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Characterization of the Saccharomyces cerevisiae RAD5 gene and protein

dc.contributor.advisorXiao, Weien_US
dc.contributor.committeeMemberKobryn, Kerrien_US
dc.contributor.committeeMemberMoore, Stanleyen_US
dc.contributor.committeeMemberDesautels, Michelen_US
dc.contributor.committeeMemberGoldie, Hughesen_US
dc.creatorBlackwell, Susanen_US
dc.date.accessioned2014-01-21T19:01:11Z
dc.date.available2014-01-21T19:01:11Z
dc.date.created2013-08en_US
dc.date.issued2013-09-23en_US
dc.date.submittedAugust 2013en_US
dc.description.abstractDNA damage tolerance (DDT) is a process utilized by cells to bypass replication blocking lesions in the DNA, preventing replication fork collapse and maintaining genomic stability and cell viability. In Saccharomyces cerevisiae DDT consists of two branched pathways. One branch allows direct replication past lesions in the DNA utilizing specific error-prone polymerases, a process known as translesion DNA synthesis (TLS). The other branch utilizes homologous recombination and template switch to replicate past damaged DNA in an error-free manner. RAD5 has traditionally been characterized as belonging to the error-free pathway of DNA damage tolerance. The protein is multi-functional, with several specific activities identified and classified to the error-free branch of DDT. However, there is also evidence for additional uncharacterized activities of the protein. The goal of this research was to determine which branches of DNA damage tolerance the uncharacterized activities of Rad5 are involved in. A two-pronged approach was utilized, elucidation of the physical interactions of the protein, and examination of the genetic interactions between RAD5 and other DDT genes. The evidence indicates that Rad5 plays a partial role in TLS and the protein is known to physically interact with Rev1, a member of the TLS pathway. We assumed this physical interaction mediates the TLS activity of Rad5. The yeast two-hybrid assay was utilized to examine the interaction between Rev1 and truncated Rad5 fragments, and the N-terminal 30 amino acids of Rad5 proved sufficient to maintain the interaction. This research sets the stage to identify key residues in Rad5 for the interaction with Rev1, and the creation of a TLS deficient rad5 mutant by targeting those key residues. Genetic interactions between RAD5 and genes required for the initiation of DDT in the cell were examined based on sensitivity to killing by various DNA damaging agents. We determined that the functions of Rad5 rely on PCNA modification, and thus do not function in a cellular process unrelated to Rad5. Potential uncharacterized functions are discussed on the basis of these results and the results of the interaction studies. Future structural and functional studies are proposed to better understand the role of Rad5 in the cell.en_US
dc.identifier.urihttp://hdl.handle.net/10388/ETD-2013-08-1139en_US
dc.language.isoengen_US
dc.subjectRAD5en_US
dc.subjectDNA damage toleranceen_US
dc.subjectPost-replication repairen_US
dc.subjectS. cerevisiaeen_US
dc.titleCharacterization of the Saccharomyces cerevisiae RAD5 gene and proteinen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentMicrobiology and Immunologyen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US

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