Replication fork collision with a DNA nick can generate a one-ended break, fostering genomic instability. The opposing fork’s collision with the nick could form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase “step arrest” nickase in mammalian cells. A Flp-nick induces two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR pathways induced by a replication-independent break and the Flp-nickase differ in their dependence on BRCA1, MRE11, and CtIP. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a Tus/Ter replication fork barrier (RFB). Flp-nickase-induced STGC remained robust and two ended. Thus, a single replication fork’s collision with a Flp-nick triggers two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of nicked DNA.

中文翻译：

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Flp 切口酶断裂复制叉处的双端重组


复制叉与 DNA 缺口的碰撞会产生单端断裂，从而促进基因组的不稳定性。相反的叉子与缺口的碰撞可以形成第二个 DNA 末端，从而通过同源重组 （HR） 进行保守修复。为了研究切口酶诱导的 HR 的机制，我们在哺乳动物细胞中开发了 Flp 重组酶“步停”切口酶。Flp 缺口诱导两端、BRCA2/RAD51 依赖性短束基因转换 （STGC）、BRCA2/RAD51 非依赖性长束基因转换和不协调的两端侵袭。由复制非依赖性断裂和 Flp 切口酶诱导的 HR 通路在对 BRCA1 、 MRE11 和 CtIP 的依赖性上有所不同。为了确定 Flp 切口酶诱导的 STGC 中第二个 DNA 末端的来源，我们使用 Tus/Ter 复制叉屏障 （RFB） 阻断了相反的叉。Flp 切口酶诱导的 STGC 保持稳健，并且 2 结束。因此，单个复制叉与 Flp 缺口的碰撞会触发两端 HR，可能反映了滞后链缺口的复制旁路。这种反应可以限制带切口的 DNA 复制过程中的基因组不稳定性。