Mitochondrial diseases are devastating disorders for which there is no cure, and no proven treatment. About 1 in 2000 children are born each year who will develop mitochondrial disease in their lifetime. Half of these will show symptoms before age 5, and 80% of these will die before age 20. The other half will develop disease between age 5 and 65. Mortality is roughly that of cancer, and can range between 5% to 50% per year after diagnosis. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much research is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genomes, either as point mutations, deletions, rearrangements, or depletion of the mtDNA. The mutation rate of the mitochondrial genome is 10-20 times greater than in the nuclear DNA and more prone to oxidative damage than nuclear DNA. Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase g (pol g), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, the pol g participates in DNA replication and DNA repair. We have cloned, overexpressed, purified, and characterized the human pol g catalytic subunit, p140, and the p55 accessory subunit. We have determined that the accessory subunit functions as a processivity factor and enhances DNA binding of the holoenzyme. We have investigated the fidelity of DNA replication by pol g with and without exonucleolytic proofreading and its p55 accessory subunit. Pol g has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Several nuclear genes involved in mitochondrial maintenance have recently been identified as disease loci for mitochondrial disorders. The pol g gene and the Twinkle gene (DNA helicase) are two genes that when mutated cause progressive external ophthalmoplegia. Additionally, other mutations in the pol g gene are associated with male infertility (causing decrease in sperm motility), Alper's syndrome, sensory ataxia and neuropathy, and Parkinson's disease. We are studying the molecular pathological effects of disease mutations in the pol g and Twinkle gene products. Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mitochondrial DNA (mtDNA). Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase g. Since Y955 is a highly conserved residue critical for nucleotide recognition among Family A DNA polymerases, we previously analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. Recently, 22 new mutations in the pol g gene in several unrelated Italian, Belgium, and Finnish pedigrees were reported to cause heritable PEO, but the functional consequences of these mutations are unknown. Five of these mutations are located in the polymerase domain and cause autosomal dominant PEO. We are analyzing the consequences of these mutations in vitro and in vivo in our group. These mutations are being produced in the cDNA and mutant protein produced in our baculovirus system. The mutant pol g proteins will be purified and characterized for polymerase and exonuclease activities and for fidelity of DNA synthesis. In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an adenosine triphosphate (ATP)-nicotinamide adenine dinucleotide (NADH) kinase, POS5, was identified that functions exclusively in mitochondria. The POS5 gene product was overproduced in E. coli and purified without a mitochondrial targeting sequence. Direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD+, with a 2-fold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA by 50-fold, as measured by the arg8m reversion assay, with no increase in nuclear mutations. Also a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases. Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. We have investigated the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts of carcinogenic diol epoxides (DE) derived from BaP as well as DE-dA adducts from benzo[c]phenanthrene (BcPh) on DNA replication by pol g with and without the p55 processivity subunit. All of the DE adducts caused erroneous purine incorporation as well as significant blockage of further primer elongation. Blockage of translesion synthesis by the BaP and BcPh DE adducts is consistent with known BaP inhibition of mtDNA synthesis and suggests that continued exposure to BaP would reduce mtDNA copy number and compromise the ATP production of the mitochondria.