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.
Huang
P
P
Department of Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Farquhar
D
D
Plunkett
W
W
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