The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase-polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.

中文翻译：

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Rad53限制了复制叉处CMG解旋酶从DNA合成中解偶联。

复制叉处DNA展开和合成的协调促进了染色体DNA的有效忠实复制。复制压力期间破坏解旋酶和聚合酶活性之间的平衡会导致叉子进展缺陷和Rad53检查点激酶的激活，这对于停滞的复制叉子的功能维持至关重要。Rad53依赖的前叉稳定机制尚不清楚。使用重组的出芽酵母复制体，我们显示前导链DNA聚合酶，Polε，dNTP耗竭以及DNA聚合酶的化学抑制作用的突变失活导致复制性DNA解旋酶CMG过度释放DNA，表明出芽酵母复制体缺乏内在机制控制叉子处的解旋酶-聚合酶偶联。重要的，