Biochemical studies of mitochondrial DNA maintenance and topology

Abstract: Mitochondria are crucial organelles in eukaryotic cells that produce the majority of adenosine trisphosphate used by cells as an energy currency to drive metabolic processes. Due to the endosymbiotic origin of mitochondria, they have their own genetic material, a small circular double-stranded molecule (mtDNA) of 16.6 kbp in size that encodes 13 essential subunits of the oxidative phosphorylation system. All other mitochondrial proteins are nuclear encoded, including the components of the dedicated enzymatic machineries used to replicate, transcribe, and translate genetic material. The organelle also contains a large number of factors needed to maintain genome integrity and proper topology. In humans, numerous disease-causing mutations affecting factors required for mtDNA maintenance and expression have been identified. These pathological changes impair mitochondrial function and cause a wide variety of symptoms, primarily affecting high-energy demand organs. In this thesis, we use a combination of in vitro biochemistry and cell biology to study the molecular mechanisms underlying mitochondrial disease associated with disturbed mtDNA maintenance. In mitochondria, topoisomerase 3a (TOP3A) is required to separate newly formed genomes after the completion of mtDNA replication, whereas the nuclear isoform of the same enzyme is required to process Holliday junctions. Here, we demonstrate that TOP3A and TOP1MT are the only topoisomerases with clear mitochondrial localization. The two topoisomerases together control the topological states of mtDNA and are required for proper genome maintenance and expression. We also characterize a number of new disease-causing mutations affecting TOP3A and causing adult-onset mitochondrial diseases. Affected patients present with a common set of phenotypes, which are caused by both the nuclear and mitochondrial function of the TOP3A enzyme. We compared the effects of these mutations with mutations in the TOP3A gene, previously shown to cause Bloom syndrome, a genetic disease characterized by short stature and predisposition to cancer. Our findings suggest a model in which the overall severity of the TOP3A mutations determines the clinical presentations. Mutations with milder effects on TOP3A activity cause adult-onset mitochondrial disease, whereas mutations with more severe consequences are responsible for Bloom-like syndromes in combination with early-onset mitochondrial disease. DNA polymerase γ is the sole polymerase required for DNA replication in human mitochondria. The enzyme is a heterotrimer, with one catalytic subunit (POLγA) and two accessory subunits (POLγB). In our work, we characterize a new disease-causing mutation in POLγA (p.F907I). In its homozygous form, this recessive mutation causes severe disease phenotypes and death in infancy. In our work, we demonstrate that the mutation specifically impairs DNA synthesis on double-stranded DNA templates. In combination with structural modeling, we conclude that F907 is required for the coordinated function of POLγ and the replicative DNA helicase TWINKLE at the replication fork. Our study also suggests that direct interactions involving F907 may be formed between these two enzymes at the active replication fork. Finally, we study EXOG, a mitochondrial nuclease with both endo- and exonuclease activities. We find that EXOG is localized to the mitochondrial intermembrane and therefore cannot be an active component of the mtDNA replication machinery, as previously reported. We demonstrate that EXOG primarily acts on RNA substrates and suggest that EXOG might be involved in RNA degradation during the stress response or apoptosis.