DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11–RAD50–NBS1 (MRN) to endonucleolytically cleave 5′-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase–nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.

中文翻译：

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PLK1 调节 CtIP 和 DNA2 在长距离 DNA 末端切除中的相互作用

DNA 双链断裂 (DSB) 修复是通过 DNA 末端切除启动的。CtIP 在短程切除中发挥作用，刺激 MRE11–RAD50–NBS1 (MRN) 以核酸内切方式切割 5' 末端 DNA，以绕过蛋白质块。CtIP 还促进 DNA2 解旋酶-核酸酶加速 MRN 下游的远程切除。在这里，使用 AlphaFold2，我们将 CtIP-F728E-Y736E 鉴定为功能分离突变体，它仍然能够熟练地与 MRN 结合，但不能刺激 DNA2 的 ssDNA 降解。因此，CtIP-F728E-Y736E 会损害与 DNA2 的物理相互作用。细胞分析显示，CtIP-F728E-Y736E 细胞表现出 DSB 依赖性染色质结合 RPA 减少、远距离切除受损以及对 DSB 诱导药物的敏感性增加。此前，CtIP被证明可以被PLK1靶向抑制远距离切除，但其潜在机制尚不清楚。我们发现 CtIP 中的 DNA2 相互作用区域包括位于 S723 的 PLK1 靶位点。CtIP 中 S723 的完整性对于刺激 DNA2 是必要的，因此体外 PLK1 对 CtIP 的磷酸化具有抑制作用，这解释了为什么 PLK1 限制远距离切除。我们的数据支持这样一个模型：CtIP 的 CDK 依赖性磷酸化激活 S 期 MRN 的切除，而 PLK1 介导的 CtIP 磷酸化会破坏 CtIP 对 DNA2 的刺激，从而减弱 G2/M 后期的长程切除。