Figure 5. Pole associated proteins are involved in the 9-1-1– independent checkpoint signaling branch. (A) Strains K699 (WT), Y799 (drc1-1), YAN21/8d (ddc1D), YFP218/1a (ddc1Ddrc1-1) and YFP62/1d (ddc1Ddpb11-1) were cultured 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 Rad53 phosphorylation was assayed by SDS- PAGE and Western blotting. (B) Ten-fold serial dilutions of overnight cultures of the strains in panel A were spotted on YPD plates supple- mented with HU at the indicated concentration. Survival was assayed by monitoring cell growth after 6 days. (C) Strains K699 (WT), YFP167/1a (dpb4D), YAN21/8d (ddc1D), YFP206/1a (ddc1Ddpb4D) and YFP62/1d (ddc1Ddpb11-1) were cultured 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 phosphorylation was assayed by western blotting. (D) The same filter was probed for Mec1 activity by testing histone H2A phosphorylation. A quantification of cH2A, using a-Rad53 cross reacting band as loading control is shown in the lower panel. The values indicate the fold increase respect to the WT G1 sample. (E) Ten-fold serial dilutions of overnight cultures of the strains in panel C were spotted on YPD plates supplemented with HU at the indicated concentration. Cell survival was assayed monitoring cell growth after 6 days. In all the relevant panels the loading control is indicated by an asterisk. doi:10.1371/journal.pgen.1002022.g005 [7,12]. Accordingly, the HU sensitivity of ddc1Ddpb11-1 double mutant cells, differently from that of a mec1-1sml1 strain, is not detectable in the case of transient HU treatment. This Sensing Replication Stress observation suggests that another Rad53 function activated by the replication checkpoint, and different from that responding to temporary fork arrest, is essential for sustaining growth in the constant presence of hydroxyurea. Indeed, ddc1D dpb11-1 double mutant cells grown in the presence of HU show defects in completing replication and accumulate replication intermediates. Moreover, ddc1D dpb11-1 cells are unable to counteract the effect of HU by upregulating ribonucleotide reductase. Interestingly, CRT1 deletion partially suppresses HU sensitivity of the double mutant strain (Figure 4E). To obtain more insights on the pathways leading to Ddc1- dependent and Dpb11-dependent activation of replication check- point and to identify possible mechanisms specific for lagging or leading strand fork arrest, we analyzed mutants in the genes coding for proteins that are known to be involved in leading strand replication. During initiation of DNA replication, Dpb11 interacts with both Sld2 and Sld3 in a phosphorylation-dependent manner, a process that is required for origin firing [26,27]. Moreover, temperature sensitive drc1-1 strains, mutated in Sld2, display the same checkpoint-deficient phenotype of dpb11-1 cells, when treated with HU at the non-permissive temperature, (Figure 5 and [25]). We tested whether Sld2 functions with Dpb11 in the same 9-1-1-independent pathway for Mec1 activation. Combining the drc1-1 allele with the DDC1 deletion, we found that ddc1D drc1- 1 double mutant cells display the same Rad53 phosphorylation defect and the same HU sensitivity of a ddc1Ddpb11-1 strain, indicating that Mec1 activation by Dpb11 also requires Sld2 (Figure 5). Mutants in the Pol2 C-terminus, the enzyme replicating the leading strand [50], are defective in the establishment of the replication checkpoint [24,50] and this protein region of Pol2 was suggested to be involved in its interaction with other three Pole subunits: the essential Dpb2 protein and the non-essential Dpb3 and Dpb4 subunits [31,45,51]. Disruption of the DPB4 gene in a ddc1D background leads to identical phenotypes to the one observed in ddc1D dpb1-1 and ddc1D drc1-1, strongly suggesting that the 9-1-1-independent pathway involves leading strand replication factors. The observations that Dpb11 acts directly on Mec1 activity [41,42] and that, in the dpb11-1 mutant, Pole seems to be normally loaded onto replication origins [52], strongly suggest that Dpb4, and possibly Sld2, function upstream of Dpb11 during checkpoint signaling. Unfortunately, it is impossible to perform a complete formal epistatic analysis as the dpb11-1 mutation also affects replication initiation and deletion of DPB4 or mutations in SLD2 are synthetic lethal when combined with the dpb11-1 allele [28,53]. In conclusion our data suggest that during exposure to hydroxyurea, two independent pathways sense replication stress and signal for Mec1 activation. The first pathway depends on 9- 1-1, which is known to be loaded at the 59 of primer-template junctions, when RPA covers ssDNA ahead of the primer [34]. During unchallenged DNA replication these structures are normally formed on the lagging strand as a consequence of discontinuous DNA synthesis, and rapidly removed by refilling polymerase activity. Inhibition of DNA polymerization by HU likely stabilizes the 59 DNA end providing the structure required for 9-1-1 loading. On the other hand, the higher processivity of leading strand synthesis makes it likely that the nearest 59 end will be far away from the site of polymerase stalling, where ssDNA is generated and the Mec1-Ddc2 complex should be recruited. The absence of such structure could prevent the 9-1-1-dependent Mec1 activation. In this case a pathway requiring the leading strand factors Dpb4, Dpb11 and Sld2 becomes relevant to induce Mec1 activation (Figure 6, center). The hypothesis that Pole, Sld2 PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1002022