Investigating the Cellular Effects of Mitochondrial DNA Damage Using Targeted Chemical Probes

Mitochondria are compartments within eukaryotic cells that produce energy to power cellular metabolism. Uniquely amongst mammalian organelles, mitochondria contain a small amount of their own genetic material (mtDNA), the proper maintenance and expression of which is essential for mitochondrial function. Mitochondrial DNA damage has been linked to several pathophysiological processes, but the cellular effects of mtDNA damage remain poorly understood. In order to better understand mtDNA damage, our group has pioneered the development of selective mitochondria-targeted DNA damaging agents using positively charged, hydrophobic delivery vectors known as mitochondria-penetrating peptides (MPPs). In my first study, an analogue of the commonly used anticancer drug cisplatin was conjugated to an MPP, yielding a mitochondria-targeted platinum DNA-damaging agent (mtPt) that induced specific damage to mtDNA. Using mtPt, I showed that selective mtDNA platinum damage induced apoptotic cell death associated with reactive oxygen species production, demonstrating that mtDNA damage is sufficient to mediate the cytotoxic activity of a platinum drug. In another work, I aimed to use mitochondria-targeted DNA damaging agents in a screen to characterize novel factors in mtDNA replication and repair. By knocking down expression of known DNA repair genes, and monitoring synergistic effects of gene knockdown on the activity of an mtDNA-oxidizing agent, I identified a series of genes with novel roles in mtDNA repair and replication. Included amongst these hits was DNA Polymerase θ (Polθ), a novel mtDNA polymerase. In a further study, I found that Polθ plays an essential role in facilitating mtDNA replication under conditions of oxidative stress. Finally, I conducted a genome-wide CRISPR screen to comprehensively identify genes whose knockout sensitizes cells to mitochondria-localized DNA damage. The hits of this screen were heavily enriched for genes involved in mitochondrial translation, implying the importance of mitochondrial protein synthesis in mediating an adaptive response to mtDNA damage. These studies demonstrate the application of mitochondria-targeted DNA damaging agents to the study of a wide range of significant areas in mitochondrial biology. Going forward, my thesis work shows mtDNA-damaging chemical probes to be essential tools for achieving better understanding of mtDNA damage in health and disease.Ph.D.2018-12-19 00:00:0