DNA Polymerase β: Structure−Fidelity Relationship from Pre-Steady-State Kinetic Analyses of All Possible Correct and Incorrect Base Pairs for Wild Type and R283A Mutant

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Abstract

The kinetic parameters (kpol, Kd app) for all possible correct and incorrect pairing between the A, T, G, and C bases were determined for wild-type (WT) rat DNA polymerase beta (pol beta) and the R283A mutant under pre-steady-state kinetic assay conditions. The base substitution fidelities of these two proteins were then determined for all 12 possible mispairs representing the first complete fidelity analysis of polymerases using pre-steady-state kinetics. The results led to several significant findings: (i) For both WT and R283A, the fidelity is determined primarily by kpol (decreases for the incorporation of incorrect nucleotides) and to a small extent by Kd app (increases for the incorporation of incorrect nucleotides). (ii) In general, the fidelity for the Y.X (incorporation of dXTP opposite template dYMP) mismatch is different from that for the X.Y mismatch, reflecting the asymmetry of the active site. (iii) The fidelity of R283A is reduced in all 12 mispairs compared to that of WT. The extent of decrease varies from 200-fold for the A.G mispair to 2.5-fold for the T.C mispair. In general, the differences in fidelity between the mutant and WT are greater for purine.purine mismatches (up to 200-fold) than purine.pyrimidine, pyrimidine. purine, or pyrimidine.pyrimidine mismatches (up to 19-fold). (iv) Overall, the decreases in the fidelity of the R283A mutant are caused mainly by changes in the values of kpol; the kpol values of correct incorporations decrease to a greater extent for the R283A mutant with respect to WT than those of incorrect incorporations. With the exception of G.C, the values of Kd app for the WT and R283A mutant remain constant for correct pairings and vary by less than a factor of 4 for incorrect pairings. (v) For WT pol beta, the Kd app of G.C (8.6 microM) is distinctly smaller than that of other correct base pairs (41-108 microM). For the R283A mutant, the kpol of G.C is higher by a factor of 15-17.

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