DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or “nicks,” which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, “runs off” the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress.

DNA 损伤阻碍复制叉的进程并威胁基因组的稳定性。在遇到大多数 DNA 加合物时，复制型 CMG 解旋酶 (CDC45-MCM2-7-GINS) 从合成点停止或解偶联，但最终恢复复制。然而，对于哺乳动物细胞中大量存在的单链断裂或“缺口”对复制的影响知之甚少。使用非洲爪蟾在鸡蛋提取物中，我们发现 CMG 与前导链模板中的缺口碰撞会产生平端双链断裂 (DSB)。此外，环绕前导链模板的 CMG “跑掉”了 DSB 的末端。相比之下，CMG 与滞后链缺口的碰撞会产生带有单链悬垂的断端。在这种情况下，CMG 沿着双链 DNA 转移到断裂处，然后通过复制终止期间使用的相同途径从染色质中泛素化和去除。我们的研究结果表明，切口是独特的危险 DNA 损伤，总是会导致复制体分解，并且它们表明 CMG 不能储存在 dsDNA 上，而细胞解决复制压力。