PROJECT SUMMARY High mutation rates in mitochondrial DNA (mtDNA) are a major cause of inherited and age-related diseases. There is a longstanding view that the mutation-prone nature of mitochondrial genomes in humans and animal models is a byproduct of the intense metabolic activity associated with oxidative phosphorylation and energy conversion. However, recent evidence has challenged this idea, and there is growing recognition that differences in the enzymatic machinery responsible for mtDNA replication and repair are a key cause of high mutation rates in human mtDNA. Variation in mtDNA replication and repair pathways may also explain why some eukaryotic lineages (e.g., plants) are able to suppress mutation rates in their mitochondrial genomes to exceptionally low levels. An overarching theme of our research program is to identify the mechanisms responsible for variation in mitochondrial mutation rates across eukaryotes and thereby help resolve the long- term uncertainties about why rates are so high in humans. To overcome the technical challenges associated with investigating rare events like de novo mutations, we have applied multiple innovative sequencing technologies that can detect new DNA sequence variants present at ultra-low frequencies, essentially capturing mutations and damaged bases as they occur and mapping them to nucleotide-level resolution. Our future work will address two major questions. First, how do plant mitochondria achieve some of the lowest rates of point mutations ever observed (less than one substitution per site per billion years)? This line of investigation will build off our recent discovery that plant-MSH1 is necessary for maintaining low mutation rates in plant organelle genomes. MSH1 is enigmatic member of the MutS gene family which was likely acquired by horizontal transfer from giant viruses. We will test the hypothesis that it is part of a novel mechanism of mismatch repair that induces double-stranded breaks followed by template-based recombinational repair. Second, how do mitochondria repair bulky DNA damage introduced by UV? We will test the hypothesis that mitochondria contain a previously unrecognized repair pathway with similarities to classic nucleotide excision repair (NER). This hypothesis is motivated by our recent observation that exposure of divergent eukaryotic model systems to UV light results in the release of mtDNA-derived oligonucleotides that carry damaged bases at consistent positions and exhibit characteristic length profiles, a hallmark of NER. Overall, this research program will help determine why mitochondrial genomes exhibit such extreme mutation rate variation across the eukaryotic tree of life.