Probing interactions between HIV-1 reverse transcriptase and its DNA substrate with backbone-modified nucleotides

Background: To gain a molecular understanding of a biochemical process, the crystal structure of enzymes that catalyze the reactions involved is extre mely helpful, Often the question arises whether conformations obtained in t his way appropriately reflect the reactivity of enzymes, however, Rates tha t characterize transitions are therefore compulsory experiments for the elu cidation of the reaction mechanism. Such experiments have been performed fo r the reverse transcriptase of the type 1 human immunodeficiency virus (HIV -1 RT). Results: We have developed a methodology to monitor the interplay between H IV-I RT and its DNA substrate. To probe the protein-DNA interactions, the s ugar backbone of one nucleotide was modified by a substituent that influenc ed the efficiency of the chain elongation in a characteristic way. We found that strand elongation after incorporation of the modified nucleotide foll ows a discontinuous efficiency for the first four nucleotides. The reaction efficiencies could be correlated with the distance between the sugar subst ituent and the enzyme, The model was confirmed by kinetic experiments with HIV-1 RT mutants. Conclusions: Experiments with HIV-1 RT demonstrate that strand-elongation e fficiency using a modified nucleotide correlates well with distances betwee n the DNA substrate and the enzyme. The functional group at the modified nu cleotides acts as an 'antenna' for steric interactions that changes the opt imal transition state. Kinetic experiments in combination with backbone-mod ified nucleotides can therefore be used to gain structural information abou t reverse transcriptases and DNA polymerases.