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.
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