Multiple deprotonation paths of the nucleophile 3′-OH in the DNA synthesis reaction

Significance To determine if there is a general base in the DNA synthesis reaction that deprotonates the nucleophile, we systematically removed potential hydrogen-bond acceptors of the nucleophile, the 3′-OH of the primer strand. We then characterized the activity of human DNA Pol η by kinetic, structural, and molecular dynamics simulation analyses. We found that no single or combined perturbations eliminate catalysis. Moreover, removal of two potential proton acceptors of the 3′-OH by mutating the conserved S113 to Ala and addition of 2′-F to the primer end rescued the defects of the S113A mutation alone. Our results support that there is no specific general base and the proton is prone to leave the O3′ upon activation by the three Mg2+ ions.