Mitochondria have their own closed circular genome. Mitochondrial DNA (mtDNA) has a 10 times greater rate of mutation than nuclear DNA. As DNA polymerase gamma is the only known DNA polymerase within the mitochondrion, it is essential for faithful replication, proofreading and repair of mtDNA. The overall goal of this proposal is to understand the fundamental mechanisms of exonuclease activity of DNA polymerase gamma in mtDNA metabolism, and to define its role in disease. Based on the following, we hypothesize that altered exonucleolytic activity of DNA polymerase gamma leads to mitochondrial dysfunction and disease. First, mutations in the exonuclease domain of DNA polymerase gamma cause devastating mitochondrial diseases. Second, polymerase domain mutations in DNA polymerase gamma that cause an error-prone phenotype can be ameliorated by functional exonuclease. Third, mouse models of proofreading-deficient DNA polymerase gamma show enhanced mtDNA mutations and accelerated ageing phenotype with reduced lifespan. Fourth, nucleoside reverse transcriptase inhibitors (NRTIs) used to control HIV infection induce mitochondrial dysfunction by inhibiting DNA polymerase gamma. Mitochondrial diseases can be caused by genetic and environmental factors. Unfortunately, there is no cure or no effective long-term treatment. As more diseases show mitochondrial defects, it is imperative that we understand how defects lead to disease. We will investigate the role of the exonuclease domain of DNA polymerase gamma in maintaining the integrity of mtDNA by:
AIM 1. To investigate the underiying consequences of disease mutations within the exonuclease domain, and how these give rise to mitochondrial dysfunction and disease.
AIM 2. To identify the critical regions within the exonuclease domain that are involved in proofreading, and to determine how efficientiy the exonuclease excises incorporated NRTIs, as well as nucleotides incorrectly incorporated opposite miscoding lesions in the template (such as 8oxoG, a marker of oxidative stress), and environmental DNA lesions (such as those caused by benzo[a]pyrene).
