Investigating the roles of cohesin acetylation and the configuration of the coiled coil domain modelled by Saccharomyces cerevisiae.

Cohesin is a protein complex involved in creating sister chromatid cohesion during mitosis and performs this role by forming topological entrapment around both chromatids. Cohesin consists of four subunits: Smc1, Smc3, Scc1 (Mcd1 in yeast), and Scc3. Cohesin is loaded onto DNA by the action of a loading complex composed of Scc2 and Scc4. Cohesin is released from the DNA by the releasing complex composed of Wapl (Rad61 in yeast) and Pds5. Both loading and releasing processes are ATP-dependent and rely on machinery present in Smc1 and Smc3. Acetylation of the cohesin subunit, Smc3, at position K112, K113 is required for successful cohesion as this abolishes the cohesin releasing activity of Wapl and likely the loading action of Scc2-Scc4. Why acetylation may abolish releasing and loading activity is not understood. However, changes to ATP binding and hydrolysis activity may be involved. Data in this study suggest that acetylation may reduce potential Scc2 dependent ATP hydrolysis activity, as acetylated cohesin mimicking forms of cohesin (smc3K112Q, K113Q) have been shown to have significantly lower activity than wild type cohesin. Further data suggests that smc3K112Q, K113Q may inhibit loading and releasing activity by promoting a different configuration between Smc3 and Smc1, forming either a rod or a ring structure. The two configurations investigated in this study, E state (Smc1-Smc3 head domains engaged) and J state (Smc1-Smc3 head domains juxtaposed) may be controlled by cohesin loading and releasing complexes via certain interaction sites located near Smc3K112, K113 and R1008. Mutations near these sites are shown to either contribute to rescuing Scc2 interaction, which is largely abolished by smc3K112Q, K113Q or interact with Scc2 itself. The interaction between the coiled coils of Smc3 and Smc1 was shown to be incompatible with certain head domain configurations via crosslinking assays, thus verifying their mutual exclusivity. The difference between these configurations determines whether ATP hydrolysis activity is possible or not, thereby controlling loading and releasing activity.