Figure 3. Low levels of Rad53 activity are sufficient to prevent replication fork breakdown. (A) HU recovery assay: K699 (WT), YFP20 (dpb11-1), YAN21/8d (ddc1D), YFP62/1d (ddc1Ddpb11-1) and YMIC5A3 (mec1-1) were synchronized in G1 with a-factor and released into fresh medium supplemented with 200 mM HU. 90 min later cells were transferred to fresh YPD + nocodazole and allowed to resume DNA replication. Progression into S phase was monitored by FACS analysis. (B) The indicated strains were synchronized in G1 with a-factor and released into 100 mM HU + nocodazole. 3 and 5 hours later cells were harvested and total DNA was analyzed by Pulse Field Gel Electrophoresis (PFGE). (C) The strains in panel A were synchronized in G1 and released in YPD supplemented with 200 mM HU. 90 min later 10-fold serial dilution were prepared and spotted onto YPD plates. The same was done with the G1-synchronised cultures as control. doi:10.1371/journal.pgen.1002022.g003 correct segregation of chromosomes. Altogether, these findings may suggest that ddc1D dpb11-1 cells are unable to counteract the effect of HU by upregulating ribonucleotide reductase (RNR). Indeed, Rad53 regulates both the timely degradation of the RNR inhibitor Sml1 and the inactivation of Crt1, which represses the transcription of RNR genes [9,10]. Consistently with this interpretation, ddc1D dpb11-1 cells show a modest delay in Sml1 degradation and, more significantly, CRT1 deletion suppresses, although not completely, the sensitivity of the double mutant strain to HU (Figure 4D, 4E). Sld2 and DNA polymerase e are required for Ddc1-independent checkpoint activation Sld2/Drc1 and Pole participate in replication checkpoint signaling [24,25]. Moreover, these factors were recently found to be part of the same pre-loading complex, together with Dpb11 and GINS [44]. An interesting possibility is that Sld2 and Pole exert their checkpoint function by controlling Dpb11-mediated Mec1 activation. To address this hypothesis we combined the drc1- 1 allele with the DDC1 deletion. As it is shown in Figure 5A, similarly to what reported above for Dpb11, Sld2 also acts in a pathway that is parallel to that involving Ddc1; indeed, residual Rad53 phosphorylation present in ddc1D cells depends on Sld2. Moreover, drc1-1 cells do not show hyperphosphorylation of Rad9 in response to HU treatment, excluding the possibility of a secondary DNA damage response (Figure S4A). In agreement with these data, deletion of DDC1 displays a synergistic sensitivity to Sensing Replication Stress HU when combined with the drc1-1 mutation and the HU sensitivity of the double mutant strain is very similar to that observed for ddc1Ddpb11-1 cells (Figure 5B). The checkpoint function of Pole appears to reside in the C- terminal domain of Pol2, which is bound, either directly or indirectly, by the three smaller subunits Dpb2, Dpb3 and Dpb4 and by Dpb11 [31,45]. To assess if Pole participates in the Dpb11 signaling branch via its minor subunits, we deleted DPB4 in combination with the DDC1 deletion. Figure 5C shows that Rad53 phosphorylation is severely impaired in the double mutant ddc1Ddpb4D, closely resembling the phenotype of a ddc1Ddpb11-1 mutant. The same effect is measured by testing H2A phosphor- ylation in HU-treated samples (Figure 5D). The signals obtained for each time-point are quantified with respect to the signal detected in G1-arrested cells, in order to compensate for the higher basal level of cH2A observed in ddc1Ddpb4D double mutant cells in the absence of any treatment. Moreover, no unscheduled DNA damage checkpoint activation occurs, since no Rad9 phosphorylation is detected in dpb4D or dpb4D ddc1D cells treated with HU (Figure S4B). Finally, the ddc1Ddpb4D strain shows an HU sensitivity similar to that found in ddc1Ddpb11-1 cells (Figure 5E). Discussion Apical checkpoint kinases (Mec1/Tel1 in budding yeast, ATR/ ATM in humans) convert a structural signal coming from damaged DNA to a phosphorylation-based signaling cascade, and a large amount of work has been devoted to clarify the underlying mechanisms. Initially, the attention was focused on the recruitment of these kinases to damaged DNA [15], based on the assumption that binding to damaged chromatin sites would lead to their activation. More recently, the finding that Dpb11/TopBP1 stimulates Mec1 activity suggests a more complex scenario [40,41,42]. In vitro data obtained in Xenopus egg and mammalian cell extracts demonstrate the ability of TopBP1 to increase Mec1 kinase activity [35,38]. The significance of this TopBP1 function does not appear to be specific for multicellular eukaryotes, since an interaction between Rad4/Cut5 and the checkpoint sensor kinase Rad3-Rad26 has also been found in S. pombe [46,47]. More recently, in S. cerevisiae cells, Dpb11 has been demonstrated to contain an ATR activation domain (AAD), which is sufficient to promote Mec1 activation in vitro [41,42]. These findings apparently contradict a previous observation that Mec1 can normally phosphorylate Ddc2 in a dpb11-1 mutant, lacking part of the AAD, after UV damage in M phase [16], while in our hands DDC1 deletion prevents Ddc2 phosphorylation (unpublished observation). Two explanations can be envisaged: in dpb11-1 mutant cells, Mec1 activity may be sufficient to phosphorylate Ddc2, while being defective towards other substrates; alternatively, Dpb11 may play only a marginal role in response to UV irradiation in M phase. We favored the second hypothesis because dpb11-1 mutant cells are mildly sensitive to UV irradiation and are proficient in the G2/M checkpoint; moreover, the 9-1-1 complex has also been identified as an activator of Mec1 in vitro [39,40] and may play a prominent role in M phase. If this assumption is correct, Dpb11 could play a role in Mec1 activation in response to a different kind of damage or in other cell cycle phases. Interestingly, it was demonstrated that the dpb11-1 temperature- sensitive mutant is defective in checkpoint activation after replication stress caused by HU treatment at the restrictive temperature (36uC), while it is only mildly sensitive to the drug at permissive temperature ([23,25] and Figure 2B). PLoS Genetics | www.plosgenetics.org 5 March 2011 | Volume 7 | Issue 3 | e1002022