Analysis of alanine point mutations in Scc3’s SCD.

<p><b>A.</b> Strain YIO81 (<i>scc3-6</i>), carrying an ectopic copy of <i>SCC3-6HA</i> (YOG3021), <i>scc3-F367A-6HA</i> (YOG3022), <i>scc3-R370A-6HA</i> (YOG3023), <i>scc3-</i>Y371A-6HA (YOG3024), <i>scc3-K372A-6HA</i> (YOG3025) or <i>scc3-D373A</i>-<i>6HA</i> (YOG3026) were grown to saturation in YPD media. Tenfold serial dilution of the strains were plated on YPD plates and grown at either permissive (23°C) or restrictive (35°C) temperature. <b>B</b>. Strains YIO81 (<i>scc3-6</i>), YOG3021 (<i>SCC3-6HA scc3-6</i>), YOG3024 (<i>scc3-Y371A-6HA scc3-6</i>), YOG3025 (<i>scc3-K372A-6HA scc3-6</i>) and YOG3026 (<i>scc3-D373A-6HA scc3-6</i>) were analyzed for sister chromatid cohesion using the GFP spot assay (n = 3). Cell cycle progression was determined by flow cytometry analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005036#pgen.1005036.s004" target="_blank">S4 Fig</a>). <b>C</b>. Co-immunoprecipitation of Mcd1 with Scc3-6HA, scc3-Y371A-6HA, scc3-K372A-6HA or scc3-D373A-6HA. Precipitation of Scc3 was done by using an anti-HA antibody.