Kinetic assays of DNA polymerase fidelity: A theoretical perspective beyond Michaelis-Menten kinetics,Physical Review E

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Kinetic assays of DNA polymerase fidelity: A theoretical perspective beyond Michaelis-Menten kinetics
Physical Review E ( IF 2.2 ) Pub Date : 2021-07-19 , DOI: 10.1103/physreve.104.014408
Qiu-Shi Li ₁ , Yao-Gen Shu ₂ , Zhong-Can Ou-Yang ₃ , Ming Li ₁

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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.

中文翻译：

DNA 聚合酶保真度的动力学分析：超越 Michaelis-Menten 动力学的理论视角

DNA 聚合酶 (DNAP) 的高保真度对于 DNA 的忠实复制至关重要。有几种定量方法可以测量 DNAP 保真度。直接计算复制产品中的错误频率给出了真正的保真度，但在实践中很难实现。然后提出并广泛采用两种生化动力学方法，即稳态测定和瞬态测定。在这些测定中，误差频率是通过使用动力学理论与测量的表观动力学速率相结合来间接估计的。然而，它是否等同于真正的保真度在理论上从未得到澄清，尤其是使用这些检测方法来量化 DNAP 的校对效率时有不同的策略，但往往会导致不一致的结果。在本文中，我们基于我们最近提出的DNAP保真度理论，对这两种动力学分析的理论基础进行了全面的考察。我们的研究表明，虽然传统的动力学分析通常可用于量化 DNAP 的鉴别效率，但仅当动力学参数满足本文将明确给出的某些约束时，它们才可用于量化 DNAP 的校对效率。这些结果可能会激发更精心设计的实验来量化 DNAP 保真度。只有当动力学参数满足本文将明确给出的一些约束时，它们才有效地量化 DNAP 的校对效率。这些结果可能会激发更精心设计的实验来量化 DNAP 保真度。只有当动力学参数满足本文将明确给出的一些约束时，它们才有效地量化 DNAP 的校对效率。这些结果可能会激发更精心设计的实验来量化 DNAP 保真度。

更新日期：2021-07-20

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