Biochemical study of the interactions between cohesin and DNA

Cohesin is a ring like SMC-kleisin complex (S-K ring) that mediates DNA interactions between chromosomes. It is responsible for the phenomenon of sister chromatid cohesion (SCC) and ensures the faithful segregation of chromosomes during mitosis. Cohesin also mediates interactions within chromosomes by organising DNA into loops in both interphase and mitosis. SCC is thought to be mediated by co-entrapment of sister DNAs within a single cohesin S-K ring. Cohesin will also entrap individual chromosomes within its S-K ring during G1, and it is thought this activity is a pre-requisite to cohesion establishment during S phase. To improve our understanding of the mechanism of the entrapment of DNA by S-K rings, I developed a protocol to measure this activity in vitro. By using a cysteine-cysteine crosslinking protocol, I was able to covalently circularise the cohesin ring on DNA and through this create protein-DNA catenations that can resist protein denaturation. This revealed that S-K entrapment was dependent on Scc3 and ATP binding, and was stimulated by Scc2 and ATP hydrolysis. I found both Scc2 and Scc3 are DNA binding proteins and discover that this DNA binding activity is essential for cohesin’s ability to entrap DNA within its S-K ring. I then set out to identify where within the cohesin ring DNA was entrapped, and through this managed to identify a novel mode of DNA interaction, whereby DNA becomes entrapped within two compartments created through cohesin binding to ATP. Entrapment within these two compartments was not associated with passage of DNA into S-K rings, and I propose entrapment of this nature may represent the first step in cohesin’s topological association with DNA.