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.
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