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DNA double-strand break repair is critical for cell viability and involves highly coordinated pathways to restore DNA integrity at the lesion. An early event during homology-dependent repair is resection of the break to generate progressively longer 3′ single-strand tails that are used to identify suitable templates for repair. Sister chromatids provide near-perfect sequence homology and are therefore the preferred templates during homologous recombination^,. To provide a bias for the use of sisters as donors, cohesin—the complex that tethers sister chromatids together—is recruited to the break^, to enforce physical proximity. Here we show that DNA breaks promote dissociation of cohesin loaded during the previous S phase in budding yeast, and that damage-induced dissociation of cohesin requires separase, the protease that dissolves cohesion in anaphase. Moreover, a separase-resistant allele of the gene coding …