Figure 1. UV-induced Mec1 activation requires the 9-1-1 complex and the Dpb11 C-terminus. (A) K699 (WT), YFP20 (dpb11-1), YAN21/8d (ddc1D), YFP62/1d (ddc1Ddpb11-1) and YMIC5A3 (mec1-1) strains were grown to mid-log phase, arrested in G1 with a- factor and subjected to UV irradiation. At the indicated time-points, protein extracts were prepared and separated by SDS-PAGE. Mec1 activation was assayed by western blotting monitoring cH2A and a- actin was used as loading control. A quantification of H2A phosphor- ylation is shown in the lower panel. The values indicate the fold increase respect to the WT untreated sample. The mec1-1 mutation is functionally equivalent to a mutation (B) The strains in panel A were arrested in M phase with nocodazole and subjected to the same treatment. Analysis and quantification of H2A phosphorylation was carried out as described above. (C) Strains K699 (WT) and YFP223 (ddc1D tel1D) were arrested in M phase with nocodazole and UV irradiated. At the indicated time-points Mec1 activation was assayed by western blotting monitoring cH2A. A quantification of the signal corresponding to H2A-S129 is shown in the lower panel. The values indicate the fold increase respect to the WT untreated samples. doi:10.1371/journal.pgen.1002022.g001 experimental conditions, ddc1D dpb11-1 double mutant cells showed a very severe defect in Rad53 phosphorylation, similar to that found in a Mec1-defective strain. These results suggest that a dpb11-1 ddc1D double mutation virtually abolishes UV-induced Sensing Replication Stress Mec1 activation differently from what previously reported [39], In addition, the double mutant strain showed synthetic lethality on HU plates (Figure 2B and [43]). To confirm that the dpb11-1 and ddc1D mutations directly affect Mec1 activity, we monitored cH2A levels in the same conditions. As shown in Figure 2C, the ddc1D and dpb11-1 mutations showed a synthetic defect in the ability to phosphorylate H2A-S129 (Figure 2D). Although displaying a severe defect in Rad53 phosphorylation, ddc1Ddpb11-1 still displays a residual low level of phosphorylated Rad53, which may be dependent upon a residual Mec1 activity. However, Figure 2E and Figure S2A show that the residual Rad53 phosphorylation in the double mutant is instead due to Tel1. Indeed, an additional mutation eliminating Tel1 function completely abolishes Rad53 phosphorylation in a dpb11-1 ddc1D strain and strongly sensitizes cells to HU treatment, as shown in Figure S2B. These findings further support the hypothesis that Mec1 cannot become activated in response to replication stress in the absence of both Ddc1 and Dpb11-AAD. To verify the possibility that in dpb11-1 mutant cells an unscheduled, Ddc1-dependent, DNA damage response is triggered as a consequence of the inability to properly activate the replication stress response, similarly to what happens in an mrc1D strain [22], we monitored DNA damage checkpoint activation looking at Rad9 hyperphosphorylation. As shown in Figure 2F, differently from what found in the mrc1D control strain, no Rad9 hyperphosphorylation was detectable in ddc1D, dpb11-1 single or double mutant strains. Consistently, rad9Ddpb11-1 double mutant cells are far less sensitive than the ddc1Ddpb11-1 strain to HU treatment (Figure 2B and [43]). Low levels of Rad53 activity are sufficient to prevent replication fork breakdown and premature entry into mitosis Rad53 kinase activity is required to stabilize stalled replication forks [7]. To verify whether the increased HU sensitivity of ddc1Ddpb11-1 double mutant cells was due to their inability to fully activate Rad53 and thus to stabilize the replisomes, we performed a recovery assay. Briefly, WT, dpb11-1, ddc1D, ddc1Ddpb11-1 and mec1-1sml1 mutant strains were blocked in G1, released and exposed to HU for 90 minutes; cells were then washed and shifted into fresh medium lacking HU and allowed to recover. As shown in the control strain mec1-1 sml1, when Rad53 activity is impaired, cells transiently exposed to HU loose the ability to resume DNA synthesis and complete DNA replication once the drug has been removed ([6] and Figure 3A). Unexpectedly, we found that not only dpb11-1 and ddc1D single mutant cells, but also the double mutant strain, which has a severe Rad53 hyperphosphorylation defect, were able to recover from the HU treatment with a WT kinetics (Figure 3A). Moreover, with lower HU concentrations, ddc1D dpb11-1 cells were capable of completing a round of DNA replication, as demonstrated by the re-entering of the replicated chromosomes in a pulsed-field gel system (Figure 3B). Another marker of checkpoint activation by HU is the arrest of cell cycle, preventing mitosis. When exposed to HU, checkpoint mutants fail to delay the onset of mitosis and display elongated spindles [14]. To address the hypothesis that ddc1D dpb11-1 cells may die as a consequence of a premature mitosis, we measured spindle length 90 minutes after HU addition. ddc1D dpb11-1 double mutant cells prevent spindle elongation in the presence of HU, a process which is clearly defective in a mec1-1 mutant strain (Figure S3A), suggesting that the replication checkpoint can delay mitotic entry in the double mutant [10]. In agreement with all these data, the HU sensitivity of ddc1D dpb11-1 double mutant cells can be observed only to chronic PLoS Genetics | www.plosgenetics.org 3 March 2011 | Volume 7 | Issue 3 | e1002022