Molecular studies on double-strand break (DSB) repair in mitosis are usually performed with enzymatically induced DSBs, but spontaneous DSBs might arise because of replication failures, for example when replication encounters nicks. To study repair of replication-born DSBs, we defined a system in Saccharomyces cerevisiae for the induction of a site-specific single-strand break. We show that a 21-base pair (bp) HO site is cleaved at only one strand by the HO endonuclease, with the resulting nick being converted into a DSB by replication during the S phase. Repair of such replication-born DSBs occurs by sister-chromatid exchange (SCE). We provide molecular evidence that cohesins are required for repair of replication-born DSBs by SCE, as determined in smc3, scc1 and scc2 mutants, but not for other recombinational repair events. This work opens new perspectives to understand the importance of single-strand breaks as a source of recombination and the relevance of cohesion in the repair of replication-born DSBs.