DNA聚合酶整合单核苷酸的全预稳态动力学分析

Q4 Chemistry
Marleen Renders, Jean-Marie Frère, Dominique Toye, Piet Herdewijn
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引用次数: 1

摘要

通过在极短的时间尺度(5 ms至10 s)和高酶浓度下测量DNA聚合酶结合反应,可以分析单个核苷酸结合的分离事件。实际上,这是使用猝灭流仪器完成的,该仪器允许酶引物/模板与核苷酸底物快速混合,之后反应被猝灭并分析。我们描述了如何滴定酶活性位点,如何通过探测实验确定聚合反应中的限速步骤,以及如何使用爆发或单次翻转实验测量表观kpol, Kd(DNA)和Kd(N)。我们包括计算聚合酶的加工性,其核苷酸结合的特异性和偏好,以及错误核苷酸结合的活化能差的方程式。讨论了数据分析,因为这是动力学分析中准确数据生成的重要组成部分。©2019 by John Wiley & Sons, Inc。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Full Pre-Steady-State Kinetic Analysis of Single Nucleotide Incorporation by DNA Polymerases

By measuring a DNA polymerase incorporation reaction on a very short time scale (5 ms to 10 s) and with a high enzyme concentration, the isolated event of a single nucleotide incorporation can be analyzed. Practically, this is done using a quench-flow instrument, which allows the rapid mixing of the enzyme-primer/template with the nucleotide substrate, after which the reaction is quenched and analyzed. We describe how to titrate the enzyme active site, how to determine, via a scouting experiment, the rate-limiting step in the polymerization reaction, and how to measure the apparent kpol, Kd(DNA), and Kd(N) using burst or single-turnover experiments. We include equations for the calculation of the processivity of the polymerase, its nucleotide incorporation specificity and preference, and the activation energy difference for the incorporation of an incorrect nucleotide. Data analysis is discussed, as this is an essential part of accurate data generation in kinetic analyses. © 2019 by John Wiley & Sons, Inc.

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来源期刊
Current Protocols in Nucleic Acid Chemistry
Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry
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期刊介绍: Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.
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