Helicase Mechanisms in DNA Replication

描述(申请人提供)：DNA复制、重组和修复是遗传信息从一代细胞传递到下一代细胞的基本过程。这些过程要求双工DMA暂时解开以形成单链中间体。解链反应是由一种名为解旋酶的酶催化的。解旋酶对核酸代谢的所有方面都是必不可少的，在这些方面需要ss核酸中间体。因此，了解这些酶发挥作用的分子机制是非常重要的。了解解旋酶催化的反应的机制细节对于我们理解为什么这些过程在各种疾病中功能失调是至关重要的，例如癌症和人类遗传病。在分子水平上研究不同的步骤将为如何调控它们提供必要的知识。反过来，这些知识将对设计有效的疾病疗法非常有用。 作为E.Coli细胞中主要的复制型解旋酶，Dna B蛋白为研究复制型解旋酶的分子机制提供了一个优秀的范型模型系统。复制的解旋酶并不是单独起作用的。在细胞中，DNAB解旋酶通过特定的复制因子DNAC蛋白与复制装置相连。DNAB-DNAC复合体构成了特定复制因子作用的基本模型，该复制因子将解旋酶连接到复制机器的其余部分，并控制酶的活性。本研究项目有三个主要目标：第一个主要目标是研究DNAB六聚体及其与DNAC蛋白的复合体的构象异质性的动力学和能量学。这一目标可以通过定量研究DNAB和DNAB-DNAC络合物的构象转变和组装反应的热力学和动力学来实现。第二个目标是确定DNAB-DNAC复合体与单链DNA和复制叉的相互作用以及双链DNA解离的机制。这一目标可以通过获得DNA识别和解离反应中涉及的各个步骤的详细动力学来实现。第三个主要目标是确定DNAB-DNAC复合体的形成动力学以及ATP、ADP和DNA在这一过程中的作用。为了实现这些目标，我们将应用稳态、寿命荧光光谱、荧光能量转移方法、快速动力学(停流、快速猝灭)方法、动态光散射和分析性超速离心法。

DESCRIPTION (provided by applicant): DMA replication, recombination, and repair are processes fundamental for the transmission of genetic information from one generation of cells to the next. These processes require that duplex DMA is transiently unwound to form a single-stranded intermediate. The unwinding reaction is catalyzed by a class of enzymes called helicases. 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 these enzymes function in performing their activities. Knowledge of the mechanistic details of the reactions catalyzed by helicases is essential for our understanding of why such processes dysfunction in various diseases; e.g.; cancer and human genetic diseases. Studying different steps on the molecular level will provide the necessary knowledge about how to regulate and control them. This knowledge, in turn, will be very useful in designing efficient therapies for diseases. As a primary replicative helicase in E. Coli cells, the DnaB protein provides an outstanding and paradigm model system to study the molecular mechanism of the replicative helicase. The replicative helicase does not act alone. In the cell, the DnaB helicase is linked to the replication apparatus through the specific replication factor, the DnaC protein. .The DnaB - DnaC complex constitutes a fundamental model of the role of a specific replication factor that connects the helicase to the rest of the replication machine and controls the activities of the enzyme. This research project has three major objectives: The first major objective is to examine the dynamics and energetics of the conformational heterogeneity of the DnaB hexamer and its complex with the DnaC protein. This objective can be achieved by quantitatively examining the thermodynamics and kinetics of conformational transitions and assembly reactions of the DnaB and DnaB-DnaC complex. The second objective is to determine the DnaB - DnaC complex interactions with the ssDNA and the replication fork and the mechanism of the dsDNA unwinding. This objective can be achieved by obtaining detailed kinetics of the individual steps involved in the DNA recognition and unwinding reactions. The third major objective is to determine the dynamics of the formation of DnaB - DnaC complexes and the role of ATP, ADP, and the DNA in this process. To achieve these goals, we will apply steady-state, lifetime fluorescence spectroscopy, the fluorescence energy transfer method, fast kinetic (stopped-flow, rapid quench-flow) methods, dynamic light scattering and analytical ultracentrifugation.