Chu Xiakun, Suo Zucai, Wang Jin
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794, United States.
Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida 32306, United States.
JACS Au. 2021 Dec 16;2(2):341-356. doi: 10.1021/jacsau.1c00368. eCollection 2022 Feb 28.
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 . 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.
在DNA聚合过程中，Y家族DNA聚合酶能够绕过各种DNA损伤，而这些损伤会阻碍复制叉的前进。人们已经充分认识到，Y家族DNA聚合酶的结构是自然进化而来以承担这一重要任务的。然而，这些蛋白质如何利用其独特的结构和构象动力学特征来进行跨损伤DNA合成的机制却鲜为人知。在这里，我们开发了基于结构的模型来研究预催化DNA聚合过程，包括DNA和核苷酸与DPO4的结合，DPO4是来自……的典型Y家族聚合酶。我们研究了DPO4的折叠与构象动力学之间的相互作用，发现DPO4首先进行解折叠（展开），然后折叠以完成功能性的“开放到闭合”构象转变。在逐步结合过程中，DNA结合通过不同类型的相互作用动态调节DPO4中的构象平衡，导致DPO4在不同DNA结合阶段具有不同的构象分布。我们观察到核苷酸结合会诱导与高自由能垒相关的DPO4-DNA复合物活性位点周围的一些接触发生调节。我们的模拟结果与实验证据相呼应，即核苷酸导致的活性位点构象变化是核苷酸掺入的限速步骤。结合局部挫折分析，我们强调了DPO4构象动力学和波动在促进DNA和核苷酸结合方面的重要性。我们的发现为与底物结合相关的DPO4构象动力学过程提供了机制性见解，并有助于理解Y家族DNA聚合酶中的“结构-动力学-功能”关系。
