Investigating the role of EXD2 in end resection, genome stability, and cancer
Thesis or Dissertation
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DNA double-strand breaks (DSBs) are highly toxic lesions, so their accurate repair is required to maintain genome stability and prevent human diseases, including cancer. DSBs are repaired by two main pathways: "error-prone" non-homologous end joining (NHEJ) and "error-free" homologous recombination (HR). DNA end resection has been proposed to play a key role in how cells regulate pathway choice; however, the molecular mechanisms of resection remain poorly defined. Resection involves the enzymatic processing of broken DNA ends, generating ssDNA overhangs to promote HR and inhibit NHEJ. The 3' - 5' exonuclease EXD2 has been identified as a novel component of the resection machinery. This thesis describes the identification and characterisation of EXD2-interacting proteins. An 11-plex tandem mass tag co-immunoprecipitation mass spectrometry screen identified novel EXD2 interactors and EXD2 post-translational modification sites (PTMs). One of the protein interactors identified is RIF1, which has been selected as a candidate for follow up work due to its known role in NHEJ (in complex with Shieldin) and replication fork stability. This thesis characterises the function of the EXD2-RIF1-Shieldin interaction by analysis of different mutants in untreated, and DNA damage inducing agent-treated conditions. Better understanding of these mechanisms is of great importance as deregulation of these pathways drives genomic instability and is the underlying cause of many devastating human syndromes, including cancer. Thus, from a clinical perspective, a greater understanding of how DNA end resection and DSB repair pathway choice are executed may pave the way for the identification of novel therapies. Therefore, this thesis contains a final chapter on the role of EXD2 in olaparib (a PARP inhibitor approved for use in the clinic) sensitivity and resistance mechanisms.
Cancer and Genome Instab
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Institute of Cancer Research (University Of London)