Fig. 3.

Efficient assembly of cohesin^ADP/AlFx–DNA complexes is enhanced by the cohesin loader and shows properties of topologically entrapped DNA. (A) Effect of cohesin loader on DNA binding of cohesin using DNA-bead assay. WT cohesin was incubated with either ATP or ADP/AlFx nucleotides with increasing amounts of loader in the DNA-bead assay described in Fig. 1B. (Upper) Cohesin bound to DNA beads detected by Western blot using anti-V5 antibodies (Psm3-3V5). Loader/cohesin ratio shown refers to the molar ratio of loader to cohesin in experiments. (Lower) Quantitation of Western blots. (B) Schematic of modified protein-bead assay to assess topologically entrapped DNA by cohesin^ADP/AlFx. Cohesin–DNA complexes were assembled using the protein-bead assay described in Fig. 2C. After purification of cohesin^ADP/AlFx–DNA complexes by immunoprecipitation, the DNA was linearized with PstI. (C) Effect of plasmid linearization on the ability of cohesin^ADP/AlFx to bind DNA. Cohesin^ADP/AlFx–DNA complexes were digested in the presence (+) or absence (−) of PstI. Cohesin was immunoprecipitated and DNA bound to cohesin^ADP/AlFx (beads) or released into the supernatant (supe) was analyzed by ethidium-stained agarose gel electrophoresis. Plasmid not incubated with cohesin, either supercoiled or linearized with PstI, was used as an electrophoresis control (left two lanes). Dotted line represents where irrelevant lanes were removed.