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). Human pol gamma readily incorporated all five analogs into DNA but with varying efficiencies. Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) 8805;ddI(didanosine) 8805;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. With the exception of terminally incorporated 3TC, the pol gamma 3-5 exonuclease was inefficient at removing these five analogs from DNA and removal required enzyme levels exceeding substrate concentrations. Even though discrimination against inserting AZT and CBV makes them only moderate inhibitors in vitro, their inefficient excision suggest AZT and CBV may persist in vivo once incorporated into mtDNA by pol gamma. Finally, we found that the exonuclease activity is inhibited by AZT-monophosphate at concentrations known to occur in cells. Thus, although these analogs exert their greatest effect by insertion and chain termination of DNA synthesis, the persistence in DNA and inhibition of proofreading activity may also contribute to mitochondrial toxicity. Recent attention has focused on understanding the health risk and consequence on children exposed to antiretroviral therapy in utero. Antiretroviral therapies based on nucleoside reverse transcriptase inhibitors (NRTIs), like zidovudine (AZT) and lamivudine (3TC), dramatically reduce mother-to-child transmission of the human immunodeficiency virus (HIV). However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mitochondrial DNA (mtDNA) damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT3TC in combination. We previously developed mouse models that emulated NRTI-induced heart damage reported in human infants. Pregnant CD-1 mice were exposed throughout gestation and their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of AZT, 3TC, AZT3TC, or vehicle control. Analyses revealed increases in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC;but not 10-week-old mice, suggesting the global effect disappeared after treatment ceased. Interestingly, ten-week-old females treated with AZT3TC had significant increases in mtDNA damage. Point mutations were elevated in 10-week-old females treated with AZT or AZT3TC, but not 3TC;no increases were seen in either gender at 4 weeks of age. Our data suggest that AZT3TC combination treatment produces greater mtDNA damage than either agent individually, and female mice appear to be more sensitive than males to AZT3TC-induced mtDNA damage. This analysis has been extended to human infants to screen for mtDNA mutations in children that were exposed in utero to antiretroviral therapy. A study was conducted to determine (i) if mitochondrial DNA (mtDNA) polymorphisms and/or mutations were detectable in healthy newborns and (ii) if prepartum AZT-based HIV-prophylaxis was associated with significant increases in mtDNA mutations in uninfected infants born to HIV-1-infected mothers. Thirteen unipolar psoralen-clamped PCR primer sets were designed to yield products with a single uniform melting temperature (61C) across all DNA segments (134 to 419 bases), together spanning 21% of the mitochondrial genome. Denaturing gradient gel electrophoresis analysis of umbilical cord tissue (where endothelial cells are the major source of DNA) showed that mtDNA sequence variants were significantly elevated nearly 4-fold in AZT-treated infants compared with unexposed controls (P<0.001), with 28 changes observed in 20/53 (38%) treated newborns (averaging 0.53 changes/subject) versus only 8 changes found in 7/55 (13%) control newborns (averaging 0.145 changes/subject). Six distinct sequence variants occurring in controls were predominately synonymous and homoplasmic, representing previously reported polymorphisms. Uninfected infants exposed to AZT-3TC and maternal HIV had a shift in mutations driven by increases in non-synonymous heteroplasmic sequence variants at polymorphic sites (10 distinct variants) and novel sites (five distinct variants). The weight of evidence suggests that prepartum AZT-based prophylaxis produces novel mtDNA mutations;nonetheless, additional research is needed to determine the degree to which fetal responses to maternal HIV infection, in the absence of antiretroviral treatment, contributes to prenatal mtDNA mutagenesis. In 2007, Yamanaka et al., reported the identification of the R964C POLG mutation associated with mitochondrial toxicity induced by D4T treatment. We sought to identify the biochemical consequence of this mutation. We utilized a pre-steady state kinetic approach to determine the effect of this mutation on incorporation of natural substrate dTTP and the active metabolite of D4T (D4T-TP). The R964C polymerase γholoenzyme demonstrated a 33% decrease in dTTP incorporation efficiency and 3-fold lower D4T-TP discrimination relative to wild-type polymerase γ, providing a mechanistic basis for genetic predisposition to NRTI toxicity. The highly potent 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA) is a promising new therapeutic for treating HIV infection currently in Phase II clinical trials. Pre-steady state kinetics were used to characterize the interaction of triphosphate form EFdA (EFdATP) with human mitochondrial DNA polymerase gamma to assess the potential for toxicity. Pol gamma incorporates EFdATP 4,300-fold less efficiently than dATP, and the excision rate is similar to that of ddATP. Thus EFdA is a poor substrate for Pol gamma, and Pol gamma-mediated host toxicity should be minimal.

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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
Prasad, Rajendra; Ça?layan, Melike; Dai, Da-Peng et al. (2017) DNA polymerase ?: A missing link of the base excision repair machinery in mammalian mitochondria. DNA Repair (Amst) 60:77-88
Çaglayan, Melike; Prasad, Rajendra; Krasich, Rachel et al. (2017) Complementation of aprataxin deficiency by base excision repair enzymes in mitochondrial extracts. Nucleic Acids Res 45:10079-10088
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
Kent, Tatiana; Rusanov, Timur D; Hoang, Trung M et al. (2016) DNA polymerase ? specializes in incorporating synthetic expanded-size (xDNA) nucleotides. Nucleic Acids Res 44:9381-9392
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
Stumpf, Jeffrey D; Saneto, Russell P; Copeland, William C (2013) Clinical and molecular features of POLG-related mitochondrial disease. Cold Spring Harb Perspect Biol 5:a011395
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

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