A critical survey on the kinetic assays of DNA polymerase fidelity from a new theoretical perspective

TL;DR

This paper develops a new theoretical framework to evaluate DNA polymerase fidelity, clarifies the limitations of existing kinetic assays, and proposes a single-molecule assay for more accurate fidelity measurement.

Contribution

It introduces a comprehensive theoretical model that clarifies the relation between different kinetic assays and the true fidelity of DNA polymerases, including a new single-molecule approach.

Findings

Steady-state and transient-state assays accurately measure true fidelity for exonuclease-deficient DNA polymerases.

These assays only conditionally measure fidelity for exonuclease-efficient DNA polymerases.

A new single-molecule assay provides precise fidelity measurements for both enzyme types.

Abstract

The high fidelity of DNA polymerase is critical for the faithful replication of genomic DNA. Several approaches were proposed to quantify the fidelity of DNA polymerase. Direct measurements of the error frequency of the replication products definitely give the true fidelity but turn out very hard to implement. 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 the steady-state or the transient-state kinetic theory combined with the measured kinetic rates. However, whether these indirectly estimated fidelities are equivalent to the true fidelity has never been clarified theoretically, and in particular there are different strategies to quantify the proofreading efficiency of DNAP but often lead to inconsistent results. The reason for all these confusions is that it's mathematically challenging to formulate a rigorous and general theory of the true fidelity. Recently we have succeeded to establish such a theoretical framework. In this paper, we develop this theory to make a comprehensive examination on the theoretical foundation of the kinetic assays and the relation between fidelities obtained by different methods. We conclude that while the steady-state assay and the transient-state assay can always measure the true fidelity of exonuclease-deficient DNA polymerases, they only do so for exonuclease-efficient DNA polymerases conditionally (the proper way to use these assays to quantify the proofreading efficiency is also suggested). We thus propose a new kinetic approach, the single-molecule assay, which indirectly but precisely characterizes the true fidelity of either exonuclease-deficient or exonuclease-efficient DNA polymerases.