Mismatched and matched dNTP incorporation by DNA polymerase beta proceed via analogous kinetic pathways.
Mismatched and matched dNTP incorporation by DNA polymerase beta proceed via analogous kinetic pathways.
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DOI：
10.1021/bi800689d
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发表时间：
2008-09-16
期刊：
影响因子：
2.9
通讯作者：
Tsai, Ming-Daw
中科院分区：
文献类型：
--
作者：
Roettger, Michelle P.;Bakhtina, Marina;Tsai, Ming-Daw
虽然DNA聚合酶β (Pol β)的匹配核苷酸结合已经得到了很好的研究，但要真正理解聚合酶的保真度，需要比较匹配和不匹配的dNTP结合途径。在这里，我们使用停止流动荧光测定和稳态荧光光谱研究了野生型（WT） Pol β和易出错的I260Q变体的错误掺入机制。在停流中，在不匹配的dNTP掺入过程中观察到两种酶的双相荧光痕迹。荧光跃迁的方向与观察到的匹配dNTP相同，尽管振幅较低。快速和慢速荧光相的分配被指定为先前确定的匹配dNTP结合的相同机制步骤。对于WT和I260Q错配的dNTP合并，反映子域关闭的快速相位速率与正确dNTP诱导的速率相当。预稳态动力学评估表明，这两种酶具有相似的正确dNTP插入谱，I260Q突变体固有的较低保真度是由于不匹配插入效率的提高。值得注意的是，与WT相比，I260Q在错配的三元配合物形成时荧光发射强度增强。动力学和稳态荧光数据都表明，I260Q对不正确的dNTP的宽松歧视是失去失稳不匹配三元配合物的能力的结果。总的来说，我们的结果提供了第一个直接证据，证明不匹配和匹配的dNTP结合是通过类似的动力学途径进行的，并支持了我们的假设，即Pol β的保真度源于不匹配的封闭三元配合物和化学过渡态的不稳定。
While matched nucleotide incorporation by DNA polymerase β (Pol β) has been well-studied, a true understanding of polymerase fidelity requires comparison of both matched and mismatched dNTP incorporation pathways. Here we examine the mechanism of misincorporation for wild-type (WT) Pol β and an error-prone I260Q variant using stopped-flow fluorescence assays and steady-state fluorescence spectroscopy. In stopped-flow, a biphasic fluorescence trace is observed for both enzymes during mismatched dNTP incorporation. The fluorescence transitions are in the same direction as that observed for matched dNTP, albeit with lower amplitude. Assignments of the fast and slow fluorescence phases are designated to the same mechanistic steps previously determined for matched dNTP incorporation. For both WT and I260Q mismatched dNTP incorporation, the rate of the fast phase, reflecting subdomain closing, is comparable to that induced by correct dNTP. Pre-steady-state kinetic evaluation reveals that both enzymes display similar correct dNTP insertion profiles, and the lower fidelity intrinsic to the I260Q mutant results from enhanced efficiency of mismatched incorporation. Notably, in comparison to WT, I260Q demonstrates enhanced intensity of fluorescence emission upon mismatched ternary complex formation. Both kinetic and steady-state fluorescence data suggest that relaxed discrimination against incorrect dNTP by I260Q is a consequence of a loss in ability to destabilize the mismatched ternary complex. Overall, our results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol β originates from destabilization of the mismatched closed ternary complex and chemical transition state.
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