| Chromosomes are protein-DNA complexes found in the nucleus of eukaryotic organisms. It is impressive how the long, linear DNA molecule is organized within ordered structures that reversibly fold and unfold within a eukaryotic chromosome. Importantly, it is the protein-DNA complex (also called chromatin), not naked DNA, that is the substrate for essential biological processes such as transcription, repair, recombination and replication. Knowing this, there is significant interest to determine the structure of chromatin and define the mechanisms through which the folding, unfolding, and condensation of chromatin occur.; The objective of this dissertation was to characterize linker histone and core histone N-termini contributions to chromatin structure and condensation. Towards this end, I utilized an in vitro chromatin model system consisting of a defined length DNA template, purified core histone octamers and linker histones. The defined length DNA template and core histone octamers are combined to form regularly spaced nucleosomal and chromatin arrays. These nucleosomal and chromatin arrays are suitable substrates for rigorous biochemical and quantitative hydrodynamic studies of chromatin structure.; The first goal of this project was to directly determine the mechanistic functions of linker histones in chromatin condensation; therefore, nucleosomal arrays were assembled with purified linker histone H5. The chromatin model system was characterized using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryo-microscopy, and nuclease digestion. These results indicate that histone H5 binding to nucleosomal arrays constrains the entering and exiting linker DNA. Furthermore, it was shown that histone H5 binding markedly stabilizes the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic folding pathway observed previously for nucleosomal arrays.; Next, the relationship between the core histone N-termini and linker histones during chromatin assembly and condensation was studied. Nuclease digestion and sedimentation studies indicate that H5 binds to both tailless and intact chromatin arrays and constrain the entering and exiting nucleosomal DNA to the same extent. Despite these similarities, chromatin arrays containing tailless histone octamers neither condense into extensively folded structures nor cooperatively oligomerize. All of these findings have essential implications for the mechanism of chromatin condensation and regulation of genomic function by chromatin. |
