Structure and Function of Nucleosome Assembly Protein 1

描述(由申请人提供)：染色质扮演着两个基本的角色，但本质上是相互排斥的。一方面，染色质是DNA致密程度极高的原因，这使得真核基因组能够适应细胞核的范围。另一方面，它必须允许DNA的局部解体，以允许细胞机器访问基因组。调节不同水平紧密染色质状态之间相互转换的活动是所有需要获得基因组DNA的过程的关键调节因素。对这些生物相关过程的机械和结构洞察是有限的。在这里，将研究核小体组装蛋白1(NAP1)家族在维持和调节染色质结构和流动性方面的作用。目标1和目标2使用X射线衍射以及生化和活体方法，解决了伴侣与组蛋白相互作用的结构和分子基础。将通过严格的动力学分析结合体内染色质结构的检测来研究NAP1介导组蛋白去除和组蛋白交换的机制和生物学作用(目标3)。最后，翻译后修饰调节后生动物中NAP1活性的新假设将在目标4中得到检验。这一提议的力量在于结合了广泛的结构、生物物理、生化和遗传学方法，以解决如何将紧凑的DNA提供给细胞机械这一非常重要的问题。

DESCRIPTION (provided by applicant): Chromatin plays two essential, yet by their very nature mutually exclusive roles. On one hand, chromatin is responsible for the extreme degree of DNA compaction that allows the eukaryotic genome to fit into the confines of the nucleus. On the other hand, it must permit the local unraveling of DNA to grant access of the cellular machinery to the genome. Activities that mediate the inter- conversion between various levels of compacted chromatin states are key regulators of all processes requiring access to genomic DNA. Mechanistic and structural insight into these biologically relevant processes is limited. Here, the role of the nucleosome assembly protein 1 (NAP1) family of histone chaperones in maintaining and modulating chromatin structure and fluidity will be investigated. Aims 1 and 2 address the structural and molecular basis for chaperone interaction with histones, using x-ray diffraction as well as biochemical and in vivo approaches. The mechanism and biological role of NAP1-mediated histone removal and histone exchange will be studied by rigorous kinetic analysis combined with in vivo examination of chromatin structure (aim 3). Finally, the novel hypothesis that a posttranslational modification regulates NAP1 activity in metazoans will be put to test in aim 4. The strength of this proposal lies in a combination of a broad spectrum of structural, biophysical, biochemical and genetic approaches to address the highly significant question of how compacted DNA is made available to the cellular machinery.