Author Summary The maintenance of genome stability is an essential process which needs a careful control. Indeed, the checkpoints are surveillance mechanisms sensing alter- ations in the integrity of the genome and preventing the replication and segregation of defective DNA molecules. The DNA integrity checkpoint is a signal transduction cascade conserved from yeast to man, and the apical factors in the pathway are protein kinases, called Mec1/ Tel1 in Saccharomyces cerevisiae and ATR/ATM in mam- mals. DNA integrity can be challenged by lesions caused by a variety of chemical/physical agents, or by replication stress caused by special DNA structures, or by a limited supply of deoxyribonucleotides (dNTPs). The mechanisms leading to checkpoint activation in response to DNA damage are better understood compared to the processes leading to activation as a consequence of replication stress. We investigated the mechanisms required for Mec1 activation in response to dNTPs depletion caused by hydroxyurea treatment. We found that Mec1 activation occurs through two independent pathways: one acting through the PCNA-like 9-1-1 complex and the second through Dpb11 and DNA polymerase e. The existence of these two pathways suggest a model possibly reflecting a DNA strand specificity in the detection of replication stress. replication origins [26,27]. Pole consists of four subunits: Pol2 and Dpb2 are essential for cell viability while Dpb3 and Dpb4 appear to be non-essential. These last two factors contains a histone-like fold motif and are also implicated in transcriptional regulation [28,29]. The Pole holoenzyme is composed of two structurally distinct domains: a globular domain, made of the N-terminus of the catalytic Pol2 subunit and a tail-like domain containing the other three factors, bound to the Pol2 C-terminus [30,31]. The catalytic subunit contains an N-terminal polymerase domain followed by a C-terminal region, where the checkpoint-defective mutations of POL2 map [24]. Surprisingly, deletion of the polymerase domain does not cause cell lethality, whereas the checkpoint domain is essential for cell viability [32]. It has been established that in response to DNA damage, the 9- 1-1 clamp is loaded onto the 59 primer-template junction adjacent to RPA-coated ssDNA [33,34]. In higher eukaryotes, 9-1-1 then recruits TopBP1 which, through an interaction with ATRIP, stimulates the ATR kinase activity [35,36,37,38]. Recent work in yeast demonstrated that Mec1 activation can proceed also through a 9-1-1-dependent, but Dpb11-independent pathway, mediated by an activation domain present in the Ddc1 subunit of the 9-1-1 complex [39]. Indeed, it has been reported that S. cerevisiae 9-1-1 can directly activate the Mec1-Ddc2 kinase in vitro [40]. The in vivo balancing between these two pathways has been recently studied, following Rad53 phosphorylation [39], which is influenced not only by Mec1 activation, but also by the Rad9 mediator [39].To determine directly the relative contributions of Ddc1 and Dpb11 to Mec1 activation in different cell cycle phases, and particularly in response to replication stress, we analyzed a direct target of Mec1 kinase, histone H2A, whose phosphorylation is not dependent upon Rad9. In this study we found that, in G1 yeast cells, Mec1 activation induced by UV irradiation completely depends on the 9-1-1 dependent pathway, whereas Dpb11 only plays a minor role. Conversely, in response to replication stress, Mec1 activation is achieved through two independent pathways which rely on the 9- 1-1 complex and Dpb11, respectively. At least one of these two Sensing Replication Stress pathways is necessary to efficiently activate Mec1 and to allow cell growth in the presence of HU. Finally, we provide evidence that the DNA polymerase e complex and Sld2 are required to establish the 9-1-1 independent branch of Mec1 activation and we suggest that this could reflect strand-specificity in detecting replication stress. Results UV-induced Mec1 activation requires 9-1-1 and the C-terminal tail of Dpb11 We have previously shown that, in M phase, Dpb11 is required to recruit the Rad9 adaptor protein to UV-damaged DNA in a pathway that is parallel to that controlled by histone modifications [16,20]. Dpb11 was also found to stimulate Mec1 kinase activity in vitro and this function appears to be modulated by its interaction with the 9-1-1 complex [41,42]. To dissect the Mec1-activation role of Dpb11 in vivo and to determine the relative contribution of Dpb11 and 9-1-1 to this mechanism in different cell cycle phases, we analyzed histone H2A phosphorylation as an assay for Mec1 activity. After UV damage H2A is phosphorylated directly on serine 129 (cH2A) by Mec1 kinase; indeed mec1-1 mutant cells fail to phosphorylate H2A after DNA damage and a strain deleted in TEL1, coding for a second sensor-kinase, does not show any significant reduction in cH2A levels (Figure S1A and S1B). We used a yeast strain carrying a C-terminal deletion of Dpb11 (D583_764) encoded by the dpb11-1 allele, which removes almost entirely the ATR Activation Domain (AAD) and a strain carrying the deletion of DDC1, the gene encoding the 9-1-1 subunit involved in Mec1 activation [40]. WT, dpb11-1, ddc1D and ddc1Ddpb11-1 cells were arrested in G1 with a-factor and in M phase with nocodazole and UV irradiated. As it is shown in Figure 1A, histone H2A is extensively phosphorylated after UV treatment in G1 and this damage-dependent modification requires the presence of a functional 9-1-1 complex, while the contribution of the AAD domain of Dpb11 is only minor. The quantification of the signal (shown in the lower panel of Figure 1A), indicates that the level of phosphorylated histone H2A (cH2A) in dpb11-1 is ,50% of that found in WT cells. In M phase cells the basal level of phosphorylated H2A-S129 is much higher (Figure S1C), and this likely influences the magnitude of the increase measured after UV-irradiation. In these conditions, Dpb11 plays a minimal role, if any, in H2A phosphorylation and also DDC1 deletion reduces cH2A only partially (,50%) (Figure 1B). However, the residual H2A phosphorylation observed in a ddc1D mutant strain is lost when TEL1 is deleted, (Figure 1C). On the other hand, deletion of TEL1in the dpb11-1 background does not significantly influence H2A phosphorylation (Figure S1D) 9-1-1 and Dpb11 act independently in signaling replication stress to the Mec1 kinase To further elucidate the balancing between 9-1-1-dependent and Dpb11-dependent Mec1 activation in S phase, we decided to analyze this process after replication stress induced by HU. This allowed us also to minimize the side effects due to the involvement of Dpb11 in Rad9 recruitment because, during HU treatment, Rad9 does not become hyperphosphorylated and is not expected to play any role in checkpoint activation [22]. WT, dpb11-1, ddc1D and ddc1Ddpb11-1 cells were synchronized in G1, released into fresh medium supplemented with 200 mM HU, and checkpoint activity was monitored by measuring Rad53 phosphorylation (Figure 2A). Differently from what found in G1 and G2 cells, strains lacking either a functional 9-1-1 complex or the Dpb11 C- terminal region were fully able to phosphorylate Rad53. In these PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1002022