Extended Data Fig. 9: Single-stranded DNA facilitates topological cohesin loading.
From: Coordination of cohesin and DNA replication observed with purified proteins
a, Cohesin and Scc2-Scc4 were incubated with ss-gapped DNA (0.2 kb) at the indicated salt and magnesium ion concentrations. Nicked circular DNA was used for comparison. Compared to dsDNA (nicked circular DNA), increased recovery of ss-gapped DNA was seen under all conditions tested. b, Quantification of cohesin-bound DNA in a (n = 4 independent experiments; means ± s.d.). c,d, Drop-out experiments using 0.2- (c) or 0.6- (d) ss-gapped DNA at the indicated salt concentrations (n = 3 independent experiments; means ± s.d.). As with dsDNA, efficient DNA recovery was only seen in the presence of both ATP and Scc2-Scc4. e, RPA inhibits cohesin loading onto ss-gapped DNA, but not onto nicked dsDNA. The indicated amount of RPA was added to the cohesin loading reaction (n = 3 independent experiments; means ± s.d.). This suggests that cohesin contacts ssDNA to convert the initial binding into a topological embrace. f, IP assays using circular dsDNA (nicked circular DNA) were carried out with the indicated amount of 0.6 kb linear ssDNA. The separated ssDNA strand showed no detectable effect on cohesin loading onto dsDNA. g, Quantification of cohesin-bound DNA in f (n = 3 independent experiments; means ± s.d.).
