The first clinically approved anti-viral drug against HIV-1 infection is zidovudine (3'azido thymidine nucleoside, AZT). AZT completely blocks viral replication in normal human peripheral blood mononuclear cells and in vitro AZT-triphosphate (AZT-TP) inhibits purified HIV-1 reverse transcriptase with a ki of 20 nM. In the cell AZT-TP has little inhibitory effect on the nuclear DNA polymerases but selectively targets ands inhibits the mitochondrial DNA polymerase. This inhibition of the mitochondrial DNA polymerase has been documented with patients undergoing antiviral drug treatment. These patients undergoing AZT treatment develop 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, possibly arising through DNA replication errors from the mitochondrial DNA polymerase. How the mitochondrial DNA polymerase makes point and deletions mutations and 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 are analyzing the unique structural features of the mitochondrial DNA polymerase gamma. Based on the homology between the S. cerevisiae DNA polymerase gamma and the bacterial DNA polymerases, we have cloned the DNA polymerase gamma genes and cDNA from S. pombe, D. melanogaster and Homo Sapiens. Alignment of these amino acid sequences with the S. cerevisiae DNA polymerase gamma shows that the S. cerevisiae DNA polymerase gamma differs by having an additional 200 amino acids in the C-terminus. The genes for these mitochondrial DNA polymerases have been mapped to their corresponding chromosomes. Monospecific polyclonal antibodies have been raised against overexpressed polypeptides of the human DNA polymerase gamma. These antibodies recognize and immunoprecipitate a 140 kDa protein from mitochondrial extracts with polymerase gamma like activity.
Showing the most recent 10 out of 26 publications
