Sensing Replication Stress Figure 4. The inability to fully activate Rad53 causes defects in the control of RNR induction. (A) 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 100 mM HU. At the indicated time-points after the release, progression into the cell cycle was monitored by FACS analysis. (B) DNA extracted from the G1-arrested cells and from the cells released for 20 hours in HU was separated by PFGE and stained with ethidium bromide. (C) A plot representing the intensity profile of the significant gel lanes in panel C. (D) K699 (WT), YFP20 (dpb11-1), YAN21/8d (ddc1D) and YFP62/1d (ddc1Ddpb11-1) strains were 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 probed with a-Sml1 antibodies to measure the levels of Sml1 and with a-actin antibodies as a loading control. (E) Ten fold serial dilutions of overnight cultures of strains K699 (WT), YFP328 (crt1D), YFP62/1d (ddc1Ddpb11-1) and YFP330 (ddc1Ddpb11- 1crt1D) were spotted on YPD plates supplemented with HU at the indicated concentration. Survival was assayed by monitoring cell growth after 6 days. doi:10.1371/journal.pgen.1002022.g004 To better understand the process of Mec1 activation in vivo after DNA damage or replication stress, we analyzed the relative functions of the two putative Mec1 activators: Dpb11 and the 9- 1-1 complex. We extended our previous analysis by monitoring, in different cell cycle phases, a direct target of Mec1 kinase (histone H2A) as marker of Mec1 activity. We found that, both in G1 and in M phase, the 9-1-1 complex is absolutely required for Mec1 activation in response to UV treatment, while the contribution of Dpb11 AAD is only partial (,50%) and restricted to G1. These in vivo findings are in agreement with the current activation model inferred from in vitro biochemical data [39], indicating that 9-1-1 can stimulate Mec1 through both Dpb11-dependent and -independent pathways in G1 (Figure 6, left). Differently, in M phase, the ATR activation domain of Dpb11 is dispensable for full Mec1 activation, which relies mainly on the presence of 9-1-1 (Figure 6, right). In fact, the residual UV-induced H2A phosphorylation detectable in the ddc1D strain, is dependent upon the Tel1 kinase (Figure 1). Different requirements for Mec1 activation in G1 and in M phase may reflect differences in CDK-controlled processing of DNA filament ends to generate the substrate detected by checkpoint factors [48,49]. To complete studying of the pathways leading to Mec1 activation in different cell cycle stages, we analyzed the contribution of Dpb11 and Ddc1 to Mec1 activation in S phase cells challenged with replication stress. HU decreases the cellular concentration of dNTPs available for DNA synthesis and yeast cells respond by activating the replication checkpoint. In vivo analysis of the phosphorylation state of two Mec1 substrates, H2A and Rad53, indicates that Dpb11 and 9-1-1 participate in Mec1 activation in response to HU treatment independently of each other in two parallel pathways. The possibility that dpb11-1 may cause problems to the replication process triggering a DNA damage response mediated by the 9-1-1 complex, similarly to what happens in mrc1D cells [22], seems unlikely. In fact, the Rad9 DNA damage-specific adaptor does not become hyperphosphorylated in both dpb11-1 and ddc1D single mutants. In agreement with such observation, rad9Ddpb11-1 cells are much less sensitive to HU than ddc1D dpb11-1 cells (Figure 2 and [43]). We report that the HU sensitivity of ddc1D dpb11-1 strain is not due to replication fork collapse or premature elongation of the mitotic spindle (Figure 3 and Figure S2), two phenotypes characteristic of mutants defective in the replication checkpoint PLoS Genetics | www.plosgenetics.org 6 March 2011 | Volume 7 | Issue 3 | e1002022