The activation of DNA-dependent kinase (DNA-PKcs) upon DNA damage contains a cascade of reactions, covering acetylation by TIP60, binding with Ku70/80, and autophosphorylation. However, how cells regulate TIP60-mediated acetylation of DNA-PKcs and the following DNA-PKcs activation upon DNA damage remains obscure. This present study reported that TIP60 is hyper-SUMOylated in normal conditions, but upon irradiation-induced DNA damage, small ubiquitin-like modifier (SUMO)-specific protease 3 (SENP3)-mediated deSUMOylation of TIP60 promoted its interaction with DNA-PKcs to form the TIP60-DNA-PKcs complex. We show that TIP60 SUMOylation is reduced quickly in response to DNA damage and the deSUMOylation of TIP60 by SENP3 is required for DNA-PKcs acetylation and its autophosphorylation. Comet and γH2AX immunofluorescence assay showed that knockdown of SENP3 impaired DNA damage repair. Using the NHEJ report system, we found that knockdown of SENP3 affected the efficiency of NHEJ. Further exploration using clonogenic survival assay, cell viability assay and cytoflow assay suggested that leaking SENP3 increased the sensitivity of tumour cells to serval DNA damage treatment. Overall, our findings revealed a previously unidentified role of SENP3 in regulating DNA-PKcs activity and DNA damage repair.

中文翻译：

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DNA-PKcs 活性和 DNA 损伤修复需要 SENP3 介导的 TIP60 去SUMO化。

DNA 依赖性激酶 (DNA-PKcs) 在 DNA 损伤后的激活包含一系列反应，包括 TIP60 的乙酰化、与 Ku70/80 的结合和自磷酸化。然而，细胞如何调节 TIP60 介导的 DNA-PKcs 乙酰化以及随后的 DNA 损伤后 DNA-PKcs 激活仍然不清楚。本研究报告称，TIP60 在正常条件下超 SUMO 化，但在辐射诱导的 DNA 损伤后，小泛素样修饰剂 (SUMO) 特异性蛋白酶 3 (SENP3) 介导的 TIP60 去 SUMO 化促进其与 DNA-PKcs 的相互作用形成 TIP60-DNA-PKcs 复合物。我们表明，TIP60 SUMOylation 迅速减少以响应 DNA 损伤，并且 SENP3 对 TIP60 的 deSUMOylation 是 DNA-PKcs 乙酰化及其自磷酸化所必需的。Comet 和 γH2AX 免疫荧光测定表明，SENP3 的敲低会损害 DNA 损伤修复。使用 NHEJ 报告系统，我们发现 SENP3 的敲低影响 NHEJ 的效率。使用克隆形成存活测定、细胞活力测定和细胞流测定的进一步探索表明，泄漏的 SENP3 增加了肿瘤细胞对服务 DNA 损伤治疗的敏感性。总的来说，我们的研究结果揭示了 SENP3 在调节 DNA-PKcs 活性和 DNA 损伤修复中的一个以前未被识别的作用。