Müller B, Restle T, Reinstein J, Goody R S
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, FRG.
Biochemistry. 1991 Apr 16;30(15):3709-15. doi: 10.1021/bi00229a017.
Succinylfluorescein-labeled dideoxyTTP has been used as a substrate for reverse transcriptase from HIV-1. On addition to the 3'-end of a primer molecule, there is a reduction of fluorescence yield of a factor of ca. 4. Release of a fluorescent DNA/DNA primer/template duplex from its complex with reverse transcriptase results in a reduction of fluorescence by a further factor of 2. The fluorescent nucleotide is incorporated somewhat less efficiently than 3'-azidoTMP and TMP, which show similar incorporation kinetics. Fluorescent chain-terminated primers have been used to investigate the interaction of normal and chain-terminated primer/template complexes with reverse transcriptase. The dissociation constant of a 36/18-mer was 0.65 nM, whereas that of the same complex after the addition of the fluorescent chain-terminating nucleotide to the primer was 3 nM at 25 degrees C. The rate of dissociation of the latter complex from the enzyme was 0.04 s-1. This was decreased by a factor of ca. 10 at high concentrations (greater than 200 microM) of the nucleotide triphosphate complementary to the next position of the template. The results obtained suggest that potent inhibition of reverse transcriptase activity in in vitro assays results from formation of a slowly dissociating complex between the enzyme and chain-terminated primer/template complexes. However, arguments are presented that lead to the conclusion that this is not the mode of inhibition in cells invaded by HIV. At the prevailing relative concentrations in this situation, chain termination resulting in incomplete transcription is likely to be the major factor.
琥珀酰荧光素标记的双脱氧胸苷三磷酸（ddTTP）已被用作HIV-1逆转录酶的底物。当添加到引物分子的3'-末端时，荧光产率降低约4倍。从其与逆转录酶的复合物中释放出荧光DNA/DNA引物/模板双链体导致荧光进一步降低2倍。荧光核苷酸的掺入效率略低于3'-叠氮胸苷一磷酸（3'-azidoTMP）和胸苷一磷酸（TMP），它们表现出相似的掺入动力学。荧光链终止引物已被用于研究正常和链终止引物/模板复合物与逆转录酶的相互作用。一个36/18-mer的解离常数为0.65 nM，而在引物中添加荧光链终止核苷酸后，相同复合物在室温下的解离常数为3 nM。后一种复合物从酶上解离的速率为0.04 s-1。在与模板下一个位置互补的三磷酸核苷酸高浓度（大于200 microM）时，该速率降低约10倍。所得结果表明，体外试验中逆转录酶活性的有效抑制是由于酶与链终止引物/模板复合物之间形成了缓慢解离的复合物。然而，也有观点认为，这不是HIV感染细胞中的抑制模式。在这种情况下的主要相对浓度下，导致转录不完全的链终止可能是主要因素。
