Structure and function of nucleosome assembly protein 1

描述(申请人提供)：真核细胞中的所有DNA都以染色质的形式存在，染色质是DNA与同等质量的蛋白质高度紧密的组合。在染色质组织的第一级，DNA的两个超螺旋缠绕在四个组蛋白的八聚体周围，形成核小体，核小体是染色质中的基本重复单位。在复制和转录过程中，细胞中存在复杂的机制来调节染色质的组装。因子介导的染色质组装过程在功能和机制水平上还不是很清楚，也不知道染色质是如何维持在其典型的高度流动状态的。我们将研究酵母核小体组装蛋白1(yNAP-1)来研究这些核心问题。特别是，将通过测定其晶体结构和在溶液中的齐聚状态来对yNAP-1的结构和功能进行详细的分子分析。将研究yNAP-1与组蛋白复合体之间的相互作用，以及组蛋白尾部修饰对yNAP-1介导的染色质组装的影响。最后，我们将研究yNAP-1与酵母核小体核心颗粒的相互作用，以及它对组蛋白亚基交换的影响。特别是，将实现以下具体目标： 1.酵母NAP-1的高分辨结构和齐聚状态 1.用X射线单晶衍射法测定了yNAP-1的高分辨结构。 1.b.测定yNAP-1在溶液中的齐聚状态。 2.酵母NAP-1与组蛋白组分的相互作用 2.a.测定yNAP-1与组蛋白复合体的化学计量比和相对结合亲和力。 2.b.研究组蛋白尾巴及其修饰在yNAP-1与组蛋白相互作用中的作用。 2.c.研究yNAP-1与酵母组蛋白变异体H_2A.Z的相互作用。 D.确定yNAP-1(或其结构域)与相关组蛋白络合物的晶体结构。 3.酵母NAP-1与核小体核心颗粒的相互作用 3.利用荧光共振能量转移技术(FRET)检测yNAP-1对核小体核心颗粒稳定性的影响。 3.b.在yNAP-1存在和不存在的情况下，测定组蛋白亚基的交换率

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