Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the clamp loader at high resolution using cryo-electron microscopy. We find that the clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

中文翻译：

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细菌和真核生物在夹钳加载机制（DNA 复制的保守过程）方面的差异

夹具加载器是五聚体 ATP 酶，可将圆形滑动夹具放置在 DNA 上，在 DNA 复制和基因组完整性中发挥作用。夹具装载机的中心活动是打开环形滑动夹具以及随后与引物-模板（p/t）连接处的结合。夹钳装载机的总体结构在整个生命周期中都是保守的，这表明它们的机制得到了保留。最近对真核生物钳装载机复制因子 C (RFC) 的结构研究表明，它利用蟹爪机制发挥作用，其中钳的打开与装载机的巨大构象变化相结合。在这里，我们使用冷冻电子显微镜以高分辨率研究夹具装载机的夹具加载机制。我们发现夹具装载机在单个枢轴点使用蟹爪运动打开夹具，而真核 RFC 装载机使用分布在整个复合体上的运动。此外，我们发现夹子的打开发生在多个步骤中，从具有螺旋构象的部分打开状态开始，并继续到令人惊讶的平面几何形状的全开夹子。最后，我们在存在 p/t 结的情况下的结构说明了夹具如何围绕 p/t 结闭合以及夹具加载器如何开始从加载的夹具上释放。我们的结果揭示了大分子机器的机械差异，这种差异在生命的所有领域中都是保守的。