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our “tool-kit” of dATP- and dGTP-β,γ substrate analogues in which the pyrophosphate leaving group (pKa4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning pKa4 values from 7.8 (CF2) to 12.3 [C(CH3)2].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 Bronsted 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 ob...