Computer Simulation Studies of the Origin of DNA

了解DNA聚合酶的保真度的来源是一个具有巨大现实意义和根本意义的问题。尽管取得了很大的实验进展，但我们仍然没有控制保真度因素的详细分子图景。在这里，我们建议使用计算机模拟方法来获得更深层次的见解，这将补充该领域的实验进展。提出了以下项目：(I)将研究正确(R)和错误(W)的引物-模板碱基对在蛋白质(聚合酶)和水溶液中的相对稳定性。这些研究将有助于确定绑定步骤对整体保真度的贡献。(Ii)各种聚合酶/DNA复合体的X射线结构将被用作计算正确(R)和错误(W)核苷酸的化学步骤的机制和激活能的起点。(Iii)分子的束缚自由能 将计算R和W的过渡态。这些计算将与相应的整体保真度直接相关。(4)探索Polb在不同构象状态下的反应途径和活化势垒。这项研究将研究W和R底物在核苷酸结合部位的调节是如何传递到催化部位的。(V)将利用不同TS类似物(TSA)的结构和能量来完善TS的理论模型。这项研究还将寻找最佳的TS类似物 并最终用于有效的药物。(6)将使用计算机模拟方法来研究基质和催化金属离子的关键修改的后果。(Vii)将在不同的聚合酶中分析不同蛋白质残基对整体保真度的贡献。所有拟议的研究都将涉及该项目的实验同行不断提供的反馈。我们希望这次合作将帮助我们对DNA聚合酶的保真度的本质发展出一个更普遍和一致的观点。

Understanding the origin of the fidelity of DNA polymerases is a problem of enormous practical and fundamental importance. Despite great experimental progress we still do not have a detailed molecular picture of the factors that control the fidelity. Here we propose to use computer simulation approaches to gain a deeper insight that will complement the experimental advances in the field. The following projects are proposed: (i) The relative stabilities of right (R) and wrong (W) primer-template base pairs will be investigated both in the protein (polymerase) and in aqueous solution. These studies will help to determine the contribution of the binding step to the overall fidelity. (ii) X-ray structures of various polymerase/ DNA complexes will be used as a starting point for calculations of the mechanisms and activation energies of the chemical step for incorporating right (R) and wrong (W) nucleotides. (iii) The binding free energy of the transition state for R and W will be calculated. These calculations will be directly related to the corresponding overall fidelity. (iv) The reaction pathway and activation barriers in different conformational states of the pol b will be explored. This study will examine how the accommodation of W and R substrates in the nucleotide binding site are transmitted to the catalytic site. (v) The structures and energies of different TS-analogues (TSAs) will be used to refine the theoretical model of the TS. This study will also search for optimal TS-analogs and eventually for effective drugs. (vi) Computer simulation approaches will be used to study the consequences of key modifications in the substrate and the catalytic metal ions. (vii) The contributions of different protein residues to the overall fidelity will be analyzed in different polymerases. All the proposed studies will involve constant feedback from the experimental counterparts of the project. We hope that this collaboration will help us to develop a more general and coherent view about the nature of DNA polymerase fidelity.