RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

中文翻译：

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依赖FUS的液-液相分离对于DNA修复的启动很重要。

RNA结合蛋白（RBP）逐渐成为细胞DNA损伤反应（DDR）的重要效应子。RBP FUS参与RNA代谢和DNA修复，并在体外经历可逆的液-液相分离（LLPS）。在这里，我们证明依赖FUS的LLPS是启动DDR所必需的。在FUS敲除细胞中使用激光微辐射，我们显示FUS是募集DDR因子KU80，NBS1和53BP1和SFPQ（与DDR有关的另一个RBP）的DNA损伤位点的必需条件。LLPS抑制剂或缺乏LLPS的FUS变体同样会损害KU80，NBS1和SFPQ的重定位。我们还表明，LLPS对于有效的γH2AX灶形成必不可少。最后，使用超分辨率结构照明显微镜，我们证明缺乏FUS会损害γH2AX纳米焦点向高阶簇的正确排列。这些发现表明，在DDR的激活和DSB修复复合物的正确组装中，对FUS依赖的LLPS的早期需求。