DNA Double Strand Break Repair Complexes in Space and Time with Applications to Druggability

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PhD project strategic theme: Biosciences for an integrated understanding of healthDouble strand breaks (DSBs) are the most dangerous forms of DNA damage. DSBs are repaired through the action of two main pathways, both of which require the assembly of multi-protein complexes in time and space: homologous recombination (HR) and non-homologous end joining (NHEJ). Cancerous cells are known to exhibit higher levels of DNA damage, thus targeting DSB repair pharmacologically is a useful form of therapy. This project will aim to build on previous structural understanding of the multiprotein complexes involved in DNA repair and exploit this information in the form structure-guided drug design as a collaboration between the Blundell and Pellegrini labs.As part of the Pellegrini Lab this project seeks to develop structural understanding of RAD51 filaments during homologous recombination. Previous work in the lab has produced the cryo-EM structure of a presynaptic RAD51 filament in complex with ssDNA, however the structure of the intermediate complex remains unknown. One aim of this project will be to solve the structure of the intermediate complex using cryo-EM. In addition, this project will focus on the functional and structural characterisation of RAD51 paralogs, little of which is known, despite their playing essential roles in HR. A variety of structural and biochemical approaches will be used to characterise the functions of these proteins in relation to RAD51 function during homologous recombination.As part of the Blundell Lab this project will build on research conducted as part of the BBSRC DTP rotation project to further explore the role of DNA Ligase IV and Artemis in NHEJ. The structure of an Artemis fragment in complex with DNA Ligase IV has been previously solved to 2.4Å resolution by the lab using X-ray crystallography, providing a framework for understanding the protein-protein interaction (PPI) site. Previous computational analysis suggests this PPI interface is highly druggable and high-throughput virtual screening (HTVS) of molecule libraries has identified candidate binders. This project will be continued through additional screening and experimental validation of leads with the aim of developing a successful inhibitor of this interaction. In addition, the project will focus on solving the structures of Ligase IV bound to its other interacting partners within the NHEJ system, notably with the DNA-PK complex. Previous work in the lab has produced the cryo-EM structure of DNA-PKcs and DNA-PK (the complex of DNA-PKcs with Ku70/80 and DNA) in monomeric and dimeric forms, establishing a workflow which can be adapted to integrate Ligase IV to produce a structure of the complex involved during the final stage of DNA blunt-end ligation.The proposed projects in a collaboration between the Pellegrini and Blundell labs integrate core structural biology with a practical application in structure-guided drug design. The dual focus on homologous recombination and non-homologous end joining provides a broad scope for investigating pathway crosstalk and decision-making and will have an emphasis on elucidating and comparing both the spatial and temporal organisation of the multiprotein complexes involved in each.