Nucleosome structure and conformational states

This dissertation describes the study of chromatin and nucleosomes by biochemical and biophysical methods. The techniques used include circular dicroism, thermal denaturation, analytical ultracentrifugation, nuclease digestion pattern, and electron microscopy.

Initially, the effect of urea on the structure of nucleosomes and chromatin was studied with the idea of to the defining different conformational states available nucleosome. It was found that there are two domains in the nucleosome which respond differently to urea: a DNA-rich outer shell and an a-helix-rich protein core. The conformation and stability of the DNR shell exhibited noncooperative changes from 0-10 M urea. The core protein, however, cooperatively lost its m-helical around 5 M urea. The observation that different domains of the nucleosome exhibited different responses to urea, a simple chemical perturbant, offers a basis for viewing the altered conformation of transcriptionally active chromatin, Modulations of the DNA-rich shell (e.g. expansion and destabilization of the DNR) by nonhistone proteins or histone modifications might occur with only minor alterations in the conformation of the protein-rich core, permitting rapid renaturation to the transcriptionally inactive chromatin configuration.

In order to study the effect of DNA sequence and base composition on nuclaosoinal structure, histone-DNR complexes containing chicken inner histones (H2A, H2B, H3, R4) and a variety of DNAs, including the synthetics poly (dA-dT) • poly(aA-dT) and poly (dg-dC) • poly (dG-dC) were reconstructed. The properties of the reconstructed complexes were very similar to those of H1- and H5-depleted chromatin. Monomer nucleosomes prepared of by digestion were these histone-DNA complexes with micrococcal nuclease clear homogeneous with respect to DHA length and exhibited nucleosomal structure as determined by circular dichroism, thermal denaturation, DNase 1 digestion, and electron microscopy. It was possible to obtain good reconstruction of a wide between chicken erythrocyte inner histones and DNA variety of G+C%.

Nucleosomes containing poly{dA-dT» gave detailed information about the internal structure of nucleosomes. DNase The average helix pitch of the DNA was determined by DNA digestion to be ~10.5 bases/turn. The helix pitch in the central region of the nucleosome, however, appeared to differ from the pitch of the DNA at the ends of the nucleosome. Thus the nucleosome appeared to have two domains of DNA differing in secondary structure. Nucleosomes containing poly(dA-dT) were also sensitive indicators of conformation changes in structure due to temperature and ionic strength.