of Work: Human mitochondrial DNA evolves faster and is more prone to oxidative damage than is nuclear DNA. Point mutations and deletions in this mitochondrial genome give rise to a wide range of mitochondrial dysfunctional diseases affecting some 50 million people in the US. These mutations may occur during replication by the DNA polymerase gamma. The DNA polymerase gamma differs from the nuclear DNA polymerases due to its sensitivity to antiviral nucleotide analogs, such as AZT and dideoxynucleotides. How the mitochondrial DNA polymerase makes point and deletion mutations and what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns are poorly understood. To address these questions we previously cloned the DNA polymerase gamma genes and cDNA from S. pombe, D. melanogaster and Homo Sapiens. The recombinant human mitochondrial DNA polymerase gamma protein has been functionally overexpressed greater than 100 fold in insect cells by a recombinant baculovirus and in E. coli. The recombinant pol gamma and two mutant derivatives were purified to homogeneity and enzymatically characterized. Using a exonuclease deficient mutant we are assessing the contribution of the proofreading and nucleotide selection. With the Wilson group we have discovered a new activity, a 5?-deoxyribose 2?- phosphatase activity intrinsic to the catalytic subunit of human DNA polymerase gamma. The implication of this in mitochondrial base excision repair is being unraveled. The putative cDNA for the second subunit of the DNA polymerase gamma was cloned, overexpressed in E. coli and baculovirus, and protein purified. We have developed a yeast genetic screen to search for new genes involved in maintaining the integrity of the mitochondrial genome. Using this screen we have identified a gene involved in copper transport that increases the mutation rate of mitochondrial DNA. The characterization of this gene and its involvement in mitochondrial DNA metabolism is being further investigated.
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