Does Cohesin Embrace DNA?

Establishment and maintenance of cohesion between sister chromatids is an essential prerequisite of proper chromosome segregation in mitosis. Newly replicated DNA molecules are held together by a multisubunit complex called cohesin. Cohesin is comprised of two Smc subunits, Smc1 and Smc3, Scc1 subunit called "kleisin", and two additional subunits, Scc3 and Pds5. Biochemical and electron microscopic studies imply that cohesin has a ring structure. Smc1 and Smc3 proteins fold into elongated molecules with a long coiled coil region flanked on one side by a "hinge" and on another by a "head" domain. The hinge serves as dimerisation domain holding Smc1 and Smc3 protamers together. The Smc1 and Smc3 heads are spanned (and presumably locked) by a "kleisin" subunit Scc1. In the yeast Saccharomyces cerevisiae, cohesin is associated with chromosomes from late G1 until the onset of anaphase. At the metaphase to anaphase transition, Scc1 is cleaved by a specific protease, separase. Upon its cleavage, cohesin leaves the chromosomes, although Scc1 proteolytic fragments remain connected together via their association with the Smc1/Smc3 heterodimer. A central question that remains unanswered is the location of DNA with regard to cohesin. According to the "ring" model, sister DNA molecules pass through the cohesin ring and are therefore "trapped" inside it. Our project will test this model experimentally. To do this we will purify a circular DNA (plasmid) associated with cohesin directly from yeast cells. We will then digest the DNA with a restriction endonuclease and test whether its association with cohesin is affected by linearization. The "ring" model predicts that cohesin will be trapped on closed circular DNA but will slide off the linear fragment. We will further proceed to cut the cohesin ring either via its Scc1 subunit or in the coiled coil region of Smc3 and test whether this also causes cohesin`s dissociation from DNA. Finally, we will attempt the linearization experiment in vivo by looping out cohesin-rich chromosome regions as covalently closed circles by means of a site-specific recombinase or as linear fragments by means of phage N15 telomere resolvase. Cohesin association with these fragments will be analysed by chromatin immunoprecipitation (CHIP). Finally we propose Scanning Force Microscopy (SFM) studies of the purified cohesin-DNA complexes.