Kinetic assays of DNA polymerase fidelity: A theoretical perspective beyond Michaelis-Menten kinetics.
Kinetic assays of DNA polymerase fidelity: A theoretical perspective beyond Michaelis-Menten kinetics.
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DOI：
10.1103/physreve.104.014408
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发表时间：
2021-07
期刊：
影响因子：
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通讯作者：
Qiu-Shi Li;Yao-Gen Shu;Z. Ou-Yang;Ming Li
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文献类型：
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作者：
Qiu-Shi Li;Yao-Gen Shu;Z. Ou-Yang;Ming Li
DNA聚合酶（DNAP）的高保真度是DNA复制的关键。有几种定量方法来测量DNAP保真度。直接计算复制产品中的错误频率可以提供真实的保真度，但实际上很难实现。两种生化动力学方法，稳态测定和瞬态测定，然后建议和广泛采用。在这些测定中，通过使用动力学理论结合测得的表观动力学速率来间接估计误差频率。然而，它是否等同于真正的保真度从未在理论上得到澄清，特别是有不同的策略使用这些检测来量化DNAP的校对效率，但往往导致不一致的结果。本文以我们最近提出的DNAP保真度理论为基础，对这两种动力学分析方法的理论基础进行了全面的探讨。我们的研究表明，虽然传统的动力学方法通常是有效的量化DNAP的识别效率，他们是有效的量化DNAP的校对效率只有当动力学参数满足一些约束条件，这将在本文中明确给出。这些结果可能会激发更多精心设计的实验来量化DNAP保真度。
The high fidelity of DNA polymerase (DNAP) is critical for the faithful replication of DNA. There are several quantitative approaches to measure DNAP fidelity. Directly counting the error frequency in the replication products gives the true fidelity but it turns out very hard to implement in practice. Two biochemical kinetic approaches, the steady-state assay and the transient-state assay, were then suggested and widely adopted. In these assays, the error frequency is indirectly estimated by using kinetic theories combined with the measured apparent kinetic rates. However, whether it is equivalent to the true fidelity has never been clarified theoretically, and in particular there are different strategies using these assays to quantify the proofreading efficiency of DNAP but often lead to inconsistent results. In this paper, we make a comprehensive examination on the theoretical foundation of the two kinetic assays, based on the theory of DNAP fidelity recently proposed by us. Our studies show that while the conventional kinetic assays are generally valid to quantify the discrimination efficiency of DNAP, they are valid to quantify the proofreading efficiency of DNAP only when the kinetic parameters satisfy some constraints which will be given explicitly in this paper. These results may inspire more carefully-designed experiments to quantify DNAP fidelity.