of Work: The first clinically approved anti-viral drug against HIV-1 infection is zidovudine (3'azido thymidine nucleoside, AZT). In the cell AZT-TP has little inhibitory effect on the nuclear DNA polymerases but selectively targets and inhibits the mitochondrial DNA polymerase. Inhibition of the mitochondrial DNA polymerase has been seen in patients undergoing AZT treatment and gives rise to a mitochondrial dysfunctional disease known as red ragged fiber disease. Red-ragged fiber disease usually is a genetic disease of the mitochondrial DNA resulting from point mutations in the mitochondrial DNA. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. In addition, the mode and effect of antiviral nucleotide analogs, such as AZT, on the inhibition and fidelity of mitochondrial DNA replication is poorly understood. To better understand the mechanism of mitochondrial DNA replication and mitochondrial toxicity of antiviral drugs we have cloned and functionally overexpressed the cDNA for the human mitochondrial DNA polymerase in the baculovirus system. The overexpressed recombinant pol gamma and two mutant derivatives were purified to homogeneity and enzymatically characterized for inhibition by antiviral analogs. Changing Tyr951 to Phe in the DNA polymerase gamma rendered the enzyme 5000 times more resistant to dideoxynucleotides with only a minor effect on polymerase activity. Structural modeling suggests that the hydroxyl group of the Tyr is necessary to stabilize the incoming nucleotide (dideoxy or deoxy) by hydrogen bonding to the beta-phosphate oxygen. We are collaborating with Dr. Bill Lewis (Cincinnati) to study the overexpression of the wild type and mutant DNA polymerase gamma proteins in a mouse transgenic system which expresses the cDNA of interest in a tissue specific manner.
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