Chromosome–Chromatin: The assembly, architecture, and the control of transcription of the eukaryotic genetic apparatus are analyzed by cytological, biochemical and biophysical techniques such as in vitro cell culture, light and electron microscopy, analytical ultracentrifugation, gel permeation chromatography, light scattering, X-ray crystallography, and microcalorimetry. We have established that the histone "core" of the nucleosome is organized as a tripartite protein entity by the assembly of two dimers of H2A-H2B, one on each side of a centrally located H3-H4 tetramer. The contact interfaces of this assembly are considered as regulatory domains in the functional transitions of chromatin and are being probed by chemical, enzymatic and biophysical probes. We determined the crystal structure of the histone octamer to ca. 2.7Å resolution and discovered the histone fold and the handshake motif. The path of the double helix over the histone octamer has been determined and found to be guided by the 12 repetitive protein paired element motifs (PEMs). Using these geometric constraints, a high resolution model for the nucleosome has been obtained and optimized through molecular dynamics simulations in collaboration with Drs. C.S. Tung and A.E. Garcia of Los Alamos. These simulations revealed that the nucleosome fluctuations are dominated by motions in the DNA backbone. The nucleosome is surrounded by a positive ion cloud with an average local density exceeding by a factor of 5-10 the bulk phase ion concentration. We also see high water density at the protein-DNA boundaries, at the DNA grooves and especially between the two apposing gyres of the nucleosomal DNA. The energetics of the assembly of the nucleosomal components are analyzed microcalorimetrically through collaborations with Prof. Ernesto Freire of the Biocalorimetry Center. The patterns and specificity of protein-DNA interactions are analyzed by chemical, topological and structural methods. Nucleosome reconstitution experiments utilizing core histone subunits (regular and acetylated), transcription factors and specific sequences of circular DNA are in progress to analyze the dynamics and specificities of nucleosome remodelling with particular emphasis on the effects of microenvironmental factors (ions, pH, hormones, etc.). The main model system for these studies is the gypsy insulator of Drosophila (in collaboration with Dr. V. Corces). Bioenergetics: The mechanism of protein thermostability and enzyme thermoactivity in Archaea are studied. Particular emphasis is focused on the role of osmolytes in protein stabilization. - Arents, G., and E.N. Moudrianakis. 1993. Topography of the histone octamer surface: Repeating structural motifs utilized in the docking of nucleosomal DNA. Proc. Natl. Acad. Sci. USA 90:10489-10493.
- Arents, G., and E.N. Moudrianakis. 1995. The histone fold: A ubiquitous architectural motif utilized in DNA compaction and protein dimerization. Proc. Natl. Acad. Sci. USA 92:11170-11174.
- Kruger, W., C.L. Peterson, A. Sil, C. Coburn, G. Arents, E.N. Moudrianakis, and I. Hershowitz. 1995. Amino Acid Substitutions in the Structured Domains of Histones H3 and H4 Partially Relieve the Requirement of the Yeast SWI/SNF Complex for Transcription. Genes Dev. 9:2770-2779.
- Karantza, V., E. Freire, and E.N. Moudrianakis. 1996. Thermodynamic studies of the core histones: pH and ionic effects on the stability of the (H3-H4)/(H3-H4)2 system. Biochemistry 35:2037-2046.
- Pruss, D., B. Bartholomew, J. Persinger, J. Hayes, G. Arents, E.N. Moudrianakis, and A.P. Wolffe. 1996. An asymmetric model for the nucleosome: A binding site for linker histones inside the DNA gyres. Science 274:614-617.
- Santisteban, M.S., G. Arents, E.N. Moudrianakis, and M.M. Smith. 1997. Histone octamer function in vivo: Mutations in the dimer-tetramer interfaces disrupt both gene activation and repression. EMBO J. 16:2493-2506.
- Bal, W., V. Karantza, E.N. Moudrianakis, and K. Kasprzak. 1999. Interaction of Nickel(II) with Histones: In vitro Binding of Nickel (II) to the Core Histone Tetramer. Arch. Biochem. Biophys. 364:161-166.
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