Structural mechanisms of sliding clamp loader ATPases

项目总结/摘要 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，协调复制体与其他细胞的作用。 流程.这些主调节器本身是由大型ATP酶机称为钳加载器调节 安装或移除DNA上的滑动夹。这个项目旨在获得原子级的理解 夹具装载机机构。这些蛋白质重塑机打开滑动夹环作为一个关键步骤， 他们的行动。我们已经发现，在关键的中间复合物-由一个开放的钳，一个ATP结合的， 钳加载器和目标DNA-蛋白质组分形成一个开放的螺旋，匹配的螺旋 DNA的对称性这种对称螺旋激活ATP水解，导致钳夹闭合和释放 加载夹具。在目标1中，我们现在将注意力转向反应的关键前两步： 夹子和DNA与复合物内腔的结合。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，以及组件如何 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，我们研究如何 夹具加载器复合体中的单个子单元变化（用Elg 1替换Rfc 1）将专用夹具 装载机装入专用卸载机。这项工作不仅将揭示一个关键蛋白质的机制和结构 参与癌症的发展，但也将提供一个蓝图，如何ATP酶机可以 重新编程以执行逆反应。 最后，在目标3中，我们探索了Ctf 18蛋白如何取代Rfc 1亚基导致组装 这是双功能的装载器和卸载器，并连接DNA复制的过程中，姐妹 染色单体凝聚力我们对这种复合物的结构和分析将揭示ATP酶机器是如何 机械上灵活，以催化正向和反向反应。此外，这项工作将提供洞察力， 这个神秘的复合体是如何将DNA复制和姐妹复制的看似不同的过程联系起来的。 染色单体凝聚力因为夹具装载机和滑动夹具是所有生命的基础， 我们从完成目标中获得的见解将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.