Helicase Mechanisms in DNA Replication

描述（由申请人提供）：DMA复制、重组和修复是遗传信息从一代细胞传递到下一代的基本过程。这些过程要求双工DMA瞬间解绕形成单链中间体。解旋反应是由一类叫做解旋酶的酶催化的。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这些过程在各种疾病中功能障碍至关重要；例如;癌症和人类遗传疾病。在分子水平上研究不同的步骤将为如何调节和控制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治疗方法非常有用。

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.