Checkpoint activation in <i>sgs1Δtop2</i>^<i>ts</i> cells is dependent on Fob1.

<p>(A) The experimental setup was as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1A</a>, except that cells were released at 34°C into nocodazole to block further cell cycling. Chromosomes were prepared from wt, <i>sgs1Δ</i>, <i>top2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i> cells and visualized after pulsed-field gel electrophoresis by EtBr staining (upper panels) and by Southern blotting with a probe (P1) specific for chr. XII (middle panels) or chr. II (lower panels). <i>M</i>. Chromosomal marker with indication of individual chromosomes to the left. Migration of individual chromosomes in the used strains is indicated to the right. FACS profiles of samples taken throughout the experiment are shown below each strain. (B) Quantification of the ratio of chr. XII to chr. II re-entering the gel. (C) Schematic illustration of the rDNA repeat unit in <i>S</i>. <i>cerevisiae</i>, indicating the 5S and 35S transcription units. <i>RFB</i>, Replication Fork Barrier. <i>ARS</i>, Autonomously Replicating Sequence. <i>Bgl</i>II restriction sites generating the <i>Bgl</i>IIA and B fragments as well as probes used for Southern blotting are indicated. (D) The experimental setup was as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1A</a>. Proteins were isolated from <i>sgs1Δtop2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>fob1Δ</i> cells at the indicated time points and processed for ISA analysis of Rad53 as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1</a>. FACS profiles are shown below each strain.