Cellular feedback systems ensure genome maintenance during DNA replication. When replication forks stall, newly replicated DNA is protected by pathways that limit excessive DNA nuclease attacks. Here we show that WEE1 activity guards against nascent DNA degradation at stalled forks. Furthermore, we identify WEE1-dependent suppression of cyclin-dependent kinase 2 (CDK2) as a major activity counteracting fork degradation. We establish DNA2 as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells. In addition, WEE1 appears to be unique among CDK activity suppressors in S phase because neither CHK1 nor p21 promote fork protection as WEE1 does. Our results identify a key role of WEE1 in protecting stalled forks, which is separate from its established role in safeguarding DNA replication initiation. Our findings highlight how WEE1 inhibition evokes massive genome challenges during DNA replication, and this knowledge may improve therapeutic strategies to specifically eradicate cancer cells that frequently harbor elevated DNA replication stress.

细胞反馈系统确保 DNA 复制期间的基因组维持。当复制叉停止时，新复制的 DNA 受到限制过度 DNA 核酸酶攻击的途径的保护。在这里，我们表明 WEE1 活动可防止在停滞的叉处新生 DNA 降解。此外，我们确定 WEE1 依赖性抑制细胞周期蛋白依赖性激酶 2 (CDK2) 作为抵消叉退化的主要活动。我们将 DNA2 确定为导致 WEE1 抑制细胞中叉过度降解的核酸酶。此外，WEE1 在 S 期的 CDK 活性抑制因子中似乎是独一无二的，因为 CHK1 和 p21 都不像 WEE1 那样促进分叉保护。我们的结果确定了 WEE1 在保护停滞叉方面的关键作用，这与其在保护 DNA 复制起始中的既定作用是分开的。