Structural mechanisms of sliding clamp loader ATPases

滑动夹加载器ATP酶的结构机制

• 批准号: 10797120
• 负责人: Brian Anthony Kelch
• 金额: $ 2.97万
• 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/10797120
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Attention; Binding; Cell Proliferation; Cell physiology; Closure by clamp; Complex; DNA; DNA Binding; DNA Structure; DNA biosynthesis; Dedications; Development; Disparate; Genome; Life; Link; Malignant Neoplasms; Mechanics; Molecular Conformation; Nature; Pharmaceutical Preparations; Process; Proteins; Reaction; SLC19A1 gene; Shapes; Sister Chromatid; Slide; Structure; Therapeutic; Work; antimicrobial; chemotherapy; cohesion; flexibility; insight; nanomachine; novel strategies

Project Summary/Abstract All life forms require a ring-shaped sliding clamp for copying 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 of open clamp, ATP-bound clamp loader and DNA, the protein components form a spiral that matches the helical symmetry of DNA. This symmetric spiral activates ATP hydrolysis leading to clamp closure and release of the clamp. In Aim 1, we now turn our attention to the critical first two steps of the reaction: opening the clamp, and then binding 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 complexes’ interior. In Aim 2, we investigate how the single subunit change in the clamp loader complex (Rfc1 replaced with Elg1) can convert 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 programmed to perform the reverse reaction. Finally, in Aim 3 we explore how replacement of the Rfc1 subunit with Ctf18 leads to an assembly that connects DNA replication to the process of sister chromatid cohesion and is bifunctional as both a loader and unloader. 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 we obtain from completing our aims will be invaluable for developing strategies for novel antimicrobial or chemotherapeutic drugs.

项目摘要/摘要 所有生命形式都需要一个环形滑动夹以复制其基因组。这些滑动夹 充当DNA复制的主要调节剂，将复制体作用与其他细胞进行协调 过程。这些主调节器本身由称为的大型ATPase机器调节 从DNA中安装或卸下滑动夹的夹具装载机。这个项目试图获得 对夹具加载机机制的原子级别的理解。这些蛋白质重塑机 打开滑动夹环作为动作的关键步骤。我们发现在钥匙中 开放夹，ATP结合的夹具装载机和DNA的中间体，蛋白质成分形成A 与DNA的螺旋对称性相匹配的螺旋。这种对称螺旋激活ATP水解 导致夹具闭合并释放夹具。在AIM 1中，我们现在将注意力转移到 反应的临界前两个步骤：打开夹具，然后结合DNA与内部结合 综合体的腔室。我们将确定夹具加载器中的构象变化 可以打开夹环的复合物，以及组件如何快速结合A 在复合物内部的紧密范围内的特定DNA结构。在AIM 2中，我们调查 夹具装载机复合物中的单个亚基如何变化（用ELG1代替RFC1）可以 将专用的夹具装载机转换为专用的卸载器。这项工作不仅会揭示 参与癌症发展的关键蛋白质的机制和结构，但也将提供 如何对ATPase机器进行编程以执行反向反应的蓝图。 最后，在AIM 3中，我们探讨了如何用CTF18替换RFC1亚基 将DNA复制连接到姐妹染色单体内聚的过程的组装和IS 双功能作为装载机和卸载器。我们对这个复合物的结构和分析 揭示ATPase机器如何在机械上灵活地催化前进和 反应。此外，这项工作将提供有关这种神秘复合体的洞察力 可以将DNA复制的看似不同的过程与姐妹染色单体内聚力联系起来。 因为夹具装载机和滑动夹是所有生命的基础，所以我们的结构见解 从完成我们的目标中获得的目标对于制定新颖的策略是无价的 抗菌或化学治疗药物。