The Effect of Magnesium Ion Concentration on the Nucleotide Specificity and Fidelity of HIV-1 Reverse Transcriptase

Like other DNA polymerases there are two Mg2+ bound to the polymerase site of HIV-1 reverse transcriptase (HIVRT) and they are thought to facilitate the incorporation of normal nucleotides through a classical two metal ion mechanism. However, the effect of Mg2+ on the fidelity of HIVRT and role of each Mg2+ on the steps governing nucleotide incorporation are unknown and although most in vitro measurements have been performed using 6 -10 mM Mg2+, it is reported that the physiological concentration of free Mg2+ may be as low as 0.25 mM. In this study, we investigated the effects of Mg2+ on nucleotide specificity by defining the kinetic parameters governing each step involved in the normal nucleotide in the incorporation pathway; namely, ground-state binding, substrate-induced conformational change, substrate release, chemistry and pyrophosphate release. One Mg2+ is chelated by the nucleotide, which binds to the enzyme as a Mg-dNTP2- complex, while the second Mg2+ binds in a separate step. Surprisingly, Mg2+ concentrations ranging from 0.25 to 10 mM did not affect the kinetics of nucleotide binding or the rate of the conformational change. Rather, the main effect of free Mg2+ is on the rate of the chemistry step. These results demonstrate that the second Mg2+ binds after the conformational change and with an apparent Kd of 4.7 mM. We also measured the fidelity of HIV-1 RT under various concentrations of Mg2+ and show that Mg2+ does not significantly affect the fidelity at concentrations ≥ 0.25 mM. We also examined the effect of Mg2+ concentration on the kinetics of DNA binding. Overall, the results from our study provide valuable information regarding magnesium concentration and nucleotide specificity.