HELICASE MECHANISMS IN DNA REPLICATION

DNA的复制、重组和修复是DNA合成的基本过程 用于将细胞的遗传信息从 下一代。 这些过程需要双链DNA处于 至少瞬时解绕以形成单链中间体。的 解旋反应由一类酶，解旋酶催化。 解旋酶反应的机械细节的知识是 这对我们理解为什么这些过程在 各种疾病，例如，癌症和人类遗传疾病。 研究 分子水平上的不同步骤应提供必要的 了解如何管理和控制它们。这些知识反过来 在设计有效的疾病治疗方法时应该非常有用。 解旋酶对于核酸代谢的各个方面都是必需的 其中需要SS核酸中间体。 因此有 对于理解 解旋酶的功能是进行它们的活性。 作为E.在大肠杆菌中，DnaB蛋白提供 解旋酶分子机理研究的一个优秀模型系统 行动上本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 ATP结合和水解的自由能被解旋， 通过DnaB解旋酶对dsDNA进行解旋。 这一目标可以通过以下方式实现： 参与ATP水解的各个步骤的详细动力学 DnaB解旋酶在其与ss、dsDNA的结合以及dsDNA的解旋中的作用。 为了实现这一目标的热力学和动力学的 还将检测酶的构象变化。 第二 主要目的是关联的动力学模型的活动， 具有负责其催化作用的结构决定因素的酶， 核酸的单向转移。 该目的可以 通过确定交互站点的拓扑结构和 在与解旋酶的复合物中的核酸的结构。 到 实现这些目标，我们将应用稳态，寿命荧光 光谱法、快速动力学（停流、快速猝灭流）方法， 分析超浓缩和各种定量物理和 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.