Structural studies of mitochondrial DNA polymerase γ

Abstract

Mitochondria are essential eukaryotic organelles that generate most of the cell’s adenosine triphosphate (ATP), the energy currency used to power cellular activities. Because mitochondria are descendants of once free-living bacteria that formed an endosymbiotic relationship with an archaeal host cell, mitochondria contain a small but well-preserved genome. Mitochondrial DNA (mtDNA) encodes 13 proteins that are crucial for ATP production, and proper maintenance of this genome is therefore essential for the cell. mtDNA replication is carried out by DNA polymerase γ (POLγ), a heterotrimeric complex composed of the catalytic subunit POLγA (POLG) and the accessory dimer POLγB (POLG2). Pathogenic POLG variants are among the most common causes of inherited mitochondrial disease, yet the underlying mechanisms remain poorly defined, and no effective therapies exist. This thesis integrates biochemical analysis, cryogenic electron microscopy (cryo-EM), cell assays, and mouse models to expand the mechanistic understanding of POLγ function and dysfunction. In Paper I, we identified small molecules that can restore polymerization activity in mutant POLγ complexes, both in vitro and in patient-derived fibroblasts. Our findings position these compounds as potential therapeutic candidates for POLG-related disease. In Paper II, we generated and characterized mouse models to study common disease-causing POLG variants. In vitro, mouse Polγ displays greater catalytic efficiency than the human enzyme, which results in milder phenotypes in mice. This observation is in part due to a more potent mouse accessory subunit, and our findings establish POLγB as a critical determinant of phenotypic severity in POLG mouse models. In Paper III, we combined cryo-EM and biochemical assays to elucidate how the small-molecule modulators identified in Paper I allosterically activate POLγ by stabilizing it in the polymerase state. Collectively, these studies provide important mechanistic insight into POLγ function and dysfunction, establish characterized mouse models, and lay the foundation for developing targeted therapies to treat mitochondrial disorders caused by POLG mutations.