The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

Summary Cohesin is a conserved, ring-shaped protein complex that topologically embraces DNA. Its central role in genome organization includes functions in sister chromatid cohesion, DNA repair, and transcriptional regulation. Cohesin loading onto chromosomes requires the Scc2-Scc4 cohesin loader, whose presence on chromatin in budding yeast depends on the RSC chromatin remodeling complex. Here we reveal a dual role of RSC in cohesin loading. RSC acts as a chromatin receptor that recruits Scc2-Scc4 by a direct protein interaction independent of chromatin remodeling. In addition, chromatin remodeling is required to generate a nucleosome-free region that is the substrate for cohesin loading. An engineered cohesin loading module can be created by fusing the Scc2 C terminus to RSC or to other chromatin remodelers, but not to unrelated DNA binding proteins. These observations demonstrate the importance of nucleosome-free DNA for cohesin loading and provide insight into how cohesin accesses DNA during its varied chromosomal activities.

The ring-shaped cohesin complex is thought to topologically hold sister chromatids together from their synthesis in S phase until chromosome segregation in mitosis. How cohesin stably binds to chromosomes for extended periods, without impeding other chromosomal processes that also require access to the DNA, is poorly understood. Budding yeast cohesin is loaded onto DNA by the Scc2–Scc4 cohesin loader at centromeres and promoters of active genes, from where cohesin translocates to more permanent places of residence at transcription termination sites. Here we show that, at the GAL2 and MET17 loci, pre-existing cohesin is pushed downstream along the DNA in response to transcriptional gene activation, apparently without need for intermittent dissociation or reloading. We observe translocation intermediates and find that the distribution of most chromosomal cohesin is shaped by transcription. Our observations support a model in which cohesin is able to slide laterally along chromosomes while maintaining topological contact with DNA. In this way, stable cohesin binding to DNA and enduring sister chromatid cohesion become compatible with simultaneous underlying chromosomal activities, including but maybe not limited to transcription.

The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2–Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large ‘hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2–Scc4 complex. Finally, using recombinantly purified Scc2–Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2–Scc4 uses its modular structure to make multiple contacts with cohesin.