Structural Studies of Nucleosomes

University Of Tennessee Knoxville, Knoxville TN

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Abstract

This research will elucidate the structural organization of genomic DNA and associated nuclear proteins using detailed models developed from crystallographic studies of our published nucleosome core particle (NCP) structure (PDB entry 1EQZ). From this structure, the NCP will be extended to near atomic resolution. A variety of defined DNA sequenced and methylated histone H3 will be used to study the variable nature of DNA-histone interactions in chromatin. In addition, 3-D structural models of modified NCP that relate to centromeric nucleosomes and nucleosomal transcription complexes will be developed. The initial objectives are to extend the current 2.5 A resolution structure of the NCP to approximately 2.0 A resolution. Target macromolecules will all used defined sequence DNA: these include the 146 base pair palindromic sequence from human alpha-satellite DNA originally pioneered by this group; an alternate palindrome composed of the other half of the original alpha-satellite DNA sequence; and alpha-satellite DNA sequences of modified length. The constitutive histone proteins of the core particles as well as the proteins involved in histone modification or DNA methylation will be derived from recombinant sources. The molecular refinement of the structures will proceed using the laboratory's 2.5A nucleosome core particle structure as the model for molecular replacement. Data collection at synchrotron light sources will be necessary to achieve the resolution levels desired. The original NCP model from this research program will lead to high- resolution information about nucleosome variants, and provide initial models of macromolecular complexes associated with histone or DNA modifications based on the crystal structures of selected transcription regulator-nucleosome assemblages. This information is of fundamental importance to understanding the structure of DNA and chromatin as well as the controlling role of nucleosomes in modulating gene expression. This information is of fundamental importance to understanding the structure of DNA and chromatin as well as the controlling role of nucleosomes in modulating gene expression. The structural information attained will contribute to an understanding of the molecular basis of carcinogenesis, transcription related genetic disease, and will provide a framework for the modeling of complex biological assemblages.