The cysteine protease separase triggers anaphase onset by cleaving chromosome-bound cohesin. In humans, separase also cleaves itself at multiple sites, but the biological significance of this reaction has been elusive. Here we show that preventing separase auto-cleavage, via targeted mutagenesis of the endogenous hSeparase locus in somatic cells, interferes with entry into and progression through mitosis. The initial delay in mitotic entry was not dependent on the G2 DNA damage checkpoint, but rather involved improper stabilization of the mitosis-inhibiting kinase Wee1. During M phase, cells deficient in separase auto-cleavage exhibited striking defects in spindle assembly and metaphase chromosome alignment, revealing an additional early mitotic function for separase. Both the G2 and M phase phenotypes could be recapitulated by separase RNA interference and corrected by re-expressing wild-type separase in trans. We conclude that separase auto-cleavage coordinates multiple aspects of the G2/M programme in human cells, thus contributing to the timing and efficiency of chromosome segregation.

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