Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma.
Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K(m) and k(cat) values for insertion of 2',3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2',3'-dideoxy-CTP (ddCTP), 2',3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3' termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.
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Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Copeland
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D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology*; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D000074002:DNA Polymerase gamma; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology; D054306:Dideoxynucleotides; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019259:Lamivudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D018119:Stavudine / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D016047:Zalcitabine / Q000378:metabolism / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate
D019380:Anti-HIV Agents; D003570:Cytidine Triphosphate; D004247:DNA; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D005090:Exodeoxyribonucleases; D019259:Lamivudine; D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; D018119:Stavudine; D013942:Thymine Nucleotides; D016047:Zalcitabine; D015215:Zidovudine; C417268:lamivudine triphosphate; C066928:carbovir triphosphate; D006160:Guanosine Triphosphate; C052873:zidovudine triphosphate