Mechanistic Analysis of Chromatin Remodeling

 描述（由申请人提供）：DNA包装到染色质中，使其在很大程度上无法进入转录、复制、重组的中心核过程 和修复。ATP依赖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中起关键作用。然而，染色质重塑马达的功能机制在很大程度上是未知的。本提案的总体目标是使用从深入研究的电机，如驱动蛋白和解旋酶的概念，了解如何一个主要的ATP依赖的染色质重塑复合物，人类ACF的功能。ACF作为二聚体马达产生均匀间隔的核小体。有序的核小体间距使得更高阶的染色质折叠和基因沉默成为可能。我们在当前资助期内的工作导致了意外的发现，ACF使用ATP在完整的组蛋白八聚体内诱导大量的扭曲。我们进一步发现，在二聚ACF内的两个ATP酶原体之间的协调依赖于核小体线索的特异性识别。在此，我们将基于这些发现来解决以下基本问题：（1）ACF如何利用ATP的能量来扭曲核小体构象，以及这种扭曲如何与核小体运动耦合？(2)两个ACF启动子的活动是如何协调的，以及在这一过程中如何使用底物线索？

 DESCRIPTION (provided by applicant): 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 how a major ATP dependent chromatin-remodeling complex, human ACF functions. ACF functions as a dimeric motor to generate evenly spaced nucleosomes. The ordered nucleosome spacing enables higher-order chromatin folding and gene silencing. Our work over the current grant period has led to the unexpected finding that ACF used ATP to induce substantial distortions within an intact histone octamer. We have further found that coordination between the two ATPase protomers within a dimeric ACF relies on specific recognition of nucleosomal cues. Here we will build on these discoveries to address the following fundamental questions: (1) How does ACF use the energy of ATP to distort nucleosome conformation and how is this distortion coupled to nucleosome movement? (2) How are the activities of the two ACF promoters coordinated and how are substrate cues used in this process?