| This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to DNA sites of binding, monovalent ions in the local environment, and conformation of the DNA molecules. Evidence obtained through gel mobility shift assays demonstrates the strong preference by the linker histone for DNA with superhelical torsion, i.e., supercoiling, and the negative cooperative mode of binding that the linker histone exhibits in association with supercoiled DNA.; The second part of the dissertation examines the location of linker histone binding on the nucleosome, and documents the pronounced tendency of the linker histone to bind to two DNA duplex strands. A preparation of homogeneous nucleosome core particles, consisting of a defined 238 base pair DNA fragment and the core histone octamer positioned precisely on this DNA, was used as a substrate for the UV-induced crosslinking of the linker histone to the DNA of this nucleosome. By site-specific labeling of a single site on the DNA of the nucleosome, the linker histone was observed crosslinked at that labeled site, confirming that the linker histone binds at the pseudo-dyad axis of the nucleosome. This evidence was used to support a model of linker histone binding to the nucleosome that invokes the association of the linker histone with no fewer than two duplex strands of DNA of the nucleosome. |
