Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

Magnesium ions play a critical role in catalysis by many enzymes and they contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ions in each step in catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. Increasing Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold by largely increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV RT binds only two metal ions prior to incorporation of a correct base-pair. MD simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. MD simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions the specificity of DNA polymerases.