Extended Data Fig. 10: Additional validiation experiments for the crosslinking-based cohesin loading assays (XL-assay).

a, Purified wild-type, 5 C, and 6 C cohesins were treated with BMOE and analysed by SDS-PAGE and CBB staining. A representative result from two independent experiments is shown. b, Drop-out experiments performed with circular nicked dsDNA. Efficient DNA shifts were seen when 6 C cohesin was incubated with both Scc2-Scc4 and ATP at a low-salt concentration, followed by BMOE crosslinking. In the “+ProK” reaction, the reaction mixture was treated with protease K after BMOE crosslinking and SDS-denaturation. This confirmed that shifted DNA species were derived from protein crosslinking. A representative result from two independent experiments is shown. c, The XL-assay was carried out with circular nicked dsDNA at the indicated salt concentrations. Like the IP assay, shifted DNA species were generated at a low-salt concentration. A representative result from two independent experiments is shown. d, Gel image of the XL assays performed with ss-gapped DNA. Consistent with the IP assay, 6 C cohesin showed more efficient DNA shifts with ss-gapped DNA than dsDNA in the presence of 100 mM KOAc. In contrast, no detectable DNA shift was seen using 5 C cohesin, confirming topological DNA loading by 6 C cohesin (lower gel). A representative result from three independent experiments is shown. e, Drop-out experiments performed with bubble DNA at 100 mM KOAc. As with dsDNA, efficient DNA shifts were observed when 6 C cohesin was incubated with Scc2-Scc4 and ATP, followed by BMOE crosslinking. Use of 5 C cohesin as well as protease K treatment confirmed topological loading of 6 C cohesin onto bubble DNA. A representative result from two independent experiments is shown.