of Work: The first clinically approved anti-viral drug against HIV-1 infection is zidovudine (3azido thymidine nucleoside, AZT). In the cell AZT-TP has little inhibitory effect on the nuclear DNA polymerases but selectively targets ands 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. To investigate the sensitivity of the human DNA polymerase to other clinically approved anti-AIDS nucleotide analogs we have produced a panel of site directed alterations in amino acids with in the active site. These amino acids were chosen based on X-ray crystallographic structure of the T7 DNA polymerase and E. coli DNA polymerase I and are believed to make contacts with the incoming nucleotide. We changed Tyr951 to Phe and Ala, Tyr955 to Phe and Ala, and Glu895 to Ala. The mutant polymerases have all been overproduced and purified to homogeneity. This panel of mutant polymerases are being screened for their sensitivity to AZT-TP, 3TC-TP, D4T-TP and ddC-TP and compared to wild type enzyme. 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. - Antiviral Agents, Dideoxynucleotides, DNA Polymerase III, HIV, Mutagenesis, Merrf Syndrome
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