Structural mechanisms of sliding clamp loader ATPases

滑动夹加载器ATP酶的结构机制

• 批准号: 10553147
• 负责人: Brian Anthony Kelch
• 金额: $ 35.18万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10553147
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Apoptosis; Architecture; Attention; Binding; Biological Models; Biopolymers; Cell Proliferation; Cell physiology; Cells; Chromatin Structure; Closure by clamp; Collaborations; Complement; Complex; Coupled; Cryoelectron Microscopy; Crystallography; DNA; DNA Binding; DNA Repair; DNA Structure; DNA biosynthesis; Dedications; Development; Disparate; Fluorescence Spectroscopy; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Homeostasis; Kinetics; Life; Link; Malignant Neoplasms; Mechanics; Methodology; Molecular Conformation; Nature; Okazaki fragments; Pathway interactions; Pharmaceutical Preparations; Process; Proteins; Reaction; Resolution; SLC19A1 gene; Science; Shapes; Sister Chromatid; Slide; Structure; Sumoylation Pathway; Testing; Therapeutic; Time; Work; antimicrobial; chemotherapy; cohesion; ds-DNA; flexibility; genome integrity; innovation; insight; molecular dynamics; nanomachine; novel; replication factor C; therapeutic target

All life forms require a ring-shaped sliding clamp to coordinate replication of their genome. These sliding clamps act as master regulators of DNA replication, coordinating replisome action with other cellular processes. These master regulators are themselves regulated by large ATPase machines called clamp loaders that either install or remove sliding clamps from DNA. This project seeks to gain an atomic-level understanding of clamp loader mechanism. These protein remodeling machines open the sliding clamp ring as a key step in their action. We have found that in the key intermediate complex—consisting of an open clamp, an ATP-bound clamp loader and the target DNA—the protein components form an open spiral that matches the helical symmetry of DNA. This symmetric spiral activates ATP hydrolysis leading to clamp closure and release of the loaded clamp. In Aim 1, we now turn our attention to the critical first two steps of the reaction: the opening of the clamp and the binding of DNA to the inner chamber of the complex. We will identify the conformational changes in the clamp loader complex that allow for opening the clamp ring, as well as how the assembly can rapidly bind a specific DNA structure in the tight confines of the complex's interior. In Aim 2, we investigate how the single subunit change in the clamp loader complex (Rfc1 replaced with Elg1) converts a dedicated clamp loader into a dedicated unloader. This work will not only reveal the mechanism and structure of a key protein involved in cancer development, but will also provide a blueprint for how an ATPase machine can be reprogrammed to perform a reverse reaction. Finally, in Aim 3 we explore how replacement of the Rfc1 subunit with the Ctf18 protein leads to an assembly that is bifunctional as both a loader and unloader, and that connects DNA replication to the process of sister chromatid cohesion. Our structures and analysis of this complex will reveal how an ATPase machine can be mechanistically flexible to catalyze both forward and reverse reactions. In addition, this work will provide insight into how this mysterious complex can link the seemingly disparate processes of DNA replication and sister chromatid cohesion. Because clamp loaders and sliding clamps are fundamental to all life, the structural insights that we obtain from completing our aims will be used for developing novel antimicrobial or chemotherapeutic drugs.

所有生命形式都需要环形滑动夹来协调其基因组的复制。这些 滑动夹作为 DNA 复制的主调节器，协调复制体的作用 其他细胞过程。这些主调节器本身由大型 ATP 酶机器调节 称为夹具装载机，用于从 DNA 上安装或移除滑动夹具。该项目旨在 获得对夹具加载机制的原子级理解。这些蛋白质重塑机器 打开滑动夹环作为其动作的关键步骤。我们发现在关键 中间复合体——由开放钳、ATP结合钳加载器和目标DNA组成—— 蛋白质成分形成一个开放的螺旋，与 DNA 的螺旋对称性相匹配。这个对称的 螺旋激活 ATP 水解，导致夹具闭合并释放加载的夹具。在目标 1 中， 现在我们将注意力转向反应的关键前两个步骤：打开夹具 以及 DNA 与复合物内室的结合。我们将鉴定构象 夹钳装载机综合体的变化允许打开夹环，以及如何 组装可以在复合物内部的紧密范围内快速结合特定的 DNA 结构。 在目标 2 中，我们研究了夹钳装载复合物中的单个亚基如何变化（Rfc1 替换为 与Elg1）将专用夹具装载机转换为专用卸载机。这项工作不仅将 揭示参与癌症发展的关键蛋白质的机制和结构，但也将 提供了如何对 ATP 酶机器进行重新编程以执行逆反应的蓝图。 最后，在目标 3 中，我们探讨了用 Ctf18 蛋白替换 Rfc1 亚基如何导致 具有装载机和卸载机双重功能的装配体，它将 DNA 复制连接到 姐妹染色单体凝聚的过程。我们对这个综合体的结构和分析将揭示 ATPase 机器如何在机械上灵活地催化正向和反向 反应。此外，这项工作将深入了解这个神秘的复合体如何联系起来 DNA 复制和姐妹染色单体凝聚的看似不同的过程。因为夹 装载机和滑动夹具是所有生命的基础，我们从中获得的结构见解 完成我们的目标将用于开发新型抗菌药物或化疗药物。