Differences in clamp loader mechanism between bacteria and eukaryotes.
Differences in clamp loader mechanism between bacteria and eukaryotes.
复制标题
细菌和真核生物之间的夹具加载机制的差异。
DOI：
10.1101/2023.11.30.569468
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Kelch,BrianA
中科院分区：
文献类型：
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作者：
Landeck,JacobT;Pajak,Joshua;Norman,EmilyK;Sedivy,EmmaL;Kelch,BrianA
钳制加载器是五聚体ATPase，它在DNA上放置圆形滑动钳子，在那里它们在DNA复制和基因组完整性方面发挥作用。夹子加载器的中心活动是打开环形滑动夹子，以及随后与引物-模板(p/t)连接的结合。夹具装载机的总体结构在整个生命周期内都是保守的，这表明它们的机构被保留了下来。最近对真核细胞钳制加载器复制因子C(RFC)的结构研究表明，它是通过蟹爪机制发挥作用的，其中钳制的打开与加载器的大量构象变化有关。在这里，我们用冷冻电子显微镜(Cryo-EM)研究了高分辨率的大肠杆菌夹持加载器的夹持加载机制。我们发现，大肠杆菌的钳夹加载器在单个枢轴点使用蟹爪运动来打开钳子，而真核细胞的RFC加载器使用分布在整个复合体的运动。此外，我们发现夹具的打开有多个步骤，从具有螺旋构象的部分打开状态开始，到以令人惊讶的平面几何形状的广泛打开的夹具进行。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启动从加载的夹具中释放。我们的结果揭示了大分子机器中的机械差异，这台机器在生命的所有领域都是保守的。
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 E. coli clamp loader at high resolution using cryo-electron microscopy (cryo-EM). We find that the E. coli 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 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.