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基本和重要的问题 实际的重要性。在这里，我们建议利用我们最新的进展，继续使用计算机 模拟方法，以获得更深入的洞察，这将补充项目1中的实验进展 3.建议使用可靠的理论工具来进行结构/保真度相关，并对其进行提炼 通过来自动力学、结合和结构实验的恒定反馈来关联。建议的项目 包括：(I)建立聚合酶中dNTP底物的PAO键的断裂机制 通过协同使用不同的理论方法，从从头算QM-MM自由能 约束DFT自由能计算和系统经验价键研究的计算(II) 改进我们对底物结合自由能的计算以及对Polpi效应的计算 这些结合能上的突变，(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！ 化学反应地点。通过计算分子间的耦合来研究变构信息的传递 不同的残基和过渡态(TS)使用关联矩阵形式。(四)探索大自然 将正确(R)和错误(W)核苷酸结合在一起的TS，包括对 蛋白质构象景观对TS性质的影响。这些研究将得到结构研究的帮助 具有正确和错误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检测(项目3)和 理论上进行了探索。这项研究将告诉我们TSA和实际TS之间的关系 并且将有助于验证所计算的TS属性。 所有上述研究都将涉及该项目的实验同行不断提供的反馈。这 合作将帮助我们对DNA聚合酶的性质形成更普遍和更连贯的看法 富达。

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.