A comparative structural study of ATP-dependent chromatin remodeling complexes

项目总结 核小体是真核生物的基本DNA包装单位。依赖于ATP的核小体重塑 复合体(“重构体”)利用三磷酸腺苷的水解来非共价改变它们的结构来调节基因组。 动力学。从酵母到人类的改构体，尽管共享一种 保守的催化亚基：一种3‘-5’dsDNA转位酶，与核小体结合并断裂组蛋白-DNA 通过将DNA推进到核小体表面来接触和改变核小体的结构。 重构体ATPase的侧翼是定义了4个亚家族的附属结构域：ISWI、CHD、SWI/SNF 和INO80。这些亚家族的复杂性不同，从单亚单位重构体(ISWI和CDH)到 大的，多亚基1MDA+复合体(SWI/SNF和INO80)。不同的亚家族催化不同的结果。 “孤儿”重构体，其易位酶不属于这4个亚家族中的任何一个，尽管它们保守 ATPase结构域，则没有得到很好的描述。这项建议的首要目标是理解 重建器功能多样性的机械基础。ISWI和CHD重建器，在 最小和最被理解的基因，提供了对它们如何调节移位酶以产生 特定的重塑结果。虽然已经提出了概念模型来解释功能 SWI/SNF和INO80重建器的专门化缺少机械性的理解。我们所知的更少 关于孤儿改造者的事。 在这个提案中，我们将处理表示机械理解的功能的模型系统 是缺乏的。对于孤儿重建器，我们将重点关注RAD26，Cockayne综合征的酿酒酵母同源基因 蛋白质B(CSB)是一种蛋白质，长期以来一直以其在转录偶联DNA修复中的作用而闻名，而且 我们最近展示了利用其DNA易位来帮助RNA聚合酶II克服转录障碍。 对于SWI/SNF重建器，我们将重点放在RSC Complex上，这是S。 能够滑动和喷射组蛋白八聚体的啤酒。对于INO80大家庭，我们将继续努力 在SWR1复合体上，其独特的能力是在不改变的情况下交换核小体中的组蛋白二聚体 它的立场。 我们将结合使用结构(低温电子显微镜)和生化方法来 了解这些不同的重构体如何调节它们的DNA转位酶来产生他们的 重塑的结果。 好了！

PROJECT SUMMARY Nucleosomes are the basic DNA packaging unit in eukaryotes. ATP-dependent nucleosome remodeling complexes (“remodelers”) use ATP hydrolysis to non-covalently alter their structure to regulate genome dynamics. Remodelers, conserved from yeast to humans, generate a wide range of products despite sharing a conserved catalytic subunit: a 3’-5’ dsDNA translocase that binds to the nucleosome and breaks histone-DNA contacts and alters the structure of nucleosomes by propelling DNA over their surface. Remodeler ATPases are flanked by accessory domains that define 4 subfamilies: ISWI, CHD, SWI/SNF and INO80. These subfamilies differ in complexity, ranging from single-subunit remodelers (ISWI and CDH), to large, multi-subunit 1MDa+ complexes (SWI/SNF and INO80). Different subfamilies catalyze different outcomes. “Orphan” remodelers, whose translocases do not belong to any of the 4 subfamilies despite their conserved ATPase domains, are less well characterized. The overarching goal of this proposal is to understand the mechanistic underpinnings of the functional diversity of remodelers. ISWI and CHD remodelers, among the smallest and best understood, have provided insights into how they regulate their translocases to produce their specific remodeling outcomes. Although conceptual models have been proposed to explain the functional specialization of SWI/SNF and INO80 remodelers a mechanistic understanding is missing. Even less is known about orphan remodelers. In this proposal, we will tackle model systems representing functions for which mechanistic understanding is lacking. For orphan remodelers, we will focus on Rad26, the S. cerevisiae ortholog of the Cockayne Syndrome protein B (CSB), a protein that has long been known for its role in Transcription Coupled DNA Repair, and that we recently showed uses its DNA translocation to help RNA Polymerase II overcome transcriptional obstacles. For the SWI/SNF remodelers, we will focus on the RSC complex, an abundant and essential remodeler from S. cerevisiae capable of both sliding and ejecting histone octamers. For the INO80 family, we will continue working on the SWR1 complex, which is unique in its ability to exchange histone dimers in a nucleosome without altering its position. We will use a combination of structural (cryo-electron microscopy) and biochemical approaches to understand how these different remodelers regulate their DNA translocases to produce their specialized remodeling outcomes. !