Ahn J, Werneburg B G, Tsai M D
Department of Chemistry, Ohio State University, Columbus 43210, USA.
Biochemistry. 1997 Feb 4;36(5):1100-7. doi: 10.1021/bi961653o.
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.
在预稳态动力学分析条件下，测定了野生型（WT）大鼠DNA聚合酶β（polβ）和R283A突变体中A、T、G和C碱基之间所有可能的正确和错误配对的动力学参数（kpol、Kd app）。然后，针对所有12种可能的错配，测定了这两种蛋白质的碱基替代保真度，这是首次使用预稳态动力学对聚合酶进行的完整保真度分析。结果得出了几个重要发现：（i）对于WT和R283A，保真度主要由kpol决定（掺入错误核苷酸时降低），在较小程度上由Kd app决定（掺入错误核苷酸时增加）。（ii）一般来说，Y.X（在模板dYMP对面掺入dXTP）错配的保真度与X.Y错配的保真度不同，这反映了活性位点的不对称性。（iii）与WT相比，R283A在所有12种错配中的保真度均降低。降低程度从A.G错配的200倍到T.C错配的2.5倍不等。一般来说，突变体与WT之间保真度的差异在嘌呤.嘌呤错配（高达200倍）中比在嘌呤.嘧啶、嘧啶.嘌呤或嘧啶.嘧啶错配（高达19倍）中更大。（iv）总体而言，R283A突变体保真度的降低主要是由kpol值的变化引起的；与WT相比，R283A突变体正确掺入的kpol值相对于错误掺入的kpol值下降幅度更大。除了G.C之外，WT和R283A突变体正确配对的Kd app值保持恒定，错误配对的Kd app值变化小于4倍。（v）对于WT polβ，G.C的Kd app（8.6 microM）明显小于其他正确碱基对的Kd app（41 - 108 microM）。对于R283A突变体，G.C的kpol高出15 - 17倍。
