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Title: Genetic analysis of DNA double-strand break mis-repair mechanisms using the human endogenous HPRT gene
Author: Ahrabi, Sara
ISNI:       0000 0004 6500 765X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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DNA double strand breaks (DSBs) are the most lethal radiation-induced lesions in response to which cells employ either the error free homologous recombination (HR) repair pathway or error prone mechanisms, such as non homologous end joining (NHEJ) and microhomology- mediated end joining (MMEJ). While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. In this thesis, I have exploited the human endogenous HPRT gene to develop a novel genetic technology to detect mutation frequencies and signatures in response to DNA DSBs in different genetic backgrounds. Using a sensitive HPRT assay we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair, we found that depletion of HR proteins, including RAD51, BRCA2, BRCA1 or SETD2, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2 - 6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection. Further, I have defined a role for the SETD2 histone methyltransferase in suppressing break-induced mutations, and have shown that CRISPR/Cas9 and ISceI- induced DSBs resulted in different repair profiles.
Supervisor: Porter, Andy ; Humphrey, Tim Sponsor: Clarendon Fund ; Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available