Chromatin structure and architecture. In the cell nucleus, DNA is packaged into chromatin. While various models describing the condensed 30-nm chromatin fiber observed in vitro have been proposed, evidence for the 30-nm fiber in vivo, except for the essentially inactive chromatin in mature avian erythrocytes, is still lacking. We are interested in understanding chromatin structure in vivo, as well as the topological constraints imposed by organizing proteins such as CTCF. We had previously shown that the N- and C-terminal domains flanking the DNA binding 11 zinc fingers of CTCF are intrinsically disordered, and found that related DNA binding proteins share this property. Current work focuses on characterizing the physical nature of these unstructured domains, identifying partners that bind with high affinity, and studying the complexes formed, with the aim of understanding how CTCF, and related proteins, regulate higher order genome organization within the eukaryotic nucleus. In related work carried out with Dr. Bai, we had shown that the binding of linker histone H5 results in chromatin arrays that are more compact than those reconstituted with linker histone H1, resulting from the on-dyad symmetric binding mode of H5. We have now identified the linker histone residues responsible for on- or off-dyad binding, demonstrating that a small number of residues in the globular domain of the linker histones play critical roles in determining the nucleosome binding mode (Zhou et al., 2016). Altogether, this work demonstrates how key residues on the linker histones regulate diverse binding, and the higher order 30-nm chromatin structure. Macromolecular assemblies of biological interest. Biological assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. In collaboration with the Craigie lab we have focused our attention on the HIV-1 strand transfer intasome. We show by analytical ultracentrifugation that the complex consists predominantly of an integrase tetramer with the branched DNA substrate, along with higher order oligomers. These studies aided in the preparation and purification of the complex, and complemented the cryo-electron microscopy structure described in Passos et al. (2017).
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