Genetic determinants of mitochondrial function and dysfunction
Hall, Brandon M.
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Maintenance of the mitochondrial genome is essential for respiration and other mitochondrial functions which are ultimately required for stability of the nuclear genome. In this work, I investigated several genetic determinants of mitochondrial DNA maintenance (UPS genes and SLX4) and determined the consequences of mitochondrial DNA depletion in budding yeast Saccharomyces cerevisiae . The highly conserved PRELI/MSF1 family of proteins in yeast (Ups proteins) is required for the maintenance of the mitochondrial genome through their pleiotropic effects on mitochondrial biology. In Chapter 2 , the contribution of Ups proteins to mitochondrial processes was elucidated, revealing roles in mitochondrial respiration, mitochondrial phospholipid metabolism and mitochondrial genome maintenance. A prominent role for Ups2p emerged in the biology of Ups protein function. Ups2p was required for optimal activity of oxidative phosphorylation complexes and maintenance of phosphatidylethanolamine levels. In addition, mitochondrial defects observed upon inactivation of UPS1 (including respiration and maintenance of cardiolipin levels) were Ups2p-dependent. In Chapter 3 , domains of Ups2p were characterized in mitochondrial processes affected by Ups2p function. These domains include the MSF1 domain which is common to all PRELI homologs and a newly identified LEA-like domain which shares significant sequence similarity with the LEA domain of human PRELID1. Functional conservation between the yeast LEA-like domain and human LEA domain is demonstrated in oxidative stress resistance. Repair of mtDNA following genotoxic stress is another important component of mitochondrial genome maintenance. In Chapter 4 , I studied a role of DNA repair protein Slx4p in the maintenance of the mitochondrial genome after exposure to a DNA-damaging agent. Slx4p exhibited mitochondrial localization. However, the mechanism of Slx4p function is unclear, as known Slx4p domains were dispensable for mtDNA stability. Mitochondrial DNA depletion is associated with alterations in DNA repair pathways invaluable in genome instability. In Chapter 5 , we identified components of multiple DNA repair pathways which were differentially regulated in ρ 0 cells. Among these DNA repair pathways, we determined the ability of DDR (DNA damage-responsive) genes and MSH (MutS homolog) mismatch repair genes to suppress nuclear genome instability generated from a mitochondrial genetic defect (in ρ 0 cells), independent of a mitochondrial metabolic defect (respiratory inhibitor-treated cells). MSH5 suppressed the rate of point mutations, as well as gross chromosomal rearrangements, in ρ 0 cells independent of respiratory deficiency. Furthermore, we identified a Mec1p (human ATR) checkpoint-dependent imbalance of nucleotide pools in ρ 0 cells which is thought to contribute to the mutator phenotype.