Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, Austin, Texas, USA.
Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, Austin, Texas, USA.
J Biol Chem. 2020 Dec 11;295(50):17265-17280. doi: 10.1074/jbc.RA120.015557. Epub 2020 Oct 5.
DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.
T7 噬菌体 DNA 聚合酶经历了大的、底物诱导的构象变化，这些变化被认为是高复制保真度的原因，但之前的研究受到构建用于定点荧光标记的 Cys-lite 变体所需突变的不利影响。在这里，我们使用正交琥珀终止密码子抑制机制优化了（7-羟基-4-香豆素基）-乙基甘氨酸的直接掺入，MS 方法验证了非天然氨基酸仅在一个位置掺入，背景最小。我们表明，单个荧光团通过正确核苷酸结合和掺入的平衡和停流动力学测量提供了检测核苷酸诱导构象变化的信号。预稳态化学猝灭方法表明，标记酶催化的 DNA 复制的动力学和保真度基本不受非天然氨基酸的影响。这些进展使得能够进行严格的分析，以确定高保真度 DNA 复制的动力学和机制基础。
