Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid.
Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid.
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DOI：
10.1016/j.jbc.2021.101451
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Johnson KA
中科院分区：
文献类型：
--
作者：
Dangerfield TL;Kirmizialtin S;Johnson KA
高保真 DNA 聚合酶以极高的特异性选择正确的核苷酸，而不是结构相似的错误核苷酸，同时保持快速的掺入率。先前的分析揭示了使用含有荧光非天然氨基酸的高保真 DNA 聚合酶变体控制正确核苷酸掺入的构象动力学和完整动力学途径。在这里，我们扩展了该分析以研究核苷酸错误掺入和错配延伸的动力学。我们报告了所有可能的错误掺入的特异性常数，并表征了错误掺入和错配延伸过程中酶的构象动力学。我们展示了基于动力学测量的自由能曲线，并讨论了不同步骤对特异性的影响。在错配掺入和随后用正确核苷酸延伸的过程中，构象变化和化学反应的速率都大大降低。然而，核苷酸解离速率增加到超过化学速率。为了研究区分错配核苷酸的结构基础，我们对聚合酶活性位点上结合正确或错配核苷酸的复合物进行了所有原子分子动力学模拟。模拟表明，由于与活性位点残基的相互作用较弱、碱基配对不理想以及引物链的 3ʹ-OH 基团与引入核苷酸的 α-磷酸基团之间的距离大幅增加，具有错配结合的酶的闭合形式大大不稳定，这解释了错误掺入率的降低。观察到的控制核苷酸错误掺入的动力学和结构机制揭示了可能适用于其他高保真 DNA 聚合酶的一般原理。
High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension with the correct nucleotide, the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. The simulations suggest that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, nonideal base pairing, and a large increase in the distance from the 3ʹ-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high-fidelity DNA polymerases.
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