In higher eukaryotes, mitochondria play multiple roles in bioenergetics, metabolism, and signaling. The mitochondrial DNA (mtDNA) genome is indispensable for mitochondrial function because it encodes protein subunits of the oxidative phosphorylation system and a set of transfer and ribosomal RNAs. mtDNA degradation is an essential mechanism in mitochondrial genomic maintenance and cell signaling. The knowledge regarding the mechanism of mtDNA degradation remains limited, representing a significant knowledge gap. Such knowledge is fundamental to the understanding of mitochondrial genomic maintenance and pathology because mtDNA degradation may contribute to the etiology of mtDNA depletion syndromes and inflammatory and immunological diseases triggered by cytosolic and cell-free mtDNA. The objective of this project is to delineate the chemical and molecular basis of damaged mtDNA degradation by identifying the proteins factors and molecular triggers in mtDNA degradation and characterizing the released mtDNA products. Addressing these critical knowledge barriers will facilitate the PI’s long-term goal of unraveling the basis of mtDNA turnover and its role in mitochondrial pathobiology. The application builds on the PI’s expertise in DNA and protein biochemistry, mechanistic enzymology, and quantitative analysis, and accelerates the progress in an exciting, productive area of research into mitochondrial biology. The proposed research is grounded in progress from the PI’s and other laboratories in the field. Recent research from the PI’s laboratory has provided strong chemical, molecular, and cellular evidence for the involvement of mitochondrial transcription factor A (TFAM) in the degradation of damaged mtDNA containing abasic (AP) sites. AP sites are ubiquitous DNA lesions and central DNA repair intermediates. Enzymology studies from the PI’s laboratory have also provided insights into catalytic and kinetic mechanisms of key proteins in mtDNA maintenance. The proposed research will focus on (i) identifying unknown protein factors in mtDNA degradation using proteomics and siRNA-based approaches, (ii) clarifying the molecular triggers of mtDNA degradation using biochemical and cellular assays, and (iii) characterizing the chemical and molecular properties of fragmented mtDNA using mass spectrometry-based methods. The expected outcome is that the results from this research will provide new insights into the molecular basis of mtDNA degradation and shed light on the characteristics of subsequent mtDNA products. The project is significant because it addresses a critical barrier in the field by providing fundamental knowledge at the molecular level. In addition, the PI’s commitment to enhancing diversity in the biomedical workforce further the significance at a minority- and Hispanic-serving institution. Considering the importance of mtDNA in cell signaling and innate immunity, new insights into mtDNA degradation will not only advance the understanding of...