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. Patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease, typically a genetic disease of the mitochondrial DNA resulting from point mutations in the mitochondrial DNA by replication errors from the mitochondrial DNA polymerase. 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 toxicity by antiviral drugs we are analyzing the unique structural features of the mitochondrial DNA polymerase gamma. We have cloned the DNA polymerase gamma genes and cDNA from S. pombe, D. melanogaster and Homo Sapiens. 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. The recombinant human mitochondrial DNA polymerase protein has been functionally overexpressed greater than 100 fold in insect cells by a recombinant baculovirus and in E. coli with and with out the mitochondrial targeting sequence. The overexpressed protein is currently being purified to homogeneity and enzymatically characterized. An exonuclease deficient site specific mutant protein of the human DNA polymerase gamma has been made and overexpressed in baculovirus. Likewise, a dideoxy-resistant DNA polymerase gamma protein has been made by site specific mutagenesis and overexpressed.

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National Institute of Environmental Health Sciences (NIEHS)
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Krasich, Rachel; Copeland, William C (2017) DNA polymerases in the mitochondria: A critical review of the evidence. Front Biosci (Landmark Ed) 22:692-709
Copeland, William C; Kasiviswanathan, Rajesh; Longley, Matthew J (2016) Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase ?. Methods Mol Biol 1351:19-26
Young, Matthew J; Copeland, William C (2016) Human mitochondrial DNA replication machinery and disease. Curr Opin Genet Dev 38:52-62
Copeland, William C; Longley, Matthew J (2014) Mitochondrial genome maintenance in health and disease. DNA Repair (Amst) 19:190-8
Copeland, William C (2014) Defects of mitochondrial DNA replication. J Child Neurol 29:1216-24
Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen et al. (2013) Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase ?. J Biol Chem 288:14247-55
Sohl, Christal D; Singh, Kamlendra; Kasiviswanathan, Rajesh et al. (2012) Mechanism of interaction of human mitochondrial DNA polymerase ? with the novel nucleoside reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine indicates a low potential for host toxicity. Antimicrob Agents Chemother 56:1630-4
Copeland, William C (2012) Defects in mitochondrial DNA replication and human disease. Crit Rev Biochem Mol Biol 47:64-74
Kasiviswanathan, Rajesh; Collins, Tammy R L; Copeland, William C (2012) The interface of transcription and DNA replication in the mitochondria. Biochim Biophys Acta 1819:970-8
Sohl, Christal D; Kasiviswanathan, Rajesh; Kim, Jiae et al. (2012) Balancing antiviral potency and host toxicity: identifying a nucleotide inhibitor with an optimal kinetic phenotype for HIV-1 reverse transcriptase. Mol Pharmacol 82:125-33

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