How does the loading complex interact with cohesin?

Cohesin is an SMC complex essential for genome organisation and the accurate segregation of sister chromatids in mitosis. The activity of this ATP dependent ring shape trimer composed of two SMC proteins and a kleisin subunit relies on two additional complexes. While the loading complex made of Scc2 and Scc4 is essential for the recruitment of cohesin at the loading sites, the releasing complex comprised of Pds5, Scc3 and Wapl is necessary to adjust the turnover of cohesin on DNA. During replication, Smc3 is acetylated and cohesion is established between the two newly replicated DNA molecules. Scc2 is essential for the ATPase activity of cohesin, its loading on chromatin and the genome reorganisation by DNA loop extrusion. In order to better understand the biological role of Scc2, the interaction between Scc2 and the different subunits of the cohesin complex was investigated using genetic mutations combined with in vivo and in vitro protein crosslinking. Scc2 makes extensive contacts with at least three regions of Scc1 whose deletion cause defects in loading and translocation of cohesin. Scc2 competes with Pds5 in order to stimulate the ATPase activity of the complex, but the mutation of the binding site in Scc1, common to Pds5 and Scc2, is not sufficient to abolish cohesin loading. By using different crosslinking strategies, accurate binding sites could be identified between Scc2 and Scc1. Scc2 was also found to interact with a conserved loop in the head of Smc1 that is essential for its engagement with Smc3’s head and ATP hydrolysis. Finally, crosslinking experiments confirmed the existence in vivo of the folded configuration of cohesin that brings the hinge into contact with the coiled coils. This configuration was observed throughout the cell cycle, independently of cohesin acetylation, suggesting that Scc2 is not involved in its stabilisation.