DNA damage tolerance and repair pathways in the mitochondrial compartment

Thesis (Ph. D.)--University of Rochester. Dept. of Biology, 2011.Mitochondria are essential organelles in nearly all eukaryotic cells. Due to their crucial function in respiration, mitochondria are frequently referred to as the powerhouses of the cell. These highly dynamic structures contain their own DNA genome, and the stability of this genome is fundamental in maintaining normal cellular function. Mutations in the human mitochondrial genome have been implicated in many diseases, including neuromuscular disorders, heart disease, and diabetes. Moreover, the accumulation of mutations in the mitochondrial genome has been shown to contribute to aging and aging-related disorders. Mutations in DNA occur as a result of errors in DNA synthesis and repair processes acting on damaged DNA. Although nuclear processes responsible for DNA replication and repair are well studied, pathways involved in mitochondrial DNA replication and repair are comparatively poorly understood. The work presented here describes the identification and characterization of mechanisms involved in DNA damage tolerance and repair in the mitochondrial compartment. In this study, I performed a screen of the Saccharomyces cerevisiae deletion collection to identify novel candidate genes involved in maintaining mitochondrial DNA integrity. Results from this analysis revealed that several proteins required for nuclear nucleotide excision repair function in mitochondrial DNA mutation avoidance after UV-induced damage. Additionally, I have demonstrated that a highly conserved nuclear protein FEN1, and its yeast homolog Rad27p, localize to the mitochondrial compartment in yeast and mice. I have completed a comprehensive genetic analysis of RAD27 mutants and describe the functional relevance in mitochondrial base excision repair as well as mitochondrial DNA maintenance. Furthermore, I have characterized the impact of translesion DNA polymerases on mitochondrial DNA stability and revealed a novel, mitochondrial-specific mechanism of mutagenesis. Lastly, I have critically examined the pathways involved in mitochondrial DNA double-strand break repair. Using novel reporter constructs, I implicate roles for the nuclear double-strand break repair proteins in generating mitochondrial DNA deletions. Moreover, I have generated a system to induce a single, specific mitochondrial double-strand break allowing for the analysis of the kinetics of this type of repair. In summary, these studies have identified novel mechanisms of maintaining mitochondrial DNA integrity, as well as, developed our current understanding of mitochondrial DNA mutagenesis