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. 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. To aid in the identification of mitochondrial DNA alterations, we have developed a highly sensitive method, called LostArc, to decipher and interrogate mitochondrial deletions from patient and animal samples. This method can detect 1 deletion event per million mtDNA genomes and is being used on the patas monkey samples to identify and quantitate the level of mtDNA deletions in patas monkeys exposed to anti-HIV analogs as compared to untreated patas monkey samples. In this last year, we have published an improved methods for the isolation of mitochondrial DNA for analysis of mtDNA deletions by LostArc. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA. This improved method will be used on the patas monkey samples treated with the anti-HIV nucleoside analogs.