9317369 Chazin The principal goals of this research are to determine the three- dimensional structure of Holliday junctions, to understand how the sequence at the junction influences structure, and to determine why the cellular recombination machinery is influenced by these differences. As a starting point, the determination of the three-dimensional structures and examination of the internal dynamics of small (32 base-pair) models of Holliday junctions is proposed using time-resolved fluorescence and nuclear magnetic resonance (NMR) spectroscopies. The selected sequences are designed to inhibit migration of the crossover junction to facilitate analysis by biophysical methods, and the results are then analyzed with the assistance of computational and molecular graphics approaches. Our objectives are formulated on the basis of critical observations indicating that the resolution of HJs in the cell and in vitro appears to be dependent on the sequence at the site of strand crossover, and that the spatial arrangement of the branches of Holliday junction is dependent on sequence. Thus, our objective extends beyond the determination of the structure of a single HJ at atomic resolution, to analysis of a series of related HJs having identical branches but different permutations of the bases at the junction. Selection of specific HJs for in-depth structural analysis is based on screening using gel mobility, thermal denaturation, and fluorescence assays. %%% Genetic recombination is a fundamental process of biology leading the variation and diversity that is essential to life. Most mechanisms proposed for recombination involve a 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. Thus, we seek an understandi ng of the molecular basis of genetic recombination from detailed knowledge of the structure of the HJ, and ultimately, of the nature of the interactions with enzymes critical to HJ recognition and processing. ***
