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

Abstract

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg 2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg 2+ 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 Mg 2+ concentration. Subsequently, the second Mg 2+ binds to the closed state of the enzyme–DNA–Mg.dNTP complex ( K d = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg 2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg 2+ concentrations. An increase of the Mg 2+ concentration from 0.25 to 10 mM increases nucleotide specificity ( k cat / K m ) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg 2+ binds very weakly ( K d ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg 2+ . Molecular dynamics 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 in the specificity of DNA polymerases.

Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication

“…In our preliminary studies, we discovered that Mg 2+ has a large effect on the binding and partitioning kinetics for various substrates, and the fractions in each site in the absence of Mg 2+ versus the fraction that binds from solution for this experiment varies greatly (data not shown). Because Mg 2+ has large effects on DNA binding ( 20 ), the fraction of DNA in each site in the absence of Mg 2+ may not be physiologically relevant. However, this experiment provides important constraints for the next stopped-flow experiment that provides additional information on the rates of transfer between sites.…”

Catalytic Magnesium as a Door Stop for DNA Sliding

“…The magnesium ions are critical for the chemical step . Recent study considered the diffusion of magnesium into the closed state . However, the desolvation step, necessary for the binding, was not examined.…”

“…Events of magnesium removal did not occur in our simulations. Mg 2+ diffusion to the active site in the closed state was explored computationally . However, the slow event of magnesium solvation or desolvation was not examined.…”

Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity

“…This mechanism seems to be adopted by enzymes that are mainly involved in DNA repair. The role of the third ion in catalysis is still a matter of dispute [ 93 ]. Also, a recent, detailed analysis of high-resolution crystal structures of PolA-type Geobacillus stearothermophilus DNA polymerase I intermediates allowed the capture of new conformations for the enzyme translocation and nucleotide pre-insertion step that underlies the conformational dynamic nature of enzymatic DNA synthesis [ 94 ].…”