HELICASE MECHANISMS IN DNA REPLICATION

DNA复制、重组和修复是基本的过程 用于将细胞的遗传信息从一个 代代相传。这些过程需要双链DNA处于 最小的瞬时解离形成单链中间体。这个 解链反应是由一类酶--解旋酶催化的。 了解解旋酶反应的机理细节是 对于我们理解为什么这样的过程在 各种疾病，例如癌症和人类遗传病。学习 分子水平上的不同步骤应该提供必要的 关于如何监管和控制它们的知识。而这些知识又反过来 在设计有效的疾病疗法方面应该非常有用。 解旋酶对核酸代谢的各个方面都是必不可少的。 其中需要SS核酸中间体。因此，它是 对于理解分子机制具有至关重要的意义 解旋酶在执行它们的活动中起作用。 作为大肠杆菌中主要的复制解旋酶，Dna B蛋白提供 一种研究解旋酶分子机制的优秀模型系统 行动。这项研究项目有两个主要目标。第一 目的是建立耦合的定量动力学模型 三磷酸腺苷结合和水解所产生的自由能 DsDNA由DNAB解旋酶催化。这一目标可以通过获得 参与ATP水解的各个步骤的详细动力学 Dna B解旋酶与ss、dsDNA的结合，以及dsDNA的解离。 为了实现这一目标，热力学和动力学 还将检测该酶的构象变化。第二 主要的目标是关联活动性的动力学模型 具有催化作用的结构决定因素的酶和 核酸的单向易位。这一目标可以是 通过确定相互作用站点的拓扑和 解旋酶复合体中核酸的结构。至 为了实现这些目标，我们将应用稳态、终身荧光 光谱学、快速动力学(停流、快速急流)方法， 分析超速离心法和各种定量的物理和化学方法 生化技术。

DNA replication, recombination, and repair are the processes fundamental for the transmission of the genetic information of the cell from one generation to the next. These processes require that duplex DNA is at least transiently unwound to form a single-stranded intermediate. The unwinding reaction is catalyzed by a class of enzymes, helicases. Knowledge of the mechanistic details of the helicase reaction is essential to our understanding of why such processes dysfunction in various diseases, e.g., cancer and human genetic diseases. Studying different steps on the molecular level should provide the necessary knowledge about how to regulate and control them. This knowledge in turn should be very useful in designing efficient therapies for diseases. The helicases are essential for all aspects of nucleic acid metabolism in which ss nucleic acid intermediates are required. Therefore, it is of fundamental importance to understand the molecular mechanism by which the helicases function in performing their activities. As the primary replicative helicase in E. coli, the DnaB protein provides an outstanding model system to study the molecular mechanism of helicase action. This research project has two major objectives. The first objective is to formulate the quantitative dynamic model of the coupling of the free energy from ATP binding and hydrolysis to be unwinding of dsDNA by the DnaB helicase. This objective can be achieved by obtaining detailed kinetics of individual steps involved in ATP hydrolysis by the DnaB helicase in its binding to ss, dsDNA, and in the unwinding of dsDNA. To achieve this objective thermodynamics and kinetics of the conformational changes of the enzyme will also be examined. The second major objective is to correlate the kinetic model of the activity of the enzyme with the structural determinants responsible for its catalysis and unidirectional tanslocation on nucleic acid. This objective can be achieved by determining the topology of interacting sites and the structure of the nucleic acid in the complex with the helicase. To achieve these goals, we will apply steady-state, lifetime fluorescence spectroscopy, fast kinetic (stop-flow, rapid quench-flow) methods, analytical ultracentrifugation and various quantitative physical and biochemical techniques.