HELICASE MECHANISMS IN DNA REPLICATION

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。这些过程要求双链DNA至少能瞬间解绕形成单链中间体。解绕反应是。由一种叫做解旋酶的酶催化。解旋酶在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，了解这些酶在发挥其活性时的分子机制是至关重要的。了解解旋酶催化反应的机理细节，对于我们理解为什么解旋酶在各种疾病（如癌症和人类遗传疾病）中不起作用至关重要。在分子水平上研究不同的步骤应该为如何调节和控制它们提供必要的知识。反过来，这些知识在设计有效的疾病治疗方法时应该非常有用。作为大肠杆菌的一级复制解旋酶，DnaB蛋白为研究解旋酶作用的分子机制提供了一个优秀的模型系统。本研究项目有三个主要目的：第一目的是确定dna解旋酶识别复制叉的机制。这一目标可以通过获得识别过程中涉及的各个步骤的详细动力学以及解旋酶和叉的构象变化动力学来实现。第二个主要目标是研究dna六聚体的构象灵活性和组装过程。这一目标可以通过定量研究由核苷酸辅因子、ssDNA结合和镁离子诱导的dna六聚体构象转变和组装过程的热力学和动力学来实现。第三个主要目标是确定分子转位酶，即DnaC蛋白，在DnaB解旋酶功能中的作用。这一目标可以通过严格分析蛋白质相互作用的能量学和三元DnaB - DnaC - ssDNA复合物的形成来实现。为了实现这些目标，我们将应用稳态、寿命荧光光谱、荧光能量转移法、快速动力学（停止流动、快速淬火流动）方法、动态光散射和分析超离心。

DNA replication, recombination, and repair are the processes fundamental for the transmission of genetic information from one generation of cells to the next. These processes require that duplex DNA is a least t:ransiently unwound to form a single-stranded intermediate. The unwinding reaction is. catalyzed by 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 ad 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. 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 three major objectives: The first objective is to determine the mechanism of the replication fork recognition by the DnaB helicase. This objective can be achieved by obtaining detailed kinetics of individual steps involved in the recognition process, and the dynamics of conformational changes of the helicase and the fork. The second major objective is to examine the conformational flexibility and the assembly process of the DnaB hexamer. This objective can be achieved by quantitatively examining the thermodynamics and kinetics of the conformational transitions and assembly process of the DnaB hexamer induced by nucleotide cofactors, ssDNA binding, and magnesium cations. The third major objective is to determine the role of the molecular translocase, the DnaC protein, in the DnaB helicase functioning. This objective can be achieved by rigorous analyses of the energetics of protein protein interactions and the formation of the ternary DnaB - DnaC - ssDNA complex. 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.