DNA polymerase beta: Structure-fidelity relationship from pre-steady-state kinetic analyses of all possible correct and incorrect base pairs for wild type and R283A mutant

The kinetic parameters (k(pol), K-d(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-stale kinetics. The results led to several significant findings: (i) For both WT and R283A, the fidelity is determined primarily by k(pol) (decreases for the incorporation of incorrect nucleotides) and to a small extent by K-d(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 fur 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 k(pol); the k(pol) 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 K-d(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 K-d(app) Of G . C (8.6 mu M) is distinctly smaller than that of other correct base pairs (41-108 mu M). For the R283A mutant, the k,ol of G C is higher by a factor of 15-17.