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dc.contributor.advisor Jinks-Robertson, Sue en_US
dc.contributor.author Mitchel, Katrina en_US
dc.date.accessioned 2012-09-04T13:15:54Z
dc.date.available 2012-09-04T13:15:54Z
dc.date.issued 2012 en_US
dc.identifier.uri http://hdl.handle.net/10161/5835
dc.description Dissertation en_US
dc.description.abstract <p>The genetic information necessary for the survival and propagation of a species is contained within a physical structure, DNA. However, this molecule is sensitive to damage arising from both exogenous and endogenous sources. DNA damage can prevent metabolic processes such as replication and transcription; thus, systems to bypass or repair DNA lesions are essential. One type of lesion in particular - the double strand break (DSB) - is extremely dangerous as inappropriate repair of DSBs can lead to deletions, mutations and rearrangements. Homologous recombination (HR) uses a template with sequence homology to the region near the DSB to restore the damaged molecule. However, this high-fidelity pathway can contribute to genome instability when recombination occurs between diverged substrates. To further our understanding of the regulation of HR during vegetative growth, we have used the budding yeast Saccharomyces cerevisiae as a model system and a plasmid-based assay to model repair of a DSB. In the first part of this work, the molecular structures of noncrossover (NCO) and crossover (CO) products of recombination were examined. While the majority of NCOs had regions of heteroduplex DNA (hDNA) on one side of the gap in the repaired allele and no change to the donor allele, most COs had two tracts of hDNA. They were present on opposite sides of the gap, one in each allele. Our results suggest that the majority of NCOs are generated through synthesis-dependent strand annealing (SDSA), and COs are the result of constrained cleavage of a Holliday junction (HJ) intermediate. To clarify the mechanisms regulating NCO production, the effects of three DNA helicases - Mph1, Sgs1 and Srs2 - on the structures of NCO events were examined. All three helicases promote NCO formation by SDSA, but Sgs1 and Srs2 also assist in NCO formation arising from an HJ-containing intermediate, consistent with HJ-dissolution. To study how CO products are generated, we have investigated the contribution of the following candidate HJ resolvases to the structures of CO events: Mus81, Yen1 and Rad1. The results suggest that Rad1 is important to normal CO formation in this assay, but Mus81 and Yen1 are largely dispensable. Together, this work advances our knowledge of how the NCO versus CO outcome is determined during HR, expanding our understanding of how mitotic recombination is regulated.</p> en_US
dc.subject Genetics en_US
dc.subject Molecular biology en_US
dc.subject DNA helicases en_US
dc.subject double strand break repair en_US
dc.subject Homologous recombination en_US
dc.subject Sgs1 en_US
dc.subject Srs2 en_US
dc.title The Molecular Structures of Recombination Intermediates in Yeast en_US
dc.type Dissertation en_US
dc.department Molecular Genetics and Microbiology en_US

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