Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1–DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD′s presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep′s activities as initiators and as helicases.

中文翻译：

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葡萄球菌自加载解旋酶将楼梯机制与域间高灵活性相结合

复制是一个至关重要的细胞过程。复制解旋酶解开 DNA，为聚合酶提供模板链并促进复制叉进程。解旋酶是使用 ATP 酶结构域将 ATP 水解与易位结合的多结构域蛋白质，但是其他结构域在易位期间可能具有的作用仍然难以捉摸。在这里，我们研究了存在于金黄色葡萄球菌致病岛 (SaPIs) 中的未被探索的自加载解旋酶，称为 Reps。我们对来自 SaPI5 (3.3 Å) 的 PriRep5、来自 SaPI1 (3.9 Å) 的 Rep1 和 Rep1-DNA 复合物 (3.1 Å) 的冷冻电镜结构表明，在两个 Reps 中，C 末端结构域 (CTD) 经历了两个不同的运动，即ATP酶域。我们在体外和体内实验证明 SaPI 编码的代表需要参与易位阶梯机制的关键氨基酸。此外，我们证明 CTD 的存在对于维持完整的 ATPase 和解旋酶活性是必要的。我们推测这种高域间灵活性将 Rep 的活动作为引发剂和解旋酶结合起来。