Defective Top2 ATP Binding and Hydrolysis Activate the Mad2-Depedent Checkpoint.

<p><b>a</b>, Cartoon describing catalytic defects in Top2^G144I which cannot bind ATP (green annotations) and Top2^E66Q which cannot hydrolyze ATP (blue annotations). (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen-1003832-g001" target="_blank">Figure 1</a> for complete Strand Passage Reaction and Key). <b>b–j</b>, Cell cycle analyses showing that Top2^G144I and Top2^E66Q activate Mad2-dependent but Rad53-independent checkpoint signaling. Analysis of the kinetics of cell cycle progression (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen-1003832-g003" target="_blank">Figure 3</a>) following depletion of Top2^deg and release from G1 synchrony in cells expressing endogenous levels of the indicated mutant Top2 proteins. (b–h) Population Assays: Histogram plots show average G2/M duration; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#s3" target="_blank">Material and Methods</a> for statistical analysis. Western blots show each Top2 mutant relative to Tub1 loading control at G1 and G2. <i>a</i> values are significantly different to <i>b</i> values in the histogram plots. Strains with the same letter are not significantly different. (i,j) Single-cell assays: <i>i</i>, plots of average spindle length versus time for single cells aligned on the x-axis at the time of SPB separation (<i>i.e.</i> at time point 12 min). Error bars show standard deviation of lengths. <i>j</i>, Histogram plots of average time interval between SPB separation and the initiation of spindle elongation in anaphase B (+/− s.e.).