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Novel Structures of RAD51 Reveal Mechanisms in DNA Damage Repair and Genomic Stability https://doi.org/10.17863/CAM.107370 Appleby, Robert Pellegrini, Luca Blundell, Thomas The RAD51 protein contributes to the maintenance of genomic stability by promoting the repair of DNA double-strand breaks and the protection of DNA replication forks. RAD51 functions alongside the tumour suppressor protein BRCA2 to catalyse DNA strand-exchange reactions which form an integral part of Homology-Directed Repair. RAD51 has been the subject of decades of research which has helped to elucidate many mechanisms underpinning its function, however numerous key questions still remain. In this thesis I present three-dimensional structures of RAD51 nucleoprotein filaments together with biochemical and biophysical data that reveal new insights into how RAD51 contributes to maintaining the stability of our genome. High-resolution structures of RAD51 filaments on single- (ss-) and double-stranded (ds-) DNA revealed the presence of a second metal cation at the ATP-binding site, which forms the basis for a mechanism of ATP-hydrolysis dependent filament disassembly, confirmed by the structure of a RAD51 filament in the presence of ADP. Here I also describe two structures of RAD51 bound to the C-terminus of BRCA2, which show how BRCA2 binds to and stabilises RAD51 filaments during DNA replication and repair. A low- resolution structure of a RAD51 synaptic filament is also presented here, which suggests that the mechanism of recombinase-catalysed strand exchange is conserved throughout the three domains of life. Furthermore, I demonstrated that RAD51 can bind to DNA damaged by base hydrolysis, based on the structure of RAD51 nucleoprotein filaments that reveal specific recognition of abasic sites. Finally, I show that RAD51 can bind RNA substrates, as established by RAD51 filament structures bound to ssRNA and a DNA : RNA hybrid. Collectively these structures and supporting biochemical experiments highlight new mechanisms of RAD51 function in both DNA repair and DNA replication. University of Cambridge 2023-11-30 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/366431 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b60d1b06-32bd-46a0-9811-c26520185399/download d5d200d004c8afcf7ddf2e906f92d8fd https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2260d8bb-4d1d-4705-9842-4d983313058f/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ BRCA2 CryoEM Homologous Recombination RAD51