The <i>smc6-56</i> mutant accumulates unresolved joint molecules but forms normal levels of recombination products.

<p>Analysis of recombination measured at the ectopic <i>URA3-ARG4</i> interval on chromosome III (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s005" target="_blank">Figure S5</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898-Sourirajan1" target="_blank">[84]</a> in strains containing the estradiol-inducible <i>NDT80</i> allele (<i>NDT80-IN</i>) under soft-shift conditions. <i>NDT80-IN</i> was induced at 7 h with 1 µM β-estradiol (+ED, arrow). Blue curves indicate <i>NDT80-IN SMC6</i> (CB2096); red <i>NDT80-IN smc6-56</i> (CB2097). (<b>A</b>) Representative Southern blot used to detect JMs after digesting with <i>XmnI</i> and probing for a region in <i>ARG4</i>. P1, P2 and JM regions based on expected sizes. (<b>B</b>) Representative Southern blot used to detect CO/NCO products at the same interval after digesting with <i>XhoI/EcoRI</i> and probing for <i>HIS4</i> sequences. P2, CO and NCO regions based on expected sizes. (<b>C</b>) Quantifications of total JM levels, NCOs and COs in percent of total DNA from blots illustrated in (A) and (B). Dotted line and arrow indicate time of <i>NDT80-IN</i> induction. Plots represent mean ± standard deviation from three independent experiments. (<b>D</b>) Two-dimensional analysis of JM species. DNA from <i>NDT80-IN SMC6</i> and <i>NDT80-IN smc6-56</i> undergoing meiosis under soft-shift conditions was isolated and subjected to two-dimensional electrophoresis as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#s5" target="_blank">Materials and Methods</a>. The interpretive panel shows the assumed identity of JM species after probing for a region which recognizes both homologs. JMs between P1×P1 are the result of random breaks and are therefore expected to give a broader signal than P2×P2 and P2×P1. Dashed line, P2×P2 IS-JM; solid line, P2×P1 IH-JM; dotted line, P1×P1 IS-JM. The enlarged panels are enhanced images of the JM region from the designated time point. Ratio of IH-JMs/ΣJMs and IS-JMs/ΣJMs given below relevant images and were calculated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s009" target="_blank">Figure S9</a>. (<b>E</b>) Quantification of total JMs in percent of total DNA from the gels represented in (D). Arrow denotes <i>NDT80</i> induction. Curves represent one experiment, with similar results from a second independent experiment presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003898#pgen.1003898.s007" target="_blank">Figure S7A</a>.