9604568 Chazin The Holliday junction (HJ) is a four-arm, four-strand DNA crossover structure formed as a requisite intermediate for genetic recombination and many repair processes in the cell. The structure and flexibility of these DNA crossovers appears to be critical to their recognition and processing into parental or recombinant products. The principle goals of this research program are to determine the three-dimensional structure and dynamics of Holliday junctions, to establish the influence of sequence, and ultimately, to understand how the cellular recombination machinery distinguishes HJs of different sequence. The Time-resolved fluorescence resonance energy transfer (tr-FRET) and NMR spectroscopy will be used to the study of 32 base pair model HJs. Computation and molecular graphics will be used to assist in interpretation and analysis of results. The combination of NMR and fluorescence spectroscopies is particularly powerful because these techniques yield information on different distance and time regimes. NMR establishes the details of local geometry while FRET is used to elucidate the overall spatial arrangement of the four helical arms. The experimental methodologies and structural principles developed in the course of these studies will be generally applicable to other nucleic acid systems. Genetic recombination is a fundamental process of biology leading to the variation and diversity that is essential to life. Most mechanisms propose for recombination involve the four-arm branched DNA intermediate termed the Holliday junction (HJ), which is cleaved by enzymes to generate either parental or recombinant products. A significant body of evidence has accumulated indicating that the structure of the HJ has a central role in determining the outcome of the recombination event. The objective of this research is to understand the molecular basis of genetic recombination from detailed knowledge of the structure of the HJ, and ultimately, of the nature of the interactions wit h enzymes critical to HJ recognition and processing.
