Cohesin rings devoid of Scc3 and Pds5 topologically embrace circular DNA.
2012-08-09T01:18:37Z (GMT) by
<p>Strains 1021 (untagged), 1813 (<i>SCC3-HA6</i>, <i>SCC1-Myc18</i>), 1625 (<i>SCC3-HA6-</i>degron, <i>SCC1-Myc18</i>), 2525 (<i>PDS5-HA6</i>, <i>SCC1-Myc18</i>) and 1818 (<i>PDS5-HA6</i>-degron, <i>SCC1-Myc18</i>) 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 <i>TRP1</i>-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 <i>SCC3-HA6</i> but not <i>SCC3-HA6</i>-degron strains could be co-immunoprecipitated with Scc3 indicating the efficient depletion of Scc3 from the minichromosomes in the <i>SCC3-HA6</i>-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 <i>PDS5-HA6</i>-degron strains under our experimental conditions.</p>