PROJECT SUMMARY Multiple debilitating disorders, such as myopathies, hearing and vision loss, are the result of defects in mitochondrial DNA (mtDNA) replication and transcription. The driving forces behind these essential processes in human mitochondria are two Pol A family polymerases – RNA polymerase mtRNAP and DNA polymerase Gamma, Polg. Structures of the macromolecular machines formed by these enzymes are the main focus of this proposal. We will investigate how mtRNAP recruits transcription factors, recognizes, binds, and melts promoter DNA, and initiates RNA synthesis using single-particle CryoEM. Because transcription generates DNA supercoiling, interactions of the transcription elongation complex with mitochondrial topoisomerase will be probed using function assays, cross-linking, and CryoEM. We will also determine the structure of replicative helicase, TWINKLE, in a complex with a fork template to elucidate the mechanism of DNA strand separation. The structure of a replisome – the complex of Polg with TWINKLE formed on a fork template – will be determined by CryoEM to elucidate the mechanism of mtDNA replication. Replication initiation at replication origin OriL requires an interplay between mtRNAP, which generates ~30 nt replication primer, and Polg. Two polymerases form a primosome complex on the OriL hairpin, the structure of which will be probed by CryoEM. Studies of the structure and function of the transcription and replication machinery are critical for understanding the regulation of mitochondrial genome expression. This, in turn, will determine our ability to influence various mitochondrial functions and, consequently, treat mitochondria-associated diseases. Human mtDNA is highly prone to somatic mutations, including point mutations and deletions, which are detrimental to mitochondrial function and cell viability. It has been found that aging mammals have increased levels of somatic mtDNA mutations, however, the biochemical mechanisms underlying the formation of mtDNA mutations and mtDNA maintenance are poorly understood. Biochemical and structural approaches will be used to fill in the large gap of knowledge associated with DNA repair in human mitochondria. This work will examine the process of proofreading by Polg using CryoEM to determine the structural basis of this mechanism. DNA repair complexes will be reconstituted using Polg and known DNA repair proteins, and their structures determined using CryoEM. Additional interaction partners of Polg that are involved in DNA repair will be identified using pulldown assays and mass spectrometry, and their activity will be probed using functional assays. We will also investigate transcription-coupled repair mechanisms by reconstituting transcription complexes on DNA scaffolds containing UV damage and determine their structure. Factors involved in DNA repair will be identified using pulldown assays from UV-treated cells and their complexes with mtRNAP subjected to CryoEM. The research...