The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested. / October 2008

The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested.

The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested.

The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases. / text