Selective action of 2',3'-didehydro-2',3'-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human DNA polymerases.
This study used DNA primer extension and sequencing gel analyses to evaluate the molecular action of 2',3'-didehydro-2',3'-dideoxythymidine triphosphate (D4TTP), in comparison with 3'-azido-2',3'-dideoxythymidine triphosphate (AZTTP), on DNA strand elongation by human immunodeficiency virus reverse transcriptases (HIV-RT) and human DNA polymerases alpha (pol alpha) and epsilon (pol epsilon) purified from T-lymphoblastoid CEM cells. D4TTP was preferentially incorporated into the T sites of the elongating DNA strand by HIV-RT and terminated DNA synthesis at the incorporation sites. The DNA chain termination activity of D4TTP was equipotent to that of AZTTP. In contrast, D4TTP was a poor substrate for pol alpha and pol epsilon. The analogue was incorporated into DNA by the human enzymes about 10,000- to 20,000-fold less efficiently than by HIV-RT, whereas the incorporation of AZTTP by pol alpha and pol epsilon was not detectable by the DNA primer extension assay. Pol epsilon, an enzyme with 3'----5'-exonuclease activity, was unable to remove the incorporated 2',3'-didehydro-2',3'-dideoxythymidine monophosphate (D4TMP) from the 3'-end of the DNA strand, whereas 3'-azido-2',3'-dideoxythymidine monophosphate was excised from DNA by pol epsilon at about 20% of the rate for normal deoxynucleotide excision. The preferential incorporation of D4TTP by HIV-RT appears to be a molecular basis for the selective anti-HIV activity of D4T, whereas the inability of pol epsilon to remove D4TMP from DNA may be related to the cytotoxicity of this compound.
Department of Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
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P
Huang
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D
Farquhar
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W
Plunkett
D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006678:HIV / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000494:pharmacology*; D015215:Zidovudine / Q000494:pharmacology; D018119:Stavudine / Q000031:analogs & derivatives
D000998:Antiviral Agents; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D019384:Nucleic Acid Synthesis Inhibitors; D012194:RNA-Directed DNA Polymerase; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D015215:Zidovudine; C053197:stavudine triphosphate; D018119:Stavudine