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.

项目总结 单元格必须在空间和时间上组织其内容。微管细胞骨架及其相关蛋白 分子马达，动力蛋白和动蛋白，是细胞用来移动从蛋白质到货物的主要系统。 组装到整个细胞器，包括细胞核。动力蛋白(~0.5MDa)是AAA+家族的一员。主动型 唯一能够运输货物的动力蛋白复合体是~4MDA，由两个动力蛋白二聚体组成 与~1.0MDa动力蛋白复合体结合，以及连接它们与货物的接头蛋白。LIS1，另一个 动力蛋白的重要调节剂，对它们的形成是必要的。在此之前，我们展示了Lis1如何调节 动力素的机械力化学。在这里，我们将重点了解90 kDa 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为包装问题提供了一种解决方案 基因组，以及一种控制获取基因组的工具。在控制染色质动力学的因素中 是依赖于三磷酸腺苷的核小体重构体，它将三磷酸腺苷的水解酶与非共价修饰结合在一起。 核小体结构。重构器和它们催化的修改都是非常不同的，即使所有 重构体使用相同的机制，并保守的催化核心，打破组蛋白-DNA接触。我是 感兴趣的是这种常见的底层机制是如何调制的，以产生广泛的 重建者有能力实现的结果。在此之前，我们重点介绍了代表以下两项的模型系统 这四个家族的“规范”重建器。在这里，我们将关注的是Rad26(CSB的酵母菌同源基因)，一个“孤儿”。 重建器，利用其类似重塑的活动作用于RNA POL II，帮助其克服障碍或启动 当障碍是DNA损伤时，转录偶联DNA修复(TCR)。我们的目标是了解如何 RAD26帮助RNA Pol II从其他障碍中识别病变，并招募下游修复因子。 我们采取以结构为导向的方法来解决基本的机械问题，并将 电子显微镜(冷冻-EM)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协作中进行测试的机械性假设 包括单分子生物物理、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力化学循环及其调节，以及功能多样性和 核小体重构体的调控。我们还对开发工具来解决我们面临的挑战感兴趣 在低温电磁管道上相遇，为低温电磁网格的准备和开发做出了重要贡献 在云中进行数据处理。我们目前正在开发提高数据效率的方法 收集。