PROJECT SUMMARY Mitochondria have retained a small circular genome (mtDNA) that encodes essential components of the elec- tron transport chain. Mutations in the mtDNA can cause devastating maternally inherited diseases, while the accumulation of somatic mtDNA mutations is linked to common pathologies. Although mtDNA mutations impact human health, the process(es) that influence their occurrence and level with the cell (i.e., heteroplasmy) remain under considerable debate, despite nearly 30 years of study. Using cutting edge sequencing methods, we have made key contributions to understanding the drivers of mtDNA mutagenesis in a variety of diseases and animal models. This includes the discovery that the types and frequencies of mutations varies considerably between organisms and tissues, evidence that deaminations arising from a single-stranded replication interemediate is the driver of mutations in most vertebrates, and strong evidence that selection of mtDNA is occurring in somatic tissues. Here, we aim to understand the cellular mechanisms that regulate mtDNA heteroplasmy and its intersection with mitochondrial quality control pathways. Testing hypotheses related to heteroplasmy and mtDNA selection has been difficult due to the reliance on bulk sequencing approaches. However, the advent of new methods, such as single-cell and ultra-high accuracy sequencing, has opened up the possibility of answering these questions. We will use a combination of these two technologies to directly assess how mitochondrial quality control mecha- nistically influences the occurrence and accumulation of mtDNA mutations. Specifically, we will pursue three main focus areas: 1) Using a modified form of scATAC-Seq adapted to quantify mtDNA mutations/heteroplasmy, we will perform experiments that will test hypotheses related to if/how mitochondrial quality control (mQC) mechanisms regulate mtDNA heteroplasmy in cells and thereby keeping deleterious mutations from exceeding a phenotypic threshold by small molecule interventions and manipulations genes known to be involved in quality control; 2) We have identified mutationally intolerant sites in mouse mtDNA. We will determine the specific molecular cause behind the presence of these apparent “immutable” sites by using cutting-edge base editing methods and then performing biochemical and in vitro assays to determine the impact of the induced mutation on mitophagy, mtDNA replication, oxidative phosphorylation, and other mitochondrial functions; 3) Develop single-cell ultra-high accu- racy long read sequencing that will allow for the simultaneous accounting of single-nucleotide variants, structural variants, and heteroplasmy. Such technology is needed in order to fully understand the biology of mtDNA mu- tations in disease. The long-term objective of our work is to define the cellular mechanisms that influence the occurrence of mtDNA mutations in the germline and somatic tissues. This work will contribute to an understandin...