Mismatched Nucleotide Rejections at the Open and Ajar States of DNA Polymerase I Ensure High Fidelity

DNA polymerases achieved high fidelity via kinetic proofreading. However, due to the lack of proper enzymatic reaction models, the mechanism has not yet been successfully applied to quantify the fidelity of polymerases. Here, we examine the multistep conformational transitions of DNA polymerase I in both binary and ternary complexes, each having three conformations: open, partially closed (ajar), and closed. The transition patterns and dwell time distributions reveal a hidden transition pathway connecting the ternary ajar state to the binary ajar state, reflecting the second rejection point of an incorrect nucleotide, in addition to the first one upon initial binding. Once the ternary complex reaches the closed state, the incorrect nucleotide can hardly be rejected directly. However, the increased closed-to-ajar reverse transition enables it to be rejected at the ternary ajar state. We constructed a reaction network connecting the dynamics of the binary and ternary complexes. It allows for quantifying the contribution of each checkpoint to the fidelity under various conditions. Our results emphasize the critical role of the ajar state in enhancing the fidelity of the polymerase.