Mechanism of cytoskeletal transport and transcription-coupled DNA repair

细胞骨架运输和转录偶联DNA修复机制

• 批准号: 10795265
• 负责人: Andres Leschziner
• 金额: $ 23.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795265
• 关键词: ATP Hydrolysis; Adaptor Signaling Protein; Address; Binding; Biochemistry; Biological Models; Biophysics; Catalytic Domain; Cell Nucleus; Cell Survival; Cells; Cellular biology; Chromatin; Collaborations; Complex; Coupled; Cryoelectron Microscopy; Cytoskeleton; DNA; DNA Polymerase II; DNA Repair; DNA lesion; Data Collection; Dynein ATPase; Family; Genetic Transcription; Genome; Histones; Kinesin; Lesion; Microtubules; Modification; Molecular Machines; Molecular Motors; Movement; Nucleosomes; Organelles; Orphan; Orthologous Gene; Outcome; Preparation; Process; Proteins; RNA; Regulation; Research; Structure; System; Techniques; Testing; Transcription Initiation; Yeasts; computerized data processing; dimer; dynactin; insight; interest; link protein; member; recruit; repaired; single molecule; tool

PROJECT SUMMARY Cells must organize their contents spatially and temporally. The microtubule cytoskeleton and its associated molecular motors, dynein and kinesin, are the main system used by cells to move cargos, ranging from protein assemblies to entire organelles, including the nucleus. Dynein (~0.5MDa) is a member of the AAA+ family. Active dynein complexes, the only ones capable of transporting cargo, are ~4MDa and consist of two dynein dimers bound to the ~1.0MDa dynactin complex, and an adaptor protein that links them to cargo. Lis1, another essential regulator of dynein, is necessary for their formation. Previously, we showed how Lis1 regulates dynein’s mechanochemistry. Here, we will focus on understanding how the 90kDa Lis1 dimer helps activate and assemble the 4MDa transport complex. Chromatin, with the nucleosome as its basic unit, provides both a solution to the problem of packaging the genome, and a tool to regulate access to it. Among the factors involved in controlling chromatin dynamics are ATP-dependent nucleosome remodelers, which couple ATP hydrolysis to the non-covalent modification of nucleosome structure. Both remodelers and the modifications they catalyze are very diverse, even though all remodelers use the same mechanism, and conserved catalytic core, to break histone-DNA contacts. I am interested in how this common underlying mechanism is modulated to result in the wide array of outcomes of which remodelers are capable. Previously, we focused on model systems representing two of the four families of “canonical” remodelers. Here, we will focus on Rad26 (the yeast ortholog of CSB), an “orphan” remodeler that uses its remodeling-like activity to act on RNA Pol II to help it overcome obstacles or initiate Transcription Coupled DNA Repair (TCR) when the obstacle is a DNA lesion. We aim to understand how Rad26 helps RNA Pol II recognize a lesion from other obstacles and recruit downstream repair factors. We take a structure-guided approach to addressing fundamental mechanistic questions, with cryo- electron microscopy (cryo-EM) as our main technique. We use the structures we generate to formulate mechanistic hypotheses that can be tested, either in house or in collaboration, using a range of techniques including single-molecule biophysics, biochemistry, and cell biology. We have made major contributions to our understanding of the mechanochemical cycle of dynein and its regulation, and to the functional diversity and regulation of nucleosome remodelers. We are also interested in developing tools to solve challenges we encounter along the cryo-EM pipeline and have made important contributions to cryo-EM grid preparation and data processing in the cloud. We are currently developing approaches to increase the efficiency of data collection.

项目概要 细胞必须在空间和时间上组织其内容。微管细胞骨架及其相关 分子马达、动力蛋白和驱动蛋白是细胞用来移动货物的主要系统，这些货物包括蛋白质 组装成整个细胞器，包括细胞核。动力蛋白 (~0.5MDa) 是 AAA+ 家族的成员。积极的 动力蛋白复合物是唯一能够运输货物的复合物，约为 4MDa，由两个动力蛋白二聚体组成 与 ~1.0MDa 动力蛋白复合物结合，以及将它们与货物连接的接头蛋白。 Lis1，另一个 动力蛋白的重要调节剂，是其形成所必需的。之前，我们展示了 Lis1 如何调节 动力蛋白的机械化学。在这里，我们将重点了解 90kDa Lis1 二聚体如何帮助激活 并组装 4MDa 运输综合体。 以核小体为基本单位的染色质，既提供了包装问题的解决方案 基因组，以及调节基因组访问的工具。参与控制染色质动力学的因素 是 ATP 依赖性核小体重塑剂，它将 ATP 水解与非共价修饰结合起来 核小体结构。重塑者和他们催化的修改都是非常多样化的，尽管所有 重塑者使用相同的机制和保守的催化核心来破坏组蛋白-DNA 接触。我是 对如何调节这种共同的基础机制以产生广泛的结果感兴趣 改造者有能力实现的结果。之前，我们重点关注代表两个的模型系统 “规范”改造者的四个家族。在这里，我们将重点关注Rad26（CSB的酵母直系同源物），一个“孤儿” 重塑者，利用其类似重塑的活性作用于 RNA Pol II，帮助其克服障碍或启动 当障碍是 DNA 损伤时进行转录耦合 DNA 修复 (TCR)。我们的目标是了解如何 Rad26 帮助 RNA Pol II 识别来自其​​他障碍的病变并招募下游修复因子。 我们采用结构引导的方法来解决基本的机械问题， 电子显微镜（冷冻电镜）是我们的主要技术。我们使用我们生成的结构来制定 可以使用一系列技术在内部或合作中测试机械假设 包括单分子生物物理学、生物化学和细胞生物学。我们为我们的事业做出了重大贡献 了解动力蛋白的机械化学循环及其调节，以及功能多样性和 核小体重塑剂的调节。我们也有兴趣开发工具来解决我们面临的挑战 沿冷冻电镜管道相遇，并为冷冻电镜网格准备和研究做出了重要贡献 云端数据处理。我们目前正在开发提高数据效率的方法 收藏。