Huang P, Farquhar D, Plunkett W
Department of Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
J Biol Chem. 1992 Feb 5;267(4):2817-22.
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.
本研究采用DNA引物延伸和测序凝胶分析，比较2′,3′-二脱氢-2′,3′-二脱氧胸苷三磷酸（D4TTP）与3′-叠氮-2′,3′-二脱氧胸苷三磷酸（AZTTP）对从T淋巴母细胞样CEM细胞中纯化的人免疫缺陷病毒逆转录酶（HIV-RT）、人DNA聚合酶α（polα）和ε（polε）介导的DNA链延伸的分子作用。D4TTP优先被HIV-RT掺入延伸的DNA链的T位点，并在掺入位点终止DNA合成。D4TTP的DNA链终止活性与AZTTP相当。相比之下，D4TTP是polα和polε的劣质底物。该类似物被人酶掺入DNA的效率比被HIV-RT掺入的效率低约10000至20000倍，而DNA引物延伸试验未检测到polα和polε对AZTTP的掺入。具有3′→5′外切核酸酶活性的polε无法从DNA链的3′末端去除掺入的2′,3′-二脱氢-2′,3′-二脱氧胸苷单磷酸（D4TMP），而polε从DNA中切除3′-叠氮-2′,3′-二脱氧胸苷单磷酸的速率约为正常脱氧核苷酸切除速率的20%。HIV-RT对D4TTP的优先掺入似乎是D4T选择性抗HIV活性的分子基础，而polε无法从DNA中去除D4TMP可能与该化合物的细胞毒性有关。
