A Transition-State Perspective on Y‑Family DNA Polymerase η Fidelity in Comparison with X‑Family DNA Polymerases λ and β

Deoxynucleotide misincorporation efficiencies can span a wide 104-fold range, from ∼10–2 to ∼10–6, depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our “tool-kit” of dATP- and dGTP-β,γ substrate analogues in which the pyrophosphate leaving group (pKa4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning pKa4 values from 7.8 (CF2) to 12.3 [C­(CH3)2]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log­(kpol) versus pKa4, for Y-family error-prone pol η and X-family pols λ and β to determine the extent to which different electrostatic active site environments alter kpol values. The apparent chemical rate constant (kpol) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show kpol values that are systematically lower than would be predicted from their pKa4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing “effective” pKa4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl