Single-molecule studies of ATP-dependent chromatin remodeling

描述(由申请人提供)：DNA作为染色质的包装调节了许多重要的细胞过程，这些过程需要获得细胞的遗传物质。负责调节染色质结构的一类主要酶是依赖于ATP的染色质重构体。这些酶在从DNA复制、修复、重组和转录到调节细胞命运决定的各种生物过程中发挥着至关重要的作用。染色质重塑酶功能障碍可导致多种癌症，如乳腺癌、肺癌、胰腺癌、前列腺癌和横纹肌样癌，以及许多多系统发育障碍。剖析染色质重塑的功能作用和开发新的治疗方法来对抗与重塑功能障碍相关的疾病，需要从机械上理解染色质重塑背后的生化和生物物理原理。我们的长期目标是建立一个详细的机制了解染色质重塑酶如何利用ATP水解的自由能来扰乱组蛋白-DNA接触，并改变核小体的位置、结构和组成。在这个项目中，我们将重点研究ISWI和SWI/SNF家族重建器的重塑机制。确定染色质重塑的机制需要对重塑反应的动力学进行定量表征。单分子技术非常适合于这一目的，因为它们允许我们实时监控复杂的分子过程，直接观察中间态，并剖析反应路径。在这个项目中，我们将 利用单分子荧光共振能量转移，结合互补生化分析，研究染色质重塑及其调控的机制。我们将解决三个具体目标。目的1：我们将研究ISWI家族重构体催化的核小体重塑动力学。特别是，我们将确定核小体在重塑过程中的结构动力学，并探索不同核小体位置的重塑行为是如何协调的。我们的目标是测试不同的机制模型，并加深我们对ISWI家族酶如何沿DNA转运核小体的理解。目的2：依赖于三磷酸腺苷的染色质重构体的活性受到多种因素的复杂调控。为此，我们计划研究几个生物相关的核小体特征，包括DNA连接子长度和组蛋白修饰/变异，如何调节ISWI的重塑活动。目的3：虽然不同家族的重塑酶具有相同的ATPase结构域，但它们表现出不同的重塑活性，并调节不同的生物学过程。在目标3中，我们将把我们的研究扩展到SWI/SNF家族重构体，并将它们与ISWI家族进行比较，目的是找出这两个主要的染色质重构体家族在核小体重塑机制上的关键共同点和差异。

DESCRIPTION (provided by applicant): The packaging of DNA as chromatin regulates many important cellular processes that require access to the cell's genetic material. One major class of enzymes responsible for regulating the structure of chromatin is the ATP-dependent chromatin remodelers. These enzymes play essential roles in a variety of biological processes ranging from DNA replication, repair, recombination and transcription to the regulation of cell fate decisions. Dysfunction of chromatin remodeling enzymes can cause a variety of cancers, such as breast, lung, pancreatic, prostate, and rhabdoid cancers, as well as a number of multisystem developmental disorders. Dissecting the functional roles of chromatin remodelers and developing novel therapies to combat diseases related to remodeler dysfunction require a mechanistic understanding of the biochemical and biophysical principles underlying chromatin remodeling. Our long-term goal is to develop a detailed mechanistic understanding of how chromatin remodeling enzymes use the free energy of ATP hydrolysis to disrupt histone- DNA contacts and alter the position, structure, and composition of nucleosomes. In this project, we will focus on investigating the remodeling mechanisms of the ISWI and SWI/SNF family remodelers. Determining the mechanisms of chromatin remodeling requires quantitative characterizations of the dynamics of the remodeling reaction. Single-molecule techniques are well suited for this purpose as they allow us to monitor complex molecular processes in real time, directly observe intermediate states, and dissect reaction pathways. In this project, we will use single-molecule fluorescence resonance energy transfer, in conjunction with complementary biochemical assays, to study the mechanisms of chromatin remodeling and its regulation. We will address three specific aims. Aim 1: We will investigate the nucleosome remodeling dynamics catalyzed by ISWI family remodelers. In particular, we will determine the structural dynamics of the nucleosome during remodeling and probe how remodeling actions at different nucleosomal sites are coordinated. We aim to test different mechanistic models and advance our understanding of how ISWI family enzymes translocate nucleosomes along DNA. Aim 2: The activity of ATP-dependent chromatin remodelers is under intricate regulation by a variety of factors. In this aim, we plan to study how several biologically relevant nucleosomal features, including DNA linker length and histone modifications/variants, regulate ISWI remodeling activity. Aim 3: While remodeling enzymes from different families share a homologous ATPase domain, they display different remodeling activities and regulate different biological processes. In Aim 3, we will extend our studies to SWI/SNF family remodelers and compare them with the ISWI family, aiming to identify key commonalities and differences in the nucleosome remodeling mechanisms used by these two major families of chromatin remodelers.