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.

Cohesin 对姐妹染色单体的凝聚至关重要，可确保染色单体与子细胞的平等分离。然而，cohesin 介导这一功能的分子机制是难以捉摸的。Scc3 是 cohesin 的四个核心亚基之一，对 cohesin 活性至关重要。然而，Scc3 对其功能域的活动和身份做出贡献的机制尚不完全清楚。在这里，我们描述了酵母 Scc3 中谷氨酸 704 (scc3-E704ins) 之后的框内五氨基酸插入突变，位于第二个犰狳重复序列的中间。突变的 cohesin-scc3-E704ins 复合物无法建立内聚力。详细的分子和遗传分析表明，突变的 cohesin 降低了对 Scc2 加载器的亲和力。这抑制了它在着丝粒和染色体臂上的富集。突变复合物在活细胞中显示出缓慢的扩散速率，表明它们在复合物中引起了重大的构象变化。对 Scc3 插入区系统突变的分析揭示了两个保守的天冬氨酸残基，它们对活性至关重要。该研究提供了对 Scc3 对 cohesin 活性的贡献以及 cohesin 在细胞周期中连接姐妹染色单体的机制的更好理解。