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: 10405228
• 负责人: Andres Leschziner
• 金额: $ 63.01万
• 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/10405228
• 关键词: ATP Hydrolysis; Adaptor Signaling Protein; Address; 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; 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.

项目摘要 细胞必须经常和临时组织其内容。微管细胞骨架及其相关 分子电动机，动力蛋白和驱动蛋白是细胞移动cargos的主要系统，范围从蛋白质 包含整个细胞器，包括核。 Dynein（〜0.5MDA）是AAA+家族的成员。积极的 Dynein复合物是唯一能够运输货物的综合体，是〜4MDA，由两个动力蛋白二聚体组成 与〜1.0MDA dynactin复合物的结合，以及将它们与货物联系起来的衔接蛋白。 Lis1，另一个 动力蛋白的基本调节剂对于它们的形成是必不可少的。以前，我们展示了LIS1如何调节 Dynein的机械化学。在这里，我们将专注于了解90KDA LIS1二聚体如何帮助激活 并组装4MDA运输综合体。 染色质，以核小体为基本单位，既可以解决包装问题的解决方案 基因组和调节访问权限的工具。在控制染色质动力学的因素中 是依赖ATP 核小体结构。重塑器和他们催化的修改都非常多样化，即​​使 远方使用相同的机制，并构成催化核心，以打破组蛋白-DNA接触。我是 对如何调制这种常见的基本机制以导致广泛的阵列感兴趣 远程有能力的结果。以前，我们专注于代表两个的模型系统 “规范”远程的四个家庭。在这里，我们将重点关注RAD26（CSB的酵母直系同源），一个“孤儿” 使用其重塑活性来对RNA POL II作用的改建器，以帮助其克服障碍或启动 当障碍物是DNA病变时，转录耦合DNA修复（TCR）。我们旨在了解如何 RAD26有助于RNA POL II识别其他障碍物的病变，并募集下游修复因子。 我们采用一种结构引导的方法来解决基本的机械问题，而冷冻 电子显微镜（Cryo-EM）作为我们的主要技术。我们使用生成的结构来制定 可以使用一系列技术在内部或协作中测试的机械假设 包括单分子生物物理学，生物化学和细胞生物学。我们为我们的主要贡献做出了重大贡献 了解动力蛋白及其调节的机械化学周期，以及功能多样性和 调节核病组偏远。我们也有兴趣开发解决挑战的工具 沿着冷冻管道遇到，并为冷冻EM电网制备做出了重要贡献 云中的数据处理。我们目前正在开发提高数据效率的方法 收藏。