Cohesin rings devoid of Scc3 and Pds5 topologically embrace circular DNA.
Strains 1021 (untagged), 1813 (SCC3-HA6, SCC1-Myc18), 1625 (SCC3-HA6-degron, SCC1-Myc18), 2525 (PDS5-HA6, SCC1-Myc18) and 1818 (PDS5-HA6-degron, SCC1-Myc18) carried the centromeric minichromosomes. (A) Yeast lysates were incubated with BglII restriction enzyme as indicated. Minichromosomes were co-immunoprecipitated with Scc1-Myc18. DNA was prepared by phenol/chloroform extraction and separated on a 1% agarose gel with ethidium bromide. Southern blot probed with a TRP1-specific probe is shown. Nicked (N), linear (L), and closed circular (C) forms of the minichromosome are indicated. (B) Minichromosomes were immunoprecipitated with anti-HA antibody. Minichromosomes from SCC3-HA6 but not SCC3-HA6-degron strains could be co-immunoprecipitated with Scc3 indicating the efficient depletion of Scc3 from the minichromosomes in the SCC3-HA6-degron strain. Since Pds5 association with minichromosomes is very salt-sensitive, they could not be co-immunoprecipitated with Pds5-HA6 in either the wild type or PDS5-HA6-degron strains under our experimental conditions.