Investigating the Conformational Dynamics of a Y-Family DNA Polymerase during Its Folding and Binding to DNA and a Nucleotide

[Image: see text] During DNA polymerization, the Y-family DNA polymerases are capable of bypassing various DNA damage, which can stall the replication fork progression. It has been well acknowledged that the structures of the Y-family DNA polymerases have been naturally evolved to undertake this vital task. However, the mechanisms of how these proteins utilize their unique structural and conformational dynamical features to perform the translesion DNA synthesis are less understood. Here, we developed structure-based models to study the precatalytic DNA polymerization process, including DNA and nucleotide binding to DPO4, a paradigmatic Y-family polymerase from Sulfolobus solfataricus. We studied the interplay between the folding and the conformational dynamics of DPO4 and found that DPO4 undergoes first unraveling (unfolding) and then folding for accomplishing the functional “open-to-closed” conformational transition. DNA binding dynamically modulates the conformational equilibrium in DPO4 during the stepwise binding through different types of interactions, leading to different conformational distributions of DPO4 at different DNA binding stages. We observed that nucleotide binding induces modulation of a few contacts surrounding the active site of the DPO4–DNA complex associated with a high free energy barrier. Our simulation results resonate with the experimental evidence that the conformational change at the active site led by nucleotide is the rate-limiting step of nucleotide incorporation. In combination with localized frustration analyses, we underlined the importance of DPO4 conformational dynamics and fluctuations in facilitating DNA and nucleotide binding. Our findings offer mechanistic insights into the processes of DPO4 conformational dynamics associated with the substrate binding and contribute to the understanding of the “structure–dynamics–function” relationship in the Y-family DNA polymerases.