Approximately 37 million people are infected by the HIV virus worldwide which approximates to 1 in every 100 adults aged 15 to 49 are infected. Of those infected, 15 million people are receiving antiretroviral therapy. Although antiviral therapy has extended the life of infected individuals, these drugs cause unwelcome side effects by inducing mitochondrial toxicity. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of mitochondrial DNA replication. As many as 20% of patients undergoing AZT treatment develop a mitochondrial muscle myopathy characteristic of red ragged fiber disease, and D4T and ddC cause neuropathy in 15-20% of patients. To prevent vertical transmission from mother to child, approximately 7 out of 10 pregnant women living with HIV receive antiretroviral therapy, despite the unknown life long effects on the child. Increasing evidence now shows that children exposed in utero to NRTI treatment used to prevent vertical transmission from mother to child, are prone to mitochondrial damage and possible future heart problems later in life (Poirier MC et al. Curr Opin Pediatr. 2015 Apr;27(2):233-239). The long-term risk for these children are unknown. The goal of this project should address the human health risks from these iatrogenic agents. 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). 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) = 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. 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. 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. Prior effort to understand the induced mitochondrial toxicity from NRTI has focused on in vitro analysis of the mitochondrial replication proteins. 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. To address the long term exposure in children, the 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 whether 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 utero and pre-natal treatment by HIV antiviral is an effective way to prevent the vertical transmission of HIV infection from mother to child, but evidence is growing that the long-term consequences of this treatment may be harmful to the child as they age. Our current research is addressing the in vivo consequences of NRTI treatment on mitochondrial DNA and mitochondrial function. We hypothesize that NRTIs may induce mitochondrial DNA mutations due to insertional events, with consequence for mitochondrial integrity and bioenergetics. To address the long term effect of in utero exposure, we are collaborating with Miriam Poirier at the NCI to study samples from her past experiment in which patas monkeys were exposed in utero to NRTIs. Using samples collected at birth, 1 and 3 years of age, that were treated in utero and pre-natally with NRTI cocktails, we will analyze the mtDNA for point mutations and deletions. Towards this effort, we have developed a novel and highly-sensitive Next Generation sequencing platform specifically designed to detect mitochondrial DNA point mutations and deletions. The sensitivity of this new NG mtDNA deletion assay can detect one mtDNA deletion event in 1 million mtDNA genomes and surpasses any current sensitivity by three orders of magnitude. This highly-sensitive NG sequencing analysis will help us to understand the post-natal consequences of in utero NRTI therapy in humans. To date, we have generated 4.3 billions sequence reads of mtDNA from 40 patas monkeys and are currently analyzing the results.
| 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 |
| Krasich, Rachel; Copeland, William C (2017) DNA polymerases in the mitochondria: A critical review of the evidence. Front Biosci (Landmark Ed) 22:692-709 |
| 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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