Support of Mitochondrial DNA Replication by Human Rad51: A Dissertation

Abstract

The function of homologous DNA recombination in human mitochondria has been a topic of ongoing debate for many years, with implications for fields ranging from DNA repair and mitochondrial disease to population genetics. While genetic and biochemical evidence supports the presence of a mitochondrial recombination activity, the purpose for this activity and the proteins involved have remained elusive. The work presented in this thesis was designed to evaluate the mitochondrial localization of the major recombinase protein in human cells, Rad51, as well as determine what function it plays in the maintenance of mitochondrial DNA (mtDNA) copy number that is critical for production of chemical energy through aerobic respiration. The combination of subcellular fractionation with immunoblotting and immunoprecipitation approaches used in this study clearly demonstrates that Rad51 is a bona fide mitochondrial protein that localizes to the matrix compartment following oxidative stress, where it physically interacts with mtDNA. Rad51 was found to be critical for mtDNA copy number maintenance under stress conditions. This requirement for Rad51 was found to be completely dependent on ongoing mtDNA replication, as treatment with the DNA polymerase gamma (Pol ϒ) inhibitor, ddC, suppresses both recruitment of Rad51 to the mitochondria following the addition of stress, as well as the mtDNA degradation observed when Rad51 has been depleted from the cell. The data presented here support a model in which oxidative stress induces a three-part response: (1) The recruitment of repair factors including Rad51 to the mitochondrial matrix, (2) the activation of mtDNA degradation systems to eliminate extensively or persistently damaged mtDNA, and (3) the increase in mtDNA replication in order to maintain copy number. The stress-induced decrease in mtDNA copy number observed when Rad51 is depleted is likely the result of failure to stabilize or repair replication forks that encounter blocking lesions resulting in further damaged to the mtDNA and its eventual degradation.