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), 1 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.