A specific DNA structure, the Holliday junction, is an intermediate in all recombination and may repair processes in the cell. Experimental evidence has been accumulating which indicates that the structure of the Holliday junction is sensitive to the DNA sequence at the branch points and that this has important biological consequences. In this project Dr. Chazin proposes to examine the physical structure and biological properties of synthetic immobile Holliday junctions by using an integrated, multi-disciplinary approach involving molecular biology, time resolved fluorescence and NMR spectroscopy, and molecular modeling/computer graphics. Time resolved fluorescence will be used to gain information on the global structure of the junctions, using fluorescence energy transfer experiments to determine the relative distances between the four arms of the junctions. NMR spectroscopy will provide detailed information on the local structure. A comparative analysis of the internal dynamics of these junctions will be made by fluorescence depolarization and NMR measurements. These data taken together will be used to solve the three-dimensional structures in solution of a series of related Holliday junctions and thereby determine how the sequence determines the geometry of the junction. These systematic studies will provide critical insights into formation of Holliday junctions, their propagation along the DNA duplexes, and the subsequent steps of junction recognition and resolution.
