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 10-fold range, from ∼10 to ∼10, 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using our "tool-kit" of dATP- and dGTP-β,γ substrate analogues in which the pyrophosphate leaving group (p K = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p K values from 7.8 (CF) to 12.3 [C(CH)]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( k) versus p K, 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 k values. The apparent chemical rate constant ( k) 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 k values that are systematically lower than would be predicted from their p K values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p K values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.