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Identification of critical residues in the carboxyl-terminal extension of the mitochondrial DNA polymerase in Saccharomyces cerevisiae

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dc.contributor.supervisor Court, Deborah A. (Microbiology) en_US
dc.contributor.author Imperial, Robin John Lester
dc.date.accessioned 2011-08-31T15:12:39Z
dc.date.available 2011-08-31T15:12:39Z
dc.date.issued 2011-08-31
dc.identifier.uri http://hdl.handle.net/1993/4798
dc.description.abstract Mip1p is the highly processive monomeric mitochondrial DNA polymerase in Saccharomyces cerevisiae. Despite differences in enzyme structure, substrate topology, and possible nucleoid interactions, Mip1p continues to be used as a model for human mitochondrial DNA polymerase (POLG) variants associated with various human mitochondrial diseases. Structurally, Mip1p functions as a monomer, whereas, the POLG holoenzyme contains a catalytic subunit (POLGA) complexed with a dimeric form of an accessory subunit (POLGB) which functions by loading the enzyme onto mitochondrial DNA and enhancing processivity. However, Mip1p does contain a 279-residue carboxyl-terminal extension (CTE) absent in the structure of POLG. The function of the CTE has not yet been determined although studies of truncation variants identify 74 N-terminal residues are essential for Mip1p wild-type activity. Furthermore, regions encompassing Mip1p residues N1033 – E1038 and Y1039 – A1049 are suggested to function in mitochondrial DNA maintenance and fidelity, respectively. This study has developed a mutagenic strategy to systematically replace the residues in the mitochondrial DNA maintenance region with glycine in order to identify residues critical for Mip1p function. Using in vivo respiratory competence and erythromycin resistance assays accompanied by an in vitro non-radioactive DNA polymerase assay, this study has identified two key residues, E1036 and D1037 that may function in the exonuclease-polymerase coupling mechanism of Mip1p. en_US
dc.rights info:eu-repo/semantics/openAccess
dc.subject mtDNA en_US
dc.subject polymerase en_US
dc.subject polg en_US
dc.subject cte en_US
dc.title Identification of critical residues in the carboxyl-terminal extension of the mitochondrial DNA polymerase in Saccharomyces cerevisiae en_US
dc.type info:eu-repo/semantics/masterThesis
dc.type master thesis en_US
dc.degree.discipline Microbiology en_US
dc.contributor.examiningcommittee Mark, Brian (Microbiology) Gietz, R. Daniel (Biochemistry and Medical Genetics) en_US
dc.degree.level Master of Science (M.Sc.) en_US
dc.description.note October 2011 en_US


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