Segregating a complete set of chromosomes into the gametes relies on exchanges of genetic material that occur during meiosis. It is only exchanges that form between the homologous chromosomes (homologs), rather than between the identical sister chromatids, that enable correct chromosome segregation. Understanding how the homologs and the sisters are distinguished requires knowledge of how they are organized relative to each other. Here, we use selective labeling of a single sister in Caenorhabditis elegans to define the organization of the sister chromatids when meiotic exchanges form. We find that pairs of sisters are well separated (resolved) throughout pachytene, despite being tethered to each other along their length. Depleting the cohesin loader NIPBLSCC-2 impairs sister resolution, suggesting that an active process—likely loop extrusion—demixes the sisters. Our work shows that meiotic exchanges form in C. elegans when the sisters and homolog are roughly the same distance from one another, suggesting that repair template choice is unlikely to rely on relative proximity.

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