The objectives of the studies proposed here are to elucidate the higher order structures adopted by the basic chromatin fiber as a function of ionic strength in vitro, and elucidate the molecular factors responsible for the stability of these structures. These studies are a continuation of our previous efforts on chromatin structure. Chromatin fibers will be imaged in air with the Scanning Force Microscope (SFM) to: i) characterize intermediate forms of the salt.induced condensation process; ii) investigate the role of the linker histones HI and H5 and their globular and tail domains on the integrity and stability of the fiber; iii) elucidate the consequences of histone acetylation on the fiber structure; and iv) follow in real time the salt-induced condensation process using the newly.developed liquid.operating capability of the SFM. The role of the linker histones H1 and H5 on the entry and exit angle of the linker DNA around the nucleosome will be investigated using trinucleosomes and H1-supercoiled DNA complexes at various ionic strengths. The participation of the various domains of HI will be addressed following the effects of partial trypsin proteolysis on the structural parameters of these systems. Using both SFM and sedimentation velocity, we will also characterize the contribution of bending and folding of the linker DNA during the salt-induced condensation process of dinucleosomes. The structural studies will be complemented with mechanical studies to determine the energetics of salt-induced condensation. We will map the net internucleosomal interaction potential at various ionic strengths using single molecule manipulation methods developed in our laboratory. The results from the above studies will be used to perform computer simulations of condensation intermediates of the fiber. These simulations will incorporate: i) the ionic strength dependence of the linker rigidity; ii) the ionic strength dependence of the entry and exit angle distributions of the linker around the nucleosome; iii) the random variability of linkers; and iv) the interaction potential among nucleosomes at a given ionic strength. The combination of structural, mechanical and simulation studies will provide insights about the molecular factors responsible for the stability of the fiber structure.
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