The physical structure of the "30 nm" chromosome fiber is a fundamental unknown of eukaryotic genetics. The fibers play an integral role in gene storage, segregation, expression, replication, recombination, damage, and repair. Detailed knowledge of the 3-dimensional structure of chromatin fibers will give insight into the essential protein and DNA contacts that stabilize chromosome structure, constraints imposed on DNA sequence and structure, and constraints placed on the enzymatic machinery that interacts with the genome. Those insights will be important to understanding the normal and abnormal regulation of growth and differentiation. The research supported by this award will focus on how nucleosome are packed into "30 nm" fibers under physiological conditions. This team of researchers proposes to determine important structural parameters of chromatin fibers by quantitative cryoEM and x-ray scattering, using specially-chosen fully hydrated chromatin molecules. In addition, the research team will make the first attempts to study the structure of chromatin designed to have homogeneous properties. We will determine the following structural parameters: 1) radial distribution of density; 2) radial distribution of protein, DNA, and counterions; 3) radial distributions of HMG14/17; and 4) diameter, axial repeat, mass per unit length, and helical symmetry satellite chromatin chosen to be homogeneous by virtue of strong phasing of nucleosomes along the repetitious DNA sequence. Our goal is to determine a model-independent, low resolution maps of protein and DNA in thick chromatin fibers, which can be combined with higher resolution crystallographic information about the nucleosome cores to determine the pattern of interactions stabilizing chromatin fibers in cells.
