TOPOLOGY GEOMETRY AND ENERGETICS OF CLOSED CIRCULAR DNA

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The general objective of this research is to understand the structure and dynamics of supercoiled DNA. During the next project period emphasis will be upon determination of the three dimensional structure of supercoiled DNA as a function of the linking number; upon prediction of the interaction of local segments that are separated by a considerable distance along the contour of the DNA; and upon elucidation of the effects on the DNA structure of wrapping on a protein surface. The specific aims are divided into five categories. (1) Description of the tertiary structure of supercoiled DNA. This project applies the finite element method from nonlinear structural mechanics, with energy minimization being used to predict the most stable configuration at each linking number. The writhe and twist will be calculated, and the results will be used to predict the hydrodynamic behavior of closed DNA and the equilibrium distribution of topoisomers. (2) Determination of the helical repeat of supercoiled DNA. This involves measurement of the effect upon the winding number of extrusion of defined palindromic sequences into cruciforms and of limited local denaturation at moderately elevated temperatures. (3) Effect of local structural elements on long range diffusion in closed DNA. This objective is to determine the effects that DNA local structures have upon long-range diffusion. The site-specific recombination diffusion step will be used as an analytical tool. Bent DNA sequences as well as protein-induced bent DNA sequences will be employed. (4) DNA wrapped on piecewise smooth surfaces. Writhe and surface twist will be calculated for wrapping of closed circular DNA on a connected series of proteins of various geometric shapes. The writhe will be analyzed into its piecewise components and into the con- tributions due to pairwise interactions, including those between the protein complexes, between the linker regions, and between the protein complexes and the linker regions. (5) Effect of protein wrapping upon the topological properties of closed circular DNA. The consequences of decomposing the linking number of protein wrapped, closed circular DNA into the sum of the surface linking and winding numbers will be examined in detail. The winding number will be measured for relaxed minichromosomes as a function of temperature, in order to determine the partition of the thermal effects into wrapping and winding components, followed by a determination of phi as a function of the superhelix density, in order to test the hypothesis that nucleosome distortion results from increases in the extent of supercoiling.