Effects of 3'-azido-3'-deoxythymidine metabolites on simian virus 40 origin-dependent replication and heteroduplex repair in HeLa cell extracts.
Although the capacity of 3'-azido-3'-deoxythymidine (AZT) triphosphate, an active metabolite of the antiviral agent zidovudine (AZT), to inhibit polymerization by a variety of purified DNA polymerases has been described, it is important to understand its effect on replication by the more complex protein assemblies responsible for DNA replication in human cells. In the present study, we have determined the effects of AZT metabolites on the efficiency of simian virus 40 origin-dependent bidirectional replication of double-stranded DNA in extracts of human HeLa cells. Replication was inhibited by AZT diphosphate and AZT triphosphate, but only at concentrations exceeding those thought to be present in vivo. However, replication was inhibited by AZT monophosphate at concentrations previously reported to accumulate in human cells cultured in the presence of AZT, suggesting that AZT monophosphate may contribute to cytotoxicity by inhibiting chromosomal replication. In an attempt to determine whether AZT treatment could have longer term mutagenic effects on cells, we also determined the effects of these AZT derivatives on replication fidelity and on the efficiency of repair of DNA substrates containing single-base mismatches. Despite the ability of a normal deoxynucleoside monophosphate to reduce the fidelity of DNA replication, presumably by reducing exonucleolytic proofreading of errors, neither the mono-, di-, nor triphosphate form of AZT reduced base substitution fidelity when present in replication reactions. Similarly, the efficiency of repair of DNA substrates containing single-base mismatches was unaffected by these compounds. However, replication fidelity was affected by perturbations in relative and absolute concentrations of deoxynucleoside triphosphate substrates similar to those reported to occur in AZT-treated cells. Thus, AZT treatment could potentially be mutagenic in vivo via reduced replication fidelity resulting from alterations in deoxynucleoside triphosphate pools.
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
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Bebenek
D C
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Thomas
J D
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Roberts
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Eckstein
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Kunkel
D000998:Antiviral Agents / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair / Q000187:drug effects*; D054306:Dideoxynucleotides; D006367:HeLa Cells; D006801:Humans; D013539:Simian virus 40 / Q000187:drug effects / Q000502:physiology; D013942:Thymine Nucleotides / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000494:pharmacology
D000998:Antiviral Agents; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; C064181:3'-azido-3'-deoxythymidine 5'-diphosphate; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C052873:zidovudine triphosphate; D004247:DNA