Local Structural Dynamics at the Metal-Centered Catalytic Site of Polymerases is Critical for Fidelity

Abstract

The fidelity of DNA polymerases (Pols) refers to their ability to incorporate the correct nucleotide into the growing strand during DNA synthesis. Each Pol operates with a certain degree of fidelity, from high (∼10-8 ∼1 error in 108 bases) to low (∼10-1 ∼1 error in 10 bases) values. The mechanistic factors behind these differences in fidelity are poorly understood. Here, we show that the formation of the Michaelis-Menten complex is critically affected by the metal-mediated dynamics of local structural features at the catalytic center of Pols. We demonstrated this by integrating recent structural and kinetics data of high-fidelity Pol β and low-fidelity Pol η with equilibrium molecular dynamics and free-energy simulations of paired and mispaired reactant complexes of these Pols. We found that local dynamics at the reaction center determines whether the nucleophile is optimally aligned to incorporate the correct (dCTP) or incorrect (dATP) nucleotide opposite a template deoxyguanosine (dG). In Pol β, local structural distortions at the catalytic site are visible only in the dG:dATP mispair complex, which energetically disfavors incorrect nucleotide addition and thus promotes high fidelity. In contrast, in Pol η we observed a more flexible base pair shape complementarity at the catalytic site. This allows reactive configurations of matched and mismatched complexes to be formed with similar ease, thus explaining the low fidelity of Pol η in line with the experimental evidence. Comparisons with other Pols suggest that these local metal-mediated structural dynamics at the reaction center of the catalytic site are crucial to modulating Pol fidelity.