Computer Simulation Studies of the Origin of DNA Polymerase

了解控制DNA聚合酶保真度的因素是一个非常重要的基础问题， 实际重要性。在这里，我们建议利用我们最近的进步，继续使用计算机 模拟方法，以获得更深入的了解，这将补充项目1的实验进展 和3.建议使用可靠的理论工具进行结构/保真度相关，并对此进行改进 通过动力学、结合和结构实验的恒定反馈进行相关。拟议项目 包括：（i）建立dNTP底物的PaO键在聚合酶中的切割机制 活性位点的协调使用不同的理论方法，从从头算QM-MM自由能 计算约束DFT自由能计算和系统的经验价键研究，（ii） 改进了我们对底物结合自由能的计算以及对聚合物影响的计算， 这些结合能的突变，（iii）探索控制聚合酶保真度的因素，| 特别注意碱基结合位点和蛋白质之间信息的变构转移。 化学反应现场变构信息传递将通过计算 不同的残基和过渡态（TS）使用相关矩阵形式主义。（四）探索自然 用于掺入正确（R）和错误（W）核苷酸的TS，包括研究 蛋白质构象景观的性质的TS。这些研究将得到结构研究的帮助 具有正确和不正确碱基对的过渡态类似物（TSA），（v）确定结构和 不同TSA的结合能将由我们的同事进行实验研究（项目3）， 从理论上探索。本研究将告诉我们的关系之间的tsa和实际的tsa 并将有助于验证计算的TS属性。 所有上述研究都将涉及该项目实验对应方的不断反馈。这 合作将有助于我们对DNA聚合酶的性质形成更普遍和一致的看法 忠诚

Understanding the factors that control the fidelity of DNA polymerases is a problem of great fundamental and practical importance. Here we propose to exploit our recent advances and to continue the use of computer simulation approaches to gain a deeper insight that will complement the experimental progress in projects 1 and 3. It is proposed to use reliable theoretical tools for structure/fidelity correlation and to refine this correlation by a constant feedback from kinetic, binding, and structural experiments. The proposed projects include: (i) Establishing the cleavage mechanism of the PaO bond of dNTP substrates in the polymerase active site by a concerted use of different theoretical approaches, ranging from ab initio QM-MM free energy calculations to constraint DFT free energy calculations and to systematic empirical valence bond studies, (ii) Refining our calculations of substrate binding free energies as well as calculations of the effects of pol pi mutations on the these binding energies, (iii) Exploring factors that control the fidelity of polymerases while| paying special attention to the allosteric transfer of information between the base binding site and the! chemical reaction site. The allosteric information transfer will be studied by calculating the coupling between different residues and the transition state (TS) using the correlation matrix formalism. (iv)Exploring the nature of the TS for incorporating right (R) and wrong (W) nucleotides, including the study of the effect of the protein conformational landscape on the nature of the TS. These studies will be helped by structural studies of transition state analogues (TSAs) with correct and incorrect base pairs, (v) Determining the structure and binding energy of different TSAs that will be examined experimentally by our coworkers (Project 3) and explored theoretically. This study will teach us about the relationship between the TSAs and the actual TS and will help in verifying the calculated TS properties. All the above studies will involve constant feedback from the experimental counterparts of the project. This collaboration will help us develop a more general and coherent view about the nature of DNA polymerase fidelity.