TY - JOUR
T1 - Selective action of 2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human DNA polymerases
AU - Huang, Peng
AU - Farquhar, David
AU - Plunkett, William
PY - 1992/2/5
Y1 - 1992/2/5
N2 - 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 α (pol α) and ε (pol ε) 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 α and pol ε. 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 α and pol ε was not detectable by the DNA primer extension assay. Pol ε0, 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 ε 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 ε to remove D4TMP from DNA may be related to the cytotoxicity of this compound.
AB - 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 α (pol α) and ε (pol ε) 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 α and pol ε. 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 α and pol ε was not detectable by the DNA primer extension assay. Pol ε0, 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 ε 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 ε to remove D4TMP from DNA may be related to the cytotoxicity of this compound.
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M3 - Article
C2 - 1370834
AN - SCOPUS:0026793636
SN - 0021-9258
VL - 267
SP - 2817
EP - 2822
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -