The DNA damage response (DDR) is essential for the maintenance of genomic stability. Protein posttranslational modifications play pivotal roles in regulating the DDR process. Here, we found that SUMOylated RNF168 undergoes liquid-liquid phase separation (LLPS), which restricts the recruitment of RNF168 to DNA damage sites, reduces RNF168-catalyzed H2A ubiquitination, restrains 53BP1 in nuclear condensates, and ultimately impairs non-homologous DNA end joining (NHEJ) repair efficiency. SENP1 was identified as a specific deSUMOylase of RNF168, and it was highly expressed in colorectal adenocarcinoma. In response to DNA damage, SENP1 decreased RNF168 SUMOylation and prevented RNF168 from forming nuclear condensates, thus promoting damage repair efficiency and cancer cell resistance to DNA damaging agents. Moreover, high SENP1 expression correlated with poor prognosis in cancer patients, and SENP1 depletion sensitized cancer cells to chemotherapy. In summary, these findings reveal DDR is suppressed by SUMOylation-induced LLPS of RNF168 and suggest that SENP1 is a potential target for cancer therapy.
