ABSTRACT While the ATP production by oxidative phosphorylation in mitochondria provides the energy for the synthesis of proteins, nucleic acids and other macromolecules, this process generates as a by-product reactive oxygen species that present a unique challenge for the circular mitochondrial genome. Notably, the accumulation of oxidative DNA damage in mitochondrial DNA inhibits the transcription of key electron transport proteins encoded by the mitochondrial genome disrupting electron transport leading to a further increase in reactive oxygen species. In addition to the reactive oxygen species generated within mitochondria, some environmental DNA damaging agents preferentially cause damage in the mitochondrial genome compared with the nuclear genome. Interestingly, although the oxidized base 8-oxoguanine is repaired, oxidative DNA damage also induces degradation of the mitochondrial genome. Since there are multiple copies of the mitochondrial genome per organelle, it has been suggested that the removal of damaged genomes by degradation serves to prevent mutations. For oxidative DNA damage, it is not known what lesion(s) triggers genome degradation and whether this reduces mutations. In Specific Aim 1, we will test the hypothesis that the MUTYH DNA glycosylase protects the mitochondrial genome from mutation by stably binding to genomes with the 8-oxoG:adenine mispairs or the 8-oxoG:abasic site repair intermediate and targeting them for degradation using unique tools and reagents developed by the PI and co-I. The proposed studies will elucidate the mechanisms that engage with oxidative DNA damage in mitochondria to either repair the damage or target the damaged genome for degradation. Specific Aim 2 builds upon a novel interaction identified between the mitochondrial DNA ligase, DNA ligase IIIa (LigIIIa) , and NDUFAB1, an accessory subunit of complex I of the electron transport chain that provides possible explanation as to why the LigIIIa inhibitor rapidly induces production of mitochondrial superoxide. We will characterize the interaction between mitochondrial LigIIIa and complex I to determine whether LigIIIa has a non-canonical role in complex I function, thereby linking mitochondrial DNA metabolism with oxidative phosphorylation. Cancer and non-malignant cells respond very differently to the dysfunction caused by inhibition of mitochondrial LigIIIa with cancer cells activating an inflammatory cell death pathway whereas non-malignant cells activate mitophagy and pro-inflammatory cell stress pathways. In Specific Aim 3, we will delineate the mechanisms and regulation of the cellular pathways that respond to mitochondrial dysfunction induced by inhibition of mitochondrial LigIIIa in cancer and non-malignant cells. Alterations in mitochondrial function that are usually associated with increased oxidative stress have been identified as the causative factor in certain human metabolic and neurodegenerative diseases and implicated in inflammation, ...