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：

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DNA聚合酶保真度的动力学测定：超越Michaelis-Menten动力学的新理论视角

DNA聚合酶（DNAP）的高保真度对于忠实复制DNA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可以得到真正的保真度，但事实证明，在实践中很难实施。然后提出并广泛采用了两种生物化学动力学方法，即稳态测定法和瞬态测定法。在这些分析中，错误频率是通过使用动力学理论与测得的表观动力学速率相结合来间接估算的。但是，从理论上讲，它是否等同于真实保真度，尤其是使用这些方法量化DNAP的校对效率的策略不同，但常常导致结果不一致。在本文中，我们基于我们最近提出的DNAP保真度理论，对这两种动力学测定的理论基础进行了全面的考察。我们的研究表明，尽管常规的动力学分析通常可以有效地定量DNAP的鉴别效率，但只有在动力学参数满足某些约束条件时（它们将在本文中明确给出），它们才有效地定量DNAP的校对效率。这些结果可能会激发出更精心设计的实验来量化DNAP保真度。仅当动力学参数满足某些约束条件时，它们才有效地量化DNAP的校对效率，这将在本文中明确给出。这些结果可能会激发出更精心设计的实验来量化DNAP保真度。仅当动力学参数满足某些约束条件时，它们才有效地量化DNAP的校对效率，这将在本文中明确给出。这些结果可能会激发出更精心设计的实验来量化DNAP保真度。