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. We have also studied and reviewed the evidence for additional DNA polymerases that may be imported into the mitochondria and affect mtDNA replication or repair. These other polymerases could be additional targets for ant-AIDS antiviral nucleotide analogs. Since 1970, the DNA polymerase gamma (PolG) has been known to be the DNA polymerase responsible for replication and repair of mitochondrial DNA, and until recently it was generally accepted that this was the only polymerase present in mitochondria. However, recent data has challenged that opinion, as several polymerases are now proposed to have activity in mitochondria. To date, their exact role of these other DNA polymerases is unclear and the amount of evidence supporting their role in mitochondria varies greatly. Further complicating matters, no universally accepted standards have been set for definitive proof of the mitochondrial localization of a protein. To gain an appreciation of these newly proposed DNA polymerases in the mitochondria, we review the evidence and standards needed to establish the role of a polymerase in the mitochondria. Employing PolG as an example, we established a list of criteria necessary to verify the existence and function of new mitochondrial proteins. We then apply this criteria towards several other putative mitochondrial polymerases. While there is still a lot left to be done in this exciting new direction, it is clear that PolG is not acting alone in mitochondria, opening new doors for potential replication and repair mechanisms.
| 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 |
| Young, Matthew J; Copeland, William C (2016) Human mitochondrial DNA replication machinery and disease. Curr Opin Genet Dev 38:52-62 |
| 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 |
| 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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