Fidelity of nucleotide incorporation by the human mitochondrial DNA polymerase

The human mitochondrial DNA polymerase (pol γ) is a nuclearly encoded enzyme, imported to the mitochondria, solely responsible for the replication of the mitochondrial genome. I have characterized the kinetics of nucleotide incorporation, determined the discrimination constants for misincorporation, and calculated the overall fidelity of this enzyme. Additionally, I have investigated the dependence of these parameters on the concentration of magnesium ion present in the reaction. There have been reports in the literature of pol γ having reverse transcriptase activity, and of attempts to determine a physiological role for this activity. Indeed, many steady-state kinetic assays of this enzyme reported in the literature are reverse transcriptase assays. I have characterized the kinetics of incorporation of the reverse transcriptase activity of polymerase γ, both in single turnover and processive polymerization assays. Additionally, I have characterized the activity of the 3′-5′ exonuclease domain on a DNA/RNA heteroduplex. For many years there has been research into the factors that contribute to polymerase discrimination. Base pair hydrogen bonding, base stacking, steric interactions, active site tightness, and factors unknown are all believed to play a role. However, recent attempts to investigate the contribution to discrimination afforded by base pair hydrogen bonding in Klenow fragment, using “shape mimic” nucleoside analogs have led to the supposition that base pairing plays little, if any, role in discrimination. In order to investigate the contribution of base pair hydrogen bonding to discrimination in pol γ, I have characterized the incorporation of natural nucleotides opposite the dT analog 2, 4-difluorotoluene deoxynucleoside (dF) and the dA analog 9- (1-aza-4-methyl-benzimidazolyl)-1′-β-2′-deoxyriboside (dQ). Additionally, the kinetic parameters of incorporation of dF opposite dT, and of dQ and 4-methylbenzimidazole (dZ) opposite dA have been determined. The rates of 3′-5′ exonuclease removal of natural nucleosides paired opposite dF and dQ have been determined as well. The mitochondrial polymerase is the sole enzyme responsible for replication of the mitochondrial genome. In the absence of accurate and efficient replication by polymerase γ, several clinical pathologies are observed including cardiac and neural myopathy, mitochondrial myopathy, anemia, and potentially fatal lactic acidosis. These symptoms are known effects of oxidative damage to the mitochondrial genome and of toxicity associated with the treatment of HIV infection with nucleotide reverse transcriptase inhibitors. Additionally, several diseases have been associated with mitochondrial genome mutation and depletion including Parkinson’s disease and Alzheimer’s disease. A full understanding of the fidelity of the mitochondrial DNA polymerase and the mechanisms by which this fidelity is insured will aid in the understanding of diseases associated with mitochondrial damage and in the design of drugs used to fight HIV, lacking the potentially fatal mitochondria based toxicities.