Refined model for primer/template binding by HIV-1 reverse transcriptase: Pre-steady-state kinetic analyses of primer/template binding and nucleotideincorporation events distinguish between different binding modes dependingon the nature of the nucleic acid substrate

The kinetic mechanism of nucleic acid substrate binding and nucleotide inco rporation by human immunodeficiency virus type 1 reverse transcriptase (HIV -1 RT) was analysed using synthetic DNA/DNA and DNA/RNA primer/templates (p /t) without predicted secondary structures in the single-stranded region. D etermination of the pre-steady-state kinetics of p/t binding by a combinati on of stopped-flow and quench flow methods indicate a branched binding mech anism for the HIV-1 RT/nucleic acid interaction. Analysis of p/t-RT associa tion by stopped-flow measurements suggest a three-step binding mode with an initial second-order step followed by two isomerisation steps with rates o f about 6 s(-1) and 0.5 s(-1) respectively. Determination of the rate-limit ing step of the association process via single turnover, single nucleotide incorporation analysis by quench flow measurements revealed two binding eve nts (the initial second-order step cannot be detected with this experimenta l set-up) with rates of 4 - 7 s(-1) and 0.4 - 0.7 s(-1), respectively, indi cating that both binding events exist in parallel. Thorough pre-steady-stat e analysis of single turnover, single nucleotide incorporation kinetics sho wed that dNTP incorporation occurs with a biphasic exponential burst follow ed by a linear phase. The exponential burst consists of a fast: phase with rates of 20 - 60 s(-1) and a slow phase with rates of 0.5 - 2 s(-1), respec tively. The relative distribution of these two burst amplitudes differs sig nificantly depending upon which substrate is used. The DNA/RNA-RT complex s hows primarily fast incorporation (>80 %) whereas less than 45 % of the DNA /DNA-RT complex incorporate dNTP rapidly. The same relative distribution of amplitudes concerning the two substrates is also found for the association process of RT and p/t. Analysis of dNTP incorporation of the preformed RT- p/t complex in the presence of a nucleic acid competitor shows no effect on the biphasic burst amplitude, however the linear phase disappears. Here, a refined model of the mechanism of RT-p/t binding is presented which is bas ed on the suggestion that two different: RT-p/t complexes are formed, i.e. a productive enyme/substrate ate complex which is capable of nucleotide inc orporation and a non-productive complex which has to undergo an isomerisati on before cNTP incorporation can occur. In addition, binding of RT to its s ubstrate can lead to a dead end complex that is not capable of dNTP incorpo ration. (C) 1999 Academic Press.