Chromosome loading of cohesin depends on conserved residues in Scc3.

Cohesin is essential for sister chromatid cohesion, which ensures equal segregation of the chromatids to daughter cells. However, the molecular mechanism by which cohesin mediates this function is elusive. Scc3, one of the four core subunits of cohesin, is vital to cohesin activity. However, the mechanism by which Scc3 contributes to the activity and identity of its functional domains is not fully understood. Here, we describe an in-frame five-amino acid insertion mutation after glutamic acid 704 (scc3-E704ins) in yeast Scc3, located in the middle of the second armadillo repeat. Mutated cohesin-scc3-E704ins complexes are unable to establish cohesion. Detailed molecular and genetic analyses revealed that the mutated cohesin has reduced affinity to the Scc2 loader. This inhibits its enrichment at centromeres and chromosomal arms. Mutant complexes show a slow diffusion rate in live cells suggesting that they induce a major conformational change in the complex. The analysis of systematic mutations in the insertion region of Scc3 revealed two conserved aspartic acid residues that are essential for the activity. The study offers a better understanding of the contribution of Scc3 to cohesin activity and the mechanism by which cohesin tethers the sister chromatids during the cell cycle.