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Title: Investigating the mechanisms responsible for DNA double-strand break-induced loss of heterozygosity in fission yeast
Author: Cullen, Jason Kingsley
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2007
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Loss of heterozygosity (LOH) is considered a causal event in the formation of many cancers, with increasing evidence suggesting that DNA double-strand breaks (DSBs) play a major role in its occurrence. Despite its prominence in cancer, however, the precise molecular mechanisms responsible for extensive LOH and how such events are suppressed in normal cells is poorly understood. To investigate the mechanisms responsible for extensive break-induced LOH in eukaryotes, this study took advantage of an assay system in which such events could be identified through screening for loss of an auxotrophic his3+ marker, found ~25kb distal to an HO-endonuclease cut site in a non-essential minichromosome in Schizosaccharomyces pombe. Studies using this system had previously shown that extensive break-induced LOH in wild-type background, whilst infrequent, was predominantly associated with large translocations resulting from both allelic crossovers during G2 phase and breakinduced replication (BIR). Such extensive loss of allele specific information was also found to require rhp55+, rhp51+, rhp54+ and mus81+. This study has identified an additional role for the MRN complex, Rad22 and RPA in such break-induced translocations, suggesting that both allelic crossovers and BIR require homologous recombination (HR) in fission yeast. Surprisingly, break-induced extensive LOH was still observed in HR mutants. In contrast to wild-type cells, however, such extensive LOH was found to arise predominantly through de novo telomere addition at, or near, the break-site. Interestingly, telomere addition was most frequently observed in a rad22Δ background that disrupts HR following end resection. Further analysis demonstrated that de novo telomere addition was also significantly increased in ku70Δ rhp55Δ cells. Moreover, overexpression of rhp51 in rhp55Δ cells led to a substantial reduction in break-induced de novo telomere addition. Together, these findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ssDNA ends. In addition to providing information on break-induced LOH this study has identified a requirement for the MRN complex in efficient repair in rhp55Δ cells, which was previously found to occur via sister chromatid recombination (SCR) or a HRdependent end-joining pathway (EJ). Interestingly, deletion of MRN components also resulted in an increase in telomere addition, providing further evidence that HR competes with telomere addition for the repair of DSBs. Overall, these findings shed light on the competitive relationships between pathways of DSB repair/misrepair in S. pombe and how such mechanisms contribute to the prevention or promotion of genome instability.
Supervisor: Humphrey, Tim Carter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Yeast ; DNA repair ; Heterozygosity ; Eukaryotic cells