The MRE11–RAD50–NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.

MRE11–RAD50–NBS1 (MRN) 复合物在细胞对 DNA 双链断裂 (DSB) 的反应中起着重要作用，这是最具细胞毒性的 DNA 损伤，并且是各种修饰和控制的目标。最近，在缺乏 RECQL4 解旋酶活性的细胞中观察到 NBS1 的赖氨酸 48 连接泛素化，导致 MRN 复合物从 DSB 位点过早分解。然而，在具有完整 RECQL4 的细胞中 DSB 反应期间这种泛素化的作用和控制仍然未知。在这里，我们发现 USP2 在 DSB 响应期间抵消了这种泛素化并稳定了 MRN 复合体。通过筛选增加 RECQL4 缺陷细胞中 MRN 复合物稳定性的去泛素化酶，USP2 被确定为一种新的去泛素化酶，它作用于 DSB 位点以抵消 NBS1 泛素化。我们确定 USP2 以依赖于 ATM 的方式被招募到 DSB 位点，ATM 是一种针对 DSB 的主要检查点激酶，并在免疫沉淀实验中与 NBS1 和 RECQL4 稳定相互作用。ATM 对 USP2 N 末端的两个关键残基进行磷酸化是将其募集到 DSB 及其与 RECQL4 的相互作用所必需的。虽然单独使 USP2 失活不会显着影响 DSB 响应，但我们发现 USP2 和 USP28（另一种影响 NBS1 泛素化的去泛素化酶）的失活会导致 MRN 复合物从 DSB 位点过早分解以及 ATM 激活和同源重组修复缺陷能力。这些结果表明，去泛素化酶抵消 NBS1 泛素化对于 MRN 复合物的稳定维持和对 DSB 的适当细胞反应至关重要。