (derived from exonucleolytic processing of double-strand breaks) binds BRCA2 along its long axis, such that one set of RAD51 monomers always binds thereon regardless of its binding polarity. These then self-assemble into RAD51-ssDNA nucleoprotein filaments that catalyse the main reaction, having been nucleated and primed for growth by BRCA2. In complementary work, it is shown that BRCA2 increases the frequency of RAD51-ssDNA filament formation, but does not impact filament length itself. It is also shown that BRCA2 initiates monodirectional (3'→5') extension of the RAD51 filament on single-stranded DNA from nucleation sites (thence at the 3' end). Furthermore, BRCA2 initiates such nucleations at multiple sites along individual ssDNA molecules, suggesting that these extend and merge into a single nucleoprotein filament, which would be most capable of catalysing the subsequent homologous pairing and strand exchange reactions. Together, the structures and biochemical data define the molecular mechanism via which BRCA2 orchestrates the formation of RAD51 nucleoprotein filaments, which constitute the central reaction intermediate in the repair of DNA double-strand breaks.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659543 |
| Date | January 2014 |
| Creators | Shahid, Taha |
| Contributors | Zhang, Xiaodong |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/25538 |
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