Computer Simulation Studies of the Origin of DNA Polymerase Fidelity

阐明控制DNA聚合酶复制保真度的因素是一个具有重要基础和实际意义的目标。为了朝着这个方向发展，我们将对野生型和突变型DNA聚合酶的催化和复制保真度进行真实的原子水平模拟，重点是DNA聚合酶B(PolB)，它是人类碱基切除修复的关键角色，并与癌症的发生有关。我们实验室的主要优势是我们在酶催化和催化景观的最先进模拟方面的综合专业知识，以及对DNA聚合酶保真度的结构和机制基础的现有实验信息的深入了解。我们的理论工具箱包括经验价键(EVB)，提供了以经验价键为参考势获得从头算量子力学/分子力学(QM/MM)自由能剖面的有效方法，以及探索构象和化学坐标之间长时间耦合的粗粒(CG)重整化方法。我们还开发并改进了有效的采样方法，使我们能够提高计算自由能的准确性。这些计算研究将与项目3中关于polB突变的动力学效应及其底物变化的生化研究相结合。我们的模拟将再现和/或预测这些变化的功能影响，并分析它们的来源。同时，我们将依靠项目1中的重要结构信息。 在原子水平上了解PolB插入新核苷酸的过渡态结构以及通过修饰活性部位来改变这种结构的延展性将极大地增加有效设计有效和选择性的PolB抑制剂的可能性。

Elucidating the factors that control the replication fidelity of DNA polymerases is a goal of great fundamental and practical importance. In order to advance in this direction we will generate realistic atomic-level simulations of the catalysis and replication fidelity of wild-type and mutant DNA polymerases, focusing on DNA polymerase B(pol B), which is a key player In human base excision repair and has been Implicated in the incidence of cancer. The main strength of our laboratories has been our combined expertise in state-of- the art simulations of enzyme catalysis and catalytic landscapes, with a deep understanding of the available experimental information on the structural and mechanistic underpinnings of the fidelity of DNA polymerases. Our theoretical toolbox includes the empirical valence bond (EVB), the paradynamics that provides effective way of obtaining ab initio quantum mechanical /molecular mechanics (QM/MM) free energy profiles using the EVB as a reference potential, as well as coarse grained (CG) renormalizatlon approaches for exploring long time coupling between the conformational and chemical coordinates. We have also develop and refined effective sampling methods that should allow us to increase the accuracy of the calculated free energies. These computational studies will be applied in concert with the biochemical studies of Project 3 of kinetic effects of mutations of pol B and of changes in its substrates. Our simulations will reproduce and/or predict the functional effects of these changes and analyze their origin. At the same time, we will rely on the important structural information from Project 1. The atomic level understanding Of the structure of the transition state for the insertion of a new nucleotide by pol B and the malleability of this structure by modifications of the active site will greatly increase the possibilities of effective design of a potent and selective inhibitor of pol B.