The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

中文翻译：

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RepC-PcrA 复合物的 DNA 切口和解旋动力学。

滚环复制是革兰氏阳性菌中携带抗生素抗性基因的小质粒复制的最常见机制。它是由复制起始蛋白 (Rep) 对双链复制起点的结合和切口启动的。然后，双链体解旋由 PcrA 解旋酶执行，其持续合成能力通过与 Rep 的相互作用而得到显着促进。Rep 和 PcrA 蛋白如何相互作用以切口和解旋双链体尚不完全清楚。在这里，我们使用磁力镊子监测 PcrA 解旋酶解旋及其与金黄色葡萄球菌质粒 pT181 起始子 RepC 切口活性的关系。我们的结果表明，PcrA 是一种高度持续性的解旋酶，容易出现随机暂停，导致平均易位率为 30 bp s-1，而在没有暂停的情况下，典型的易位速度为 50 bp s-1。单链DNA结合蛋白不影响PcrA易位速度，但略微增加其持续合成能力。对 RepC 切口所需的 DNA 超螺旋程度以及 RepC 切口和 DNA 解旋之间的时间的分析表明，RepC 和 PcrA 在切口之前在 DNA 结合位点上形成蛋白质复合物。提出了一个合理化这些发现的综合模型。