Structural mechanisms of sliding clamp loader ATPases

滑动夹加载器ATP酶的结构机制

• 批准号: 10092193
• 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 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092193
• 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; DNA; DNA Binding; DNA Repair; DNA Structure; DNA biosynthesis; Development; Ensure; 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; Sister Chromatid; Slide; Structure; Testing; Time; Work; antimicrobial; chemotherapy; cohesion; ds-DNA; flexibility; genome integrity; innovation; insight; molecular dynamics; nanomachine; novel; replication factor C

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.

所有的生命形式都需要一个环形的滑动夹来协调它们基因组的复制。这些 滑动夹作为DNA复制的主要调节器，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 称为夹加载器，它可以安装或移除DNA上的滑动夹。本项目谋求 获得原子级的理解钳加载机制。这些蛋白质重塑机器 打开滑动夹环是其动作的关键步骤。我们发现在钥匙里 中间复合物-由开放钳、ATP结合的钳加载器和靶DNA组成- 蛋白质组分形成与DNA的螺旋对称性相匹配的开放螺旋。此对称 螺旋激活ATP水解，导致夹钳闭合和负载夹钳的释放。在目标1中， 我们现在将注意力转向反应的关键的前两步：打开夹钳 以及DNA与复合物内室的结合。我们将确定构象 允许打开夹紧环的夹紧加载器复合体的变化，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。 在目的2中，我们研究了在钳装载复合物中单个亚基的变化（Rfc 1被替换 使用Elg 1）将专用夹具装载机转换为专用卸载机。这项工作不仅将 揭示了参与癌症发展的关键蛋白质的机制和结构，但也将 为如何重新编程ATP酶机器以执行逆反应提供了蓝图。 最后，在目标3中，我们探索了Ctf 18蛋白如何取代Rfc 1亚基， 一种具有装载和卸载双重功能的组装体，它将DNA复制连接到 姐妹染色单体凝聚的过程。我们对这个复合体的结构和分析将揭示 ATP酶机器如何在机械上灵活地催化正向和反向 反应.此外，这项工作将提供深入了解如何这个神秘的复杂可以链接 DNA复制和姐妹染色单体凝聚的看似不同的过程。因为夹钳 装载机和滑动夹是所有生命的基础，我们从结构上获得的见解， 完成我们的目标将用于开发新的抗微生物或化疗药物。