Sensing Replication Stress Figure 2. Dpb11 and 9-1-1 independently activate Mec1 after replication stress. (A) K699 (WT), YFP20 (dpb11-1), YAN21/8d (ddc1D), YFP62/ 1d (ddc1Ddpb11-1), YFP125/6d (mrc1D) and YMIC5A3 (mec1-1) strains were grown to mid-log phase, synchronized in G1 with a-factor and released into fresh medium supplemented with 200 mM HU. At the indicated time-points, protein extracts were prepared and separated by SDS-PAGE. Rad53 activation was assayed as the phosphorylation-dependent shift of the protein. (B) 103–104 cells from overnight cultures of the strains analyzed in panel A and YFP74 (rad9D) and YFP161/5C (rad9Ddpb11-1) were spotted on YPD plates supplemented with HU at the indicated concentrations. Cell survival was assayed after 2–7 days. (C) The same filter in panel A was probed for Mec1 activation by analyzing the level of histone H2A phosphorylation. (D) Quantification of cH2A in the experiment shown in panel C, using a-Rad53 cross-reacting band as loading control. The values indicate the fold increase respect to the WT G1 sample. (E) Strains K699 (WT), YMIC6C3 (tel1D), YFP62/1d (ddc1Ddpb11-1) and YFP230 (ddc1Ddpb11- 1tel1D) were synchronized in G1 and released into fresh medium supplemented with 200 mM HU. At the indicated time-points Rad53 and H2A phosphorylation were assayed by western blotting. A quantification of the signal is shown in the lower panel, using a-Rad53 cross-reacting band as loading control. The numbers indicate the fold increase respect to the WT G1 sample. (F) The same extracts of panel A were probed with a-Rad9 antibodies to determine the extent of Rad9 hyperphosphorylation. In all the relevant panels the loading control is indicated by an asterisk. doi:10.1371/journal.pgen.1002022.g002 exposure to the drug, while it is virtually undetectable if cells are transiently exposed to HU (Figure 3C). The inability to fully activate Rad53 causes defects in the control of RNR induction ddc1D dpb11-1 mutant cells exhibit extremely low levels of Mec1 and Rad53 activation and, despite being sensitive to exposure to even low concentrations of HU (Figure 2B), they do not show some of the most common phenotypes observed in replication checkpoint defective cells. To better characterize the sensitivity to the drug, we monitored cell growth in the presence of 100 mM HU. The single and double mutant ddc1D dpb11-1 yeast strains were synchronized in G1, released into fresh medium supple- mented with HU and cell cycle progression followed by FACS analysis. The double mutant ddc1D dpb11-1 showed a small delay in progressing through S-phase in the presence of HU, compared to WT and single mutant cells. Significantly, at late times (20 hours) after the release, a large fraction of double mutant cells appeared to be arrested at different stages of S-phase, while WT and single mutant cells had regained a FACS profile with 1C and 2C peaks (Figure 4A). Consistently, PFGE analysis of genomic DNA prepared from the various strains 20 hours after release from HU showed that in ddc1D dpb11-1 double mutant cells most of the DNA fails to enter the gel, suggesting the presence of branched intermediates (Figure 4B, 4C). It is important to note that, differently from what found in a mec1-1 strain, the ddc1D dpb11-1 strain did not accumulate cells with a,1C DNA content, or low molecular weight DNA fragments (Figure 4A–4C) indicating a PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1002022