Topological in vitro loading of the budding yeast cohesin ring onto DNA

(A) Purification scheme for the cohesin tetramer complex. Size exclusion chromatography was the final purification step. Fractions from the column were analyzed by SDS–PAGE, followed by Coomassie Blue staining. Peak fractions corresponding to the cohesin tetramer complex were pooled and used for biochemical experiments described in this article. The cohesin tetramer complex contained a substoichiometric contamination from chaperones of the Hsp70 family (Ssa1-4, Ssb1-2, identified by mass spectrometry), indicated by “*”. The UV absorbance elution profile from the size exclusion column is also shown, together with the profiles of size markers used to calibrate the column. (B) As (A), but the purification scheme and final size exclusion chromatography step of the Scc2–Scc4 cohesin loader complex purification are shown.

(A) DNA binding of cohesin to covalently closed circular DNA (CCC) and to linear DNA (L) was measured using an electrophoretic mobility shift assay. (B) DNA binding of the Scc2–Scc4 cohesin loader complex, next to the Scc2C fragment, was analyzed in an electrophoretic mobility shift assay. (C) Dose-dependent stimulation of cohesin loading by the cohesin loader. Gel image and quantification of the recovered DNA from cohesin loading assays performed with the indicated concentrations of the Scc2–Scc4 complex. The mean values and standard deviations from three independent experiments are shown. (D) Stimulation of ATP hydrolysis by the Scc2C fragment. Rates of ATP hydrolysis were determined in reactions containing cohesin and DNA, without or with the cohesin loader or with the Scc2C fragment. The mean values and standard deviations from three independent experiments are shown.

15 pmol of tetrameric cohesin was diluted to 150 nM in cohesin loading buffer (35 mM Tris–HCl pH 7.0, 20 mM NaCl, 0.5 mM MgCl₂, 13.3% glycerol, 0.5 mM ATP, 0.003% Tween, 1 mM TCEP) to mimic the conditions of a cohesin loading reaction. The final cohesin concentration was somewhat higher than in a typical loading reaction, which was required to detect the protein in the following analysis. The sample was separated on a Superose 6 Increase 10/300 GL column equilibrated in R buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.5 mM TCEP) or was first incubated at 29°C for 120 min and then separated. In both samples, cohesin eluted at the expected size of the tetrameric complex (compare Fig S1A). At later elution volumes, the absorptions of detergent and ATP become apparent. The positions of the separation peaks of blue dextran (>2 MD), thyroglobulin (660 kD), aldolase (158 kD), carbonic anhydrase (29 kD) and aprotinin (6.5 kD), under the same conditions, are shown as a reference.

(A) Gel image of input DNA of various topologies and of recovered DNA following cohesin loading. (B) Topological DNA loading promoted by Scc2C was assessed by DNA release following linearization with PstI. Supernatant (S) and bead-bound fractions (B) were retrieved following PstI or mock treatment. DNA in both fractions was analyzed by agarose gel electrophoresis. CCC, covalently closed circular DNA; L, linear DNA; NC, nicked circular DNA; RC, relaxed circular DNA.