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

项目总结/摘要 该提案涉及组蛋白伴侣FACT和Spt 6用于促进组装的机制， 染色质的分解和修复。了解染色质是如何形成和维持的， 理解转录、DNA复制和修复的调节。酿酒酵母是 作为一个模型系统，具有强大的遗传，生物化学和基因组工具可供研究 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 这些工具的优点，同时使用广泛的方法。FACT使用多种组蛋白- 结合结构域将核小体转化为替代结构形式（重组核小体） DNA和组蛋白都更容易接近。它不需要ATP水解， 来自染色质重塑。重组是可逆的，所以FACT也可以从核小体中组装核小体。 松散结合的DNA和组蛋白来构建染色质。Spt 6还结合组蛋白、DNA和核小体， 但不引起重组。比较这两种伴侣的活动和功能将有所帮助 我们了解这些因素中的每一个如何既能阻止组蛋白相互作用，又能促进组蛋白相互作用。 适当的时间，但每一个有独特的生理作用，另一个不能执行。事实已 被认为是一种管家因子，通过松开每个 核小体，以允许聚合酶通过染色质。这一观点受到了挑战 我们正在用FACT建立和维持染色质的模型来取代它， 在一个精确优化的结构中，并在转录或复制等干扰后恢复这种形式。 该提案的一个关键目标是继续挑战现有模式，并促进提出和 测试新的。目标1解决了关于是什么驱动FACT的差异定位的问题， spt 6，以及它们如何促进或抑制局部核小体周转，从而在全基因组范围内塑造染色质结构。 重组可以促进核小体的组装或拆卸，但我们不知道是什么， 重新组织的形式看起来像什么或什么影响它是如何解决的。目标2使用两个不同的单个 与Ha和Studitsky实验室合作的分子方法，以及我们建立的和新兴的 生物化学测定检查核小体，组蛋白和分子伴侣的特征， 重组。目的3研究DNA结合模块的作用，这是所有FACT复合物所共有的， 但其结构因物种而异。这些方法将共同为持久的 关于组蛋白伴侣使用的机制的问题，以及关于它们的新问题， 生理作用。

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.