Structure and function of nucleosome assembly protein 1

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DESCRIPTION (provided by applicant): All DNA in eukaryotic cells exists in the form of chromatin, a highly compacted assembly of DNA with an equal mass of proteins. At the first level of chromatin organization, two superhelical turns of DNA are wrapped around an octamer of four histone proteins to form the nucleosome, the basic repeating unit in chromatin. Elaborate machinery exists in the cell to mediate chromatin assembly during replication and transcription. The process of factor-mediated chromatin assembly is not well understood at a functional and mechanistic level, nor is it known how chromatin is maintained in its typical highly fluid state. Nucleosome Assembly Protein 1 from yeast (yNAP-1) will be studied to investigate these central questions. In particular, the structure and function of yNAP-1 will be analyzed in molecular detail by determining its crystal structure and its oligomerization state in solution. The interaction between yNAP-1 and histone complexes, and the effect of histone tail modifications on yNAP-1 - mediated chromatin assembly will be investigated. Finally, the interaction of yNAP-1 with the yeast nucleosome core particle, and its effect on the exchange of histone subunits will be investigated. In particular, the following specific aims will be pursued: 1. High-resolution structure and oligomerization state of yeast NAP-1 1.A. Determine the high-resolution structure of yNAP-1 by x-ray crystallography. 1.B. Determine the oligomerization state of yNAP-1 in solution. 2. Interaction of yeast NAP-1 with histone components 2.A. Determine the stoichiometry and relative binding affinity of yNAP-1 for histone complexes. 2.B. Investigate the role of histone tails and their modifications in the interaction between yNAP-1 and histones. 2.C. Characterize the interaction of yNAP-1 with the yeast histone variant H2A.Z. 2.D. Determine the crystal structure of yNAP-1 (or domains thereof) with the relevant histone complexes. 3. Interaction of yeast NAP-1 with the nucleosome core particle 3.A. Measure the effect of yNAP-1 on the stability of the nucleosome core particle, using fluorescence resonance energy transfer (FRET). 3.B. Determine the rate of exchange of histone subunits in the presence and absence of yNAP-1