The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.

检查点激酶Rad53在复制压力下被激活，以防止前叉塌陷，这是必不可少的但尚不清楚的过程。在这里，我们显示Rad53在复制压力下耦合前导链和滞后链的合成。在dNTP耗竭的rad53-1细胞中，复制性DNA解旋酶MCM和前链DNA聚合酶Polε移出DNA合成位点，可能解开了模板DNA。值得注意的是，DNA合成沿着后链比前导链进一步进行，导致暴露了长链的单链前导模板。体内dNTPs水平升高可抑制rad53-1细胞中的不对称DNA合成，并且在低dNTP浓度下， Polε的活性比滞后链聚合酶Polδ受到更大的损害。因此，我们建议Rad53通过协调DNA的解链与两条链的合成来防止复制压力下过量ssDNA的产生。