SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1(-/-) cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2 alpha(+/-) mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2 alpha is SUMOylated during mitosis, the TOP2 alpha(+/-) mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1(-/-) cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons. (c) 2012 Elsevier Inc. All rights reserved.