DNA聚合酶的工作机理研究

DNA polymerases (DNA pols) are molecular motors that interact with DNA. They play critic roles in DNA replications and DNA repairs. Recently, with the advent of new experimental methods such as single-molecule techniques, florescence, high-resolution imaging, etc., more and more kinetic and dynamic properties of DNA pols have been revealed. However, the detailed working mechanism of DNA pols remains elusive. The current research proposal is to study the working mechanism of DNA pols such as A-family bacteriophage T7 DNA pol, X-family DNA pols beta, mu and lambda, Y-family Dbh and Dpo4. We will use molecular dynamics to simulate the dynamics of conformational changes of DNA pols of different families induced by the change of the nucleotide state. We will also calculate and compare the interaction strengths of the DNA pols with the DNA substrate under different nucleotide states. Based on these results of conformational changes and interaction-strength changes, we will build up the working model for the DNA pols and the coupled mathematical equations for translocation of the DNA pols along the DNA substrate and the enzymatic reactions during one nucleotide-addition cycle. Using the model and the equations, we will explain the intriguing but unexplained dynamic properties of DNA pols. We will quantitatively compare our theoretical results with the available experimental data. Moreover, we will present predicted results, which will be verified by our assays.

DNA聚合酶是作用于核酸的分子马达，在DNA复制、修复等过程中起着关键作用。近年来，随着单分子、荧光、超分辨成像等新技术的发展，DNA聚合酶的特性被越来越多地揭示出来。但其工作的物理、化学动力学机制仍然存在很多谜。本项目主要研究不同家族DNA聚合酶（如：A家族T7聚合酶，X家族聚合酶beta、mu和lambda，Y家族聚合酶Dbh和Dpo4）工作的分子机理。研究内容包括：利用分子动力学全原子计算分析上述不同家族DNA聚合酶的构象随化学状态的变化动力学。计算和确定聚合酶与DNA相互作用势随化学状态的变化规律。在分析这些构象及作用势的变化规律的基础上，建立不同家族聚合酶沿DNA模板运动的物理和数学模型。解释关于DNA聚合酶一些至今没有解释的特性。定量比较理论所得结果与已发表的实验结果，并预言新的结果，指导进一步实验研究。

利用全原子分子动力学模拟研究了DNA聚合酶和RNA 聚合酶工作特性及动力学特性。主要内容包括：DNA 聚合酶手指子域转动的动力学特性，DNA聚合酶I中5’内切酶域与主心域之间的时空协调的动力学特性。单体T7 RNA聚合酶和多体Pol II RNA聚合酶转录过程中沿DNA模板移动的机理，单体T7 RNA聚合酶转录过程中对底物NTP的选择机制。另外，研究了无结构多肽链的弹性，研究了驱动蛋白沿微管的运动机理，研究了核糖体沿信息RNA移动的机理，研究了DNA解旋酶解旋DNA工作机理。