Program of DNA replication in cells operating with a single cyclin B-CDK fusion protein.

<p><b>A)</b> Experimental design for determining the replication profiles in Cdc13-Cdc2 and <i>Control</i> cells. Top: Schematic for Cdc13-Cdc2 cells. For array experiments, 12 mM HU was added 10 min after inhibitor removal (Release). Cells were then harvested 60 min following the addition of HU (70 min after release from G2), after S phase is completed in a sample without HU (see <i>B</i>). Bottom: Schematic for <i>cdc25-22</i> cells. For array experiments, HU was added at the time of the return to permissive temperature. Cells were then harvested 90 min following the shift to 25°C, when S phase is completed in a sample without HU (see <i>B</i>). B: Block. <b>B)</b> DNA content analysis of Cdc13-Cdc2 and <i>Control</i> cells undergoing a synchronous S phase. Experimental procedures were as in <i>A</i>, but without HU treatment. These assays allowed the determination of the timing of sampling when using HU. Samples in which cells are undergoing DNA replication are shown in dark gray. Cdc13-Cdc2 cells arrested in G2 with a 2C DNA content (B: Block as in <i>A</i>) enter S phase ~30 min after release from inhibitor treatment. A 4C peak then appears, and cell division occurs while S phase is finishing, resulting in a 2C peak. <i>Control</i> cells initially arrested in G2 with a 2C DNA content (B: Block as in <i>A</i>) enter S phase at ~40 min following the release from G2. This leads to the appearance of a 4C peak, which is resolved upon cytokinesis shortly after the genome is duplicated (profiles prior to S phase and after cell division, which occurs ~100 min after release, are not shown). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#sec009" target="_blank">Materials and Methods</a> for additional details for the interpretation of flow cytometry profiles. <b>C</b>) Origin usage profiles of <i>Control</i> (black) and Cdc13-Cdc2 (red) cells. x-axis: chromosome coordinates, y-axis: origin efficiencies. For full detailed profiles, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.s001" target="_blank">S1A Fig</a>. <b>D)</b> Detailed view of origin efficiencies in a representative region of the genome for <i>Control</i> (black) and Cdc13-Cdc2 (red) cells. Data are as in <i>C</i>. x-axis: chromosome coordinates, y-axis: origin efficiencies. <b>E)</b> Comparison of origin efficiencies in Cdc13-Cdc2 vs. <i>Control</i> cells. The efficiencies of the 598 origins common to both replication programs are shown. x-axis: efficiency in the <i>Control</i> program, y-axis: efficiency in the Cdc13-Cdc2 program. The dashed line represents efficiencies if they were identical in the two backgrounds. <b>F)</b> Regional profiles of replication domains in <i>Control</i> (black, scale on left y-axis) and Cdc13-Cdc2 (red, scale on right y-axis) cells. The averages of origin efficiencies were determined for continuous windows of ~250 kb (1000 probes) across the genome. x-axis: chromosome coordinates, y-axis: average origin efficiencies. Note that for comparison, the Cdc13-Cdc2 profile is shown on a scale that is shifted to adjust for the 4.2% difference in overall average origin efficiency between the two backgrounds (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007214#pgen.1007214.s001" target="_blank">S1B Fig</a>). This does not alter the absolute efficiency scales on the y-axes, which each span 50% efficiency. Replication efficiency domains are clearly apparent: for instance, the right arm of chromosome II (2.5–3.5 Mb) comprises a low efficiency region, whereas the right arm of chromosome I (3–4 Mb) represents a high efficiency region. The Spearman’s rank correlation coefficient (ρ) comparing the replication programs in the two backgrounds is shown and demonstrates a very strong correlation. ***: p-value < 0.001.