The long range goal of this project is to understand the nature of the forces and the chemical basis of specificity responsible for the structural organization of the eukaryotic genetic material, and the regulation of differential gene expression at the molecular level. For the next five years we want to focus on the biophysical chemistry of the assembly of the octamer histone core of the nucleosome. The interdisciplinary work described here is aimed at the quantitative analysis of this histone octamer. This complex, composed of two of each of the four polypeptides of the """"""""inner histones"""""""" H2A, H2B, H3 and H4, is responsible for the primary compaction of the DNA double helix in all eukaryotic organisms and must also be involved at least in some aspect of the regulation of chromosomal replication and transcription.
The specific aims of this project are: a) to provide a thorough analysis of the assembly of this octameric protein complex and to determine the energetics of the association reactions; b) to characterize the contact interfaces among the subunits of the histone octamer in order to elucidate the temporal and spatial specificities involved in the functional regulation of this fundamental and universal protein entity; c) to enzymatically alter these interfaces and correlate these structural changes with changes in the functional properties of this chromatin both in normal and deregulated growth; and d) to link the findings of this study with the results of our crystallographic analysis of the histone octamer at 3.5 A resolution. This part of the work is done in collaboration with Dr. W. Love, and it is presented here solely to aid in the analysis of its scientific impact on the overall project. No funds are requested for it. We also have on-going studies on the solution physicochemical properties of the histone octamer and its subunits. We will continue to investigate their association behavior by ultracentrifugation, gel-permeation and chromatographic techniques, microcalorimetry, CD-spectroscopy, hydrogen-exchange, and modern biochemical and enzymatic techniques. The results of the proposed studies will further the understanding of the properties of the genetic material and hopefully elucidate one of the ways its deregulation can lead to uncontrolled transcription and proliferation, and are thus relevant to the areas of Cell Biology, Genetics, and Carcinogenesis.
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