Structural mechanisms of sliding clamp loader ATPases

项目摘要/摘要 所有的生命形式都需要一个环形的滑动夹子来复制它们的基因组。这些滑动夹子 作为DNA复制的主要调节者，与其他细胞协调复制体的活动 流程。这些主调节器本身受大型ATPase机器的监管，称为 从DNA上安装或移除滑动夹子的钳位装载机。这个项目寻求的是 对夹具装载机机构有原子级的理解。这些蛋白质重塑机器 打开滑动夹环作为他们行动的关键一步。我们在钥匙里发现了 作为开放钳制、三磷酸腺苷结合钳制加载器和DNA的中间环节，蛋白质组分形成一种 与DNA的螺旋对称性相匹配的螺旋。这种对称的螺旋激活了三磷酸腺苷的水解 从而导致夹具关闭并松开夹具。在目标1中，我们现在将注意力转向 关键的前两步反应：打开夹子，然后将DNA结合到内部 建筑群的密室。我们将确定钳夹加载器中的构象变化 允许打开夹环的复合体，以及组件如何快速绑定 在复合体内部的严密范围内有特殊的DNA结构。在目标2中，我们调查 钳位加载器复合体(用Elg1取代Rfc1)中的单个亚基如何变化 将专用夹具装载机改装成专用卸料机。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 如何对ATPase机器进行编程以执行反向反应的蓝图。 最后，在目标3中，我们探索了用Ctf18替换Rfc1亚基如何导致 将DNA复制连接到姐妹染色单体凝聚过程的组件，并且是 既是装载机又是卸货机的两用设备。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转发和 反向反应。此外，这项工作将提供对这个神秘的情结如何 可以将DNA复制和姐妹染色单体凝聚这两个看似完全不同的过程联系起来。 由于夹具装载机和滑动夹具是所有生命的基础，我们的结构见解 从完成我们的目标中获得的东西对于制定小说的策略将是无价的 抗菌药物或化疗药物。

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.