Biochemical, Genetic, and Genomic Analysis of Nucleosome Reorganization by FACT

项目摘要/摘要 该提案涉及组蛋白伴侣Fact和Spt6用来促进组装的机制， 染色质的拆解和修复。了解染色质是如何形成和维持的是 了解转录、DNA复制和修复的调控。酿酒酵母是 用作模型系统，拥有强大的遗传、生化和基因组工具可用于研究 所有真核生物共有的基本过程。这里提出的高度协作的方法充分利用了 通过同时使用广泛的方法来利用这些工具。FACT使用多个组蛋白- 结合结构域将核小体转换为另一种结构形式(重组的核小体) 其中DNA和组蛋白都更容易获得。它在没有ATP水解的情况下做到了这一点，区分了它 来自染色质改变剂。重组是可逆的，所以FACT也可以将核小体组装成 松散地结合DNA和组蛋白来构建染色质。Spt6还与组蛋白、DNA和核小体结合， 但不会导致重组。比较这两个监护人的活动和功能会有所帮助 我们了解这些因素如何既能阻止组蛋白相互作用，又能在 适当的时间，然而每个人都有其他人不能发挥的独特的生理作用。事实是 被视为管家因素，通过放松转录和DNA复制促进转录和DNA复制 遇到允许聚合酶通过染色质的核小体。这一观点受到了挑战 我们正在用一个模型来取代它，在这个模型中，事实建立和维持染色质 在精确优化的体系结构中，并在转录或复制等干扰后恢复这种形式。 这项提案的一个关键目标是继续挑战现有模式，并为提出和 测试新款。目标1解决的问题是，是什么推动了事实和事实的差异本地化 Spt6，以及它们如何促进或抑制核小体局部周转，以塑造全基因组的染色质结构。 重组可以促进核小体的组装或拆解，但我们不知道 重组后的表单是什么样子的，或者是什么影响了它的解析方式。Aim 2使用两个不同的单项来检查这一点 分子方法在与Ha和Studitsky实验室的合作中，以及我们现有的和新兴的 检测影响核小体、组蛋白和伴侣蛋白的特征的生化分析 重组。目标3检查了所有事实复合体共有的DNA结合模块的作用， 但其结构因物种而异。这些方法结合在一起将为持久化提供答案 关于组蛋白伴侣蛋白使用的机制的问题，以及关于其 生理角色。

Project Summary/Abstract This proposal addresses mechanisms used by the histone chaperones FACT and Spt6 to promote assembly, disassembly, and repair of chromatin. Understanding how chromatin is formed and maintained is central to understanding regulation of transcription, DNA replication, and repair. The yeast Saccharomyces cerevisiae is used as a model system that has powerful genetic, biochemical, and genomic tools available to study fundamental processes common to all eukaryotes. The highly collaborative approach proposed here takes full advantage of these tools by using a broad range of methods simultaneously. FACT uses multiple histone- binding domains to convert nucleosomes into an alternative structural form (the reorganized nucleosome) in which both the DNA and the histones are more accessible. It does this without ATP hydrolysis, distinguishing it from chromatin remodelers. Reorganization is reversible, so FACT can also assemble nucleosomes out of loosely associated DNA and histones to construct chromatin. Spt6 also binds histones, DNA, and nucleosomes, but does not induce reorganization. Comparing the activities and functions of these two chaperones will help us understand how each of these factors can both block histone interactions and promote them at the appropriate times, yet each has unique physiological roles that the other cannot perform. FACT has been viewed as a housekeeping factor that promotes transcription and DNA replication by loosening each nucleosome encountered to allow polymerases to progress through chromatin. This view has been challenged and we are in the process of replacing it with a model in which FACT acts to establish and maintain chromatin in a precisely optimized architecture, and to restore this form after disturbances like transcription or replication. A key goal of this proposal is to continue challenging the existing model and to contribute to proposing and testing the new one. Aim 1 addresses questions about what drives the differential localization of FACT and Spt6, and how they promote or inhibit nucleosome turnover locally to sculpt chromatin structure genome-wide. Reorganization can promote assembly or disassembly of nucleosomes, but we do not know what the reorganized form looks like or what influences how it is resolved. Aim 2 examines this using two distinct single molecule methods in collaborations with the Ha and Studitsky labs, as well as our established and emerging biochemical assays examining the features of nucleosomes, histones, and chaperones that affect reorganization. Aim 3 examines the role of the DNA binding module that is common to all FACT complexes, but whose architecture varies among species. Together these approaches will provide answers to persistent questions about the mechanisms used by histone chaperones, as well as new questions about their physiological roles.