Mechanistic Analysis of Chromatin Remodeling

描述(由申请人提供)： 项目摘要将DNA包装到染色质中，使其在转录、复制、重组和修复等核心核过程中很大程度上无法获得。依赖于ATP的染色质重塑马达在增加染色质内的DNA访问以及产生促进转录抑制的高阶染色质结构方面发挥着关键作用。然而，染色质重塑马达的作用机制在很大程度上还不清楚。这项提议的总体目标是使用从运动蛋白和解旋酶等研究良好的马达中学到的概念来了解主要的依赖于ATP的染色质重塑复合体-人类ACF是如何发挥作用的。ACF产生均匀分布的核小体，以实现更高级别的染色质折叠和基因沉默。我们在上一次授予期间的工作表明，ACF作为一个二聚体马达发挥作用，其中每个ATPase亚单位轮流与核小体的两侧接合。在这里，我们将以这些发现为基础来解决以下问题：(1)ACF中两个ATPase亚基的活性是如何协调的？(2)ACF如何在其反应机制中识别和使用特定的核小体特征？(3)ACF活性是如何受到相邻核小体的存在调节的？

DESCRIPTION (provided by applicant): PROJECT SUMMARY The packaging of DNA into chromatin makes it largely inaccessible for the central nuclear processes of transcription, replication, recombination and repair. ATP-dependent chromatin remodeling motors play key roles in both increasing DNA access within chromatin as well in generating higher-order chromatin structures that promote transcriptional repression. However, the mechanisms by which chromatin remodeling motors function are largely unknown. The overall goal of this proposal is to use concepts learnt from well-studied motors such as kinesin and helicases to understand the how a major ATP- dependent chromatin-remodeling complex, human ACF functions. ACF generates evenly spaced nucleosomes to enable higher-order chromatin folding and gene silencing. Our work over the last grant period has shown that ACF functions as a dimeric motor in which each ATPase subunit takes turns engaging either side of a nucleosome. Here we will build on these discoveries to address the following questions: (1) How are the activities of the two ATPase subunits in ACF coordinated? (2) How does ACF recognize and use specific nucleosomal features in its reaction mechanism? (3) How is ACF activity regulated by the presence of adjacent nucleosomes?