RNA helicase DHX9 is essential for genome stability by resolving aberrant R-loops. However, its regulatory mechanisms remain unclear. Here we show that SUMOylation at lysine 120 (K120) is crucial for DHX9 function. Preventing SUMOylation at K120 leads to R-loop dysregulation, increased DNA damage, and cell death. Cells expressing DHX9 K120R mutant which cannot be SUMOylated are more sensitive to genotoxic agents and this sensitivity is mitigated by RNase H overexpression. Unlike the mutant, wild-type DHX9 interacts with R-loop-associated proteins such as PARP1 and DDX21 via SUMO-interacting motifs. Fusion of SUMO2 to the DHX9 K120R mutant enhances its association with these proteins, reduces R-loop accumulation, and alleviates survival defects of DHX9 K120R. Our findings highlight the critical role of DHX9 SUMOylation in maintaining genome stability by regulating protein interactions necessary for R-loop balance.

RNA 解旋酶 DHX9 通过解决异常 R 环而对于基因组稳定性至关重要。然而，其监管机制仍不清楚。在这里，我们表明赖氨酸 120 (K120) 处的 SUMO 化对于 DHX9 功能至关重要。阻止 K120 处的 SUMOylation 会导致 R 环失调、DNA 损伤增加和细胞死亡。表达不能被 SUMO 化的 DHX9 K120R 突变体的细胞对基因毒性剂更敏感，并且这种敏感性通过 RNase H 过表达而减轻。与突变体不同，野生型 DHX9 通过 SUMO 相互作用基序与 R 环相关蛋白（如 PARP1 和 DDX21）相互作用。 SUMO2 与 DHX9 K120R 突变体的融合增强了其与这些蛋白质的结合，减少了 R 环积累，并减轻了 DHX9 K120R 的生存缺陷。我们的研究结果强调了 DHX9 SUMOylation 通过调节 R 环平衡所需的蛋白质相互作用来维持基因组稳定性的关键作用。