6th International AIDS Conference San Francisco, California, USA — June 20-23, 1990

Int Conf AIDS 1990 Jun 20-23; 6:316 (abstract no. 1011)
Muller B, Restle T, Goody RS; Abteilung Biophysik, Max-Planck Institute fur medizinische Forschung, Heidelberg, FRG

Succinyl-fluorescein ddTTP can be used to terminate template-directed oligonucleotide extension by reverse transcriptase. This occurs with a kcat/Km value of ca. 10(4) M(-1)s(-1) at 25 degrees C and leads to a 75% quenching of fluorescence of the terminator. Dissociation of the fluorescent primer/template occurs at a rate of 6x10(-3) s(-1) and leads to a further quenching of ca. 50%. Titration of a complex between template, fluorescent primer and reverse transcriptase with the triphosphate of the nucleoside which would have been incorporated next (in the absence of chain termination) leads to a large increase in fluorescence (ca. 150%, depending on conditions) and a dissociation constant for dGTP of 20 muM. In contrast, deoxynucleoside monophosphates bind in a base specific manner with a large decrease in fluorescence (dissociation constant for dGMP 50 muM). Binding of dGTP and dGMP appears to be partially competitive. The affinity of the fluorescent primer/template complex used (??) for reverse transcriptase was 4.1 nM in the absence of dGTP and 1.2 nM at dGTP concentrations which are saturating for its specific binding site. A significant but relatively small reduction in the rate of the primer/template complex was only seen at much higher dGTP concentrations (1.4x10(-3) s(-1) at 500 muM), suggesting that at normal substrate concentrations a "dead end" complex between template, terminated primer, reverse transcriptase and a deoxynucleoside triphosphate does not occur.

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