HUMAN MITOCHONDRIAL DNA POLYMERASE WITH ANTI-HIV NUCLEOTIDES

Environmental Health Sciences

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Abstract

Summary of Work: There are more than 33.6 million people infected by the HIV virus worldwide with 5.6 million new cases expected this year. 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. 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 based on X-ray crystallographic structure of the T7 DNA polymerase and E. coli DNA polymerase I. These amino acids 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 purified mutant polymerases have been screened for their sensitivity to AZT-TP, 3TC-TP, D4T-TP, ddC-TP, and Carbovir and compared to wild type enzyme. The removal of these analogs from the 3? end of DNA by the DNA polymerase gamma proofreading function has also been tested. We show that removal of these analogs from DNA is extremely inefficient.