Human Mitochondrial Dna Polymerase With Anti-hiv Nucleotides

National Institute Of Environmental Health Sciences

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Abstract

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 have been documented and characterized in my laboratory. We now know what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to mitochondrial toxicity. We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2-3-dideoxy-TTP (ddTTP), 3-azido-TTP (AZT-TP), 2-3-dideoxy-CTP (ddCTP), 2-3didehydro-TTP (D4T-TP), (-)-2,3-dideoxy-3-thiacytidine (3TC-TP), and carbocyclic 2,3-didehydro-dGTP (CBV-TP). Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) = ddI(didanosine) = D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol gamma utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. We have also identified genetic variants of the mitochondrial DNA polymerase that increases the susceptibility of these NRTI to cause mitochondrial toxicity and identified critical amino acids in the mitochondrial DNA polymerase that allow for insertion of these NRTIs into mitochondrial DNA. To enhance and expand our program we are now addressing the in vivo consequences of NRTI treatment on mitochondrial DNA and mitochondrial function using cell and animal models. Our current goal for this project is to determine the effect of nucleoside reverse transcriptase inhibitors on mitochondrial DNA integrity by looking at cell growth, mtDNA copy number, and if the NRTIs have the ability to cause mtDNA mutations in vivo. A mitochondrial DNA lesion assay and deep sequencing is being used to see if any mutations are present within the mitochondrial DNA and to see what kind of mutations are forming in the mitochondrial DNA. Specifically, HepG2 are chronically treated with a combination of NRTIs and mtDNA assessed for mtDNA integrity and bioenergetics. In collaboration with Miriam Poirier at the NCI, will are evaluating mitochondrial DNA for mutations and deletions from patas monkeys that have been exposed in utero to NRTIs. Pregnant patas monkeys were exposed with human equivalent doses of AZT, 3TC, abacavir and nevirapine. Tissues were collected at birth, 1 and 3 years of age and will be analyzed by next generation sequencing for point mutations and deletions in mitochondrial DNA. This analysis will help us to understand the long term consequences of NRTI treatment on children exposed in utero to antiretroviral therapy.