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.

项目摘要 细胞必须在空间和时间上组织它们的内容。微管细胞骨架及其相关的 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， 整个细胞器，包括细胞核。动力蛋白（约0.5MDa）是AAA+家族的成员。活性 动力蛋白复合物是唯一能够运输货物的复合物，分子量约为4 MDa，由两个动力蛋白二聚体组成 结合到~1.0MDa dynactin复合物，以及连接它们与货物的接头蛋白。LIS 1，另一个 动力蛋白的重要调节剂，是它们形成所必需的。之前，我们展示了Lis 1如何调节 动力蛋白的机械化学在这里，我们将专注于了解90 kDa的Lis 1二聚体如何帮助激活 组装4 MDa转运复合体 以核小体为基本单位的染色质， 基因组，以及一个工具来调节访问它。在控制染色质动力学的因素中， 是ATP依赖性核小体重塑剂，其将ATP水解偶联到 核小体结构重塑剂和它们催化的修饰都是非常多样的，尽管所有的 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 感兴趣的是，这种共同的基本机制是如何调制，以导致广泛的 重塑者所能达到的效果在此之前，我们主要关注代表以下两个方面的模型系统： 四个家族的“典型”重塑者。在这里，我们将重点放在Rad 26（CSB的酵母直系同源物），一个“孤儿”， 一种重塑剂，利用其重塑样活性作用于RNA Pol II，以帮助其克服障碍或启动 转录偶联DNA修复（TCR），当障碍物是DNA损伤时。我们的目标是了解 Rad 26帮助RNA Pol II识别来自其他障碍物的损伤并募集下游修复因子。 我们采取结构导向的方法来解决基本的机械问题，与低温， 电子显微镜（cryo-EM）作为我们的主要技术。我们用生成的结构来表达 可以在内部或合作中使用一系列技术进行测试的机制假设 包括单分子生物物理学、生物化学和细胞生物学。我们为我们的发展做出了重大贡献。 了解动力蛋白的机械化学循环及其调节，以及动力蛋白的功能多样性， 调节核小体重塑。我们也有兴趣开发工具来解决我们面临的挑战， 沿着遇到低温EM管道，并为低温EM网格制备做出了重要贡献， 数据处理在云端。我们目前正在开发提高数据效率的方法 收藏.