HELICASE MECHANISMS IN DNA REPLICATION

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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.