Structural mechanisms of sliding clamp loader ATPases

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 滑动夹作为DNA复制的主要调节器，协调复制体的活动 其他细胞过程。这些主调节器本身受大型ATPase机器的监管 所谓的夹子加载器，它可以安装或移除DNA的滑动夹子。这一项目旨在 对夹具装载机机构有一个原子级的了解。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 中间复合体-由开放的钳、结合三磷酸腺苷的钳夹加载器和靶DNA组成- 蛋白质组分形成一个开放的螺旋，与DNA的螺旋对称性相匹配。这种对称性 螺旋激活三磷酸腺苷的水解，导致钳夹的关闭和被加载的钳位的释放。在目标1中， 现在我们将注意力转向反应的前两个关键步骤：夹子的打开 以及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具环的变化，以及如何 组装可以在复合体内部的严密范围内快速结合特定的DNA结构。 在目标2中，我们研究了钳位加载器复合体(Rfc1被取代)中单个亚基的变化 使用Elg1)将专用夹具装载机转换为专用卸料机。这项工作不仅将 揭示一种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机器重新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蛋白替换Rfc1亚单位如何导致 作为加载器和卸载器的两种功能的组件，并将DNA复制连接到 姐妹染色单体凝聚的过程。我们对这个建筑群的结构和分析将揭示 ATPase机器如何在机械上灵活地催化前进和后退 反应。此外，这项工作将提供对这个神秘的情结如何联系起来的洞察 看似完全不同的DNA复制和姐妹染色单体凝聚的过程。因为夹具 装载机和滑动夹具是所有生命的基础，我们从这些结构中获得的洞察力 完成我们的目标将用于开发新型抗菌药物或化疗药物。

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.