of Work: There are more than 36 million people infected by the HIV virus worldwide where 5.3 million new infections occurred during 2000. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 2.6 million people, the highest number since the epidemic began, died from AIDS this year. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As much as 40% of patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease. The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. We previously evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase gamma (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. The structural elements responsible for this inhibition and the amino acids that interact with these analogs are unknown. In efforts to understand why pol gamma is so sensitive to antiviral nucleotide analogs, we have changed three key amino acids believed to make contacts with the incoming nucleotide. These changes were based on the X-ray crystallographic structure of T7 DNA polymerase and E. coli DNA polymerase I. We changed Tyr951 to Phe and Ala, Tyr955 to Phe and Ala, and Glu895 to Ala. These overproduced, purified mutant gamma polymerases are being screened for their sensitivities to the antiviral nucleotide analogs AZT-TP, 3TC-TP, D4T-TP, ddC-TP, and carbovir and compared with wild-type pol gamma. Additionally, steady state kinetic analysis and DNA binding properties are being determined for these mutant pol gamma proteins. Incorporation of antiviral nucleotides is being assayed using oligonucleotide substrates as previously described.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES065080-08
Application #
6673218
Study Section
(LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2002
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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 (2014) Defects of mitochondrial DNA replication. J Child Neurol 29:1216-24
Copeland, William C; Longley, Matthew J (2014) Mitochondrial genome maintenance in health and disease. DNA Repair (Amst) 19:190-8
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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