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协调它们基因组的复制。这些滑块 钳子作为DNA复制的主要调节器，与其他细胞协调复制体的活动 流程。这些主调节器本身由称为钳位装载机的大型ATPase机器进行调节 在DNA上安装或移除滑动夹子。这个项目寻求获得原子级别的理解 夹持式装载机机构。这些蛋白质重塑机器打开滑动夹环是 他们的行动。我们发现，在关键的中间复合体中--由一个开放的钳，一个结合的ATP--组成 钳制加载器和靶DNA-蛋白质成分形成一个开放的螺旋，与螺旋相匹配 DNA的对称性。这种对称的螺旋激活了三磷酸腺苷的水解，导致钳夹关闭和释放 装填的夹具。在目标1中，我们现在将注意力转向反应的前两个关键步骤：打开 钳制和DNA与复合体内腔的结合。我们将确定构象 夹具加载器复合体中允许打开夹持环的变化，以及组件如何 在复合体内部的严密范围内迅速结合特定的DNA结构。在目标2中，我们调查了 钳位加载器复合体中的单亚基改变(用Elg1取代Rfc1)转换专用钳位 将装载机装入专用卸载机。这项工作不仅将揭示一种关键蛋白质的机制和结构 参与癌症的发展，但也将为ATPase机器如何成为 重新编程以执行反向反应。 最后，在目标3中，我们探索了用Ctf18蛋白替换Rfc1亚单位如何导致组装 这是作为加载器和卸载器的双重功能，并将DNA复制与SISTER过程联系起来 染色单体凝聚力。我们对这个复合体的结构和分析将揭示ATPase机器如何 机械上灵活，既能催化正向反应，也能催化反向反应。此外，这项工作将提供洞察力 这个神秘的复合体是如何将看似完全不同的DNA复制和姐妹过程联系起来的 染色单体凝聚力。由于夹具装载机和滑动夹具是所有生命的基础，因此结构 我们从完成目标中获得的见解将被用于开发新的抗菌剂或 化疗药物。

Project Summary/Abstract 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.