Sensing Replication Stress Figure 6. A model for 9-1-1 and Dpb11 function in Mec1 activation. After UV irradiation in G1, Mec1 is activated by the 9-1-1 complex both directly and through the Dpb11 C-terminus (right); in M phase Mec1 activation is achieved mainly through the 9-1-1 complex, independently of Dpb11 (left). In S phase Dpb11 and the 9-1-1 complex signal replication stress to Mec1 independently from each other, likely because the detection of replication stress occurs independently on the leading and lagging strands. 9-1-1 complex could signal replication stress on the lagging strand, where the 59 ends necessary for its loading are generated as the result of discontinuous replication. Dpb11, instead, could signal replication stress on the leading strand together with the interacting Pole. doi:10.1371/journal.pgen.1002022.g006 and Dpb11 work together in sensing replication stress is supported by the recent finding that an unstable complex containing Dpb11, Sld2, Pole and GINS is formed at the beginning of S-phase [44]. Moreover, the demonstration that under unstressed conditions Pole acts on the leading strand while Pold works on the lagging strand [50,54] supports the hypothesis that Pole and its interacting subunits may function in sensing replication stress on the leading strand, while the 9-1-1 complex may be more important to detect lagging strand fork arrest. Additional work will be needed to confirm this model and to identify the mechanisms leading to Dpb11 recruitment at the sites of replication fork stalling, since Dpb11 appears to co-localize with Pole during initiation of DNA replication, but not during elongation [52]. Materials and Methods Yeast strains All of the strains used in this work are derivatives of W303 (K699 [MATa ade2-1 trp1-1 can1-100 leu2-3,12 his3- 11,15 ura3]) and are listed in Table 1. Deletion strains were generated by using the one-step PCR system [55] or by genetic crossing. Cell cycle synchronization and HU treatment Cells were grown overnight at 25uC to a concentration of 56106 cells/ml and arrested in G1 with 5 mg/ml a-factor for three hours. 60 ml of cultures were spun and resuspended in the same volume of YPD supplemented with HU (200 mM or 100 mM, depending on the experiment). 20 ml samples were taken every 30 minutes after the release. In the case of untreated samples cells were released in fresh YPD +10 mg/ml nocodazole and every 5 minutes samples were taken for SDS-PAGE and FACS analysis. Cell cycle arrest and DNA damage treatments Cells were grown in YPD medium at 25uC to a concentration of 56106 cells/ml and arrested with nocodazole or a-factor (20 mg/ ml). 50 ml of cultures were spun, resuspended in 500 ml of sterile water, and plated on a Petri dish (14-cm diameter). Rapidly, a 15 ml untreated sample was taken. Plates were irradiated at 75 J/ m2 and cells were resuspended in 50 ml of YPD + nocodazole or a factor. Three 15 ml samples were taken every 10 minutes after irradiation. SDS page, western blot, and quantification Trichloroacetic acid protein extracts [56] were separated by SDS-PAGE; for the analysis of Rad9 phosphorylation, NuPAGE Tris-Acetate 3–8% gels (Invitrogen) were used following the manufacturer’s instructions. Western blotting was performed with anti-Rad53, anti-H2A-S129 (Abcam #15083), anti-Actin (Sigma #A2066), anti-Sml1 and anti-Rad9 antibodies, using standard techniques. Values of phospho-H2A levels were obtained by quantifying the signal in the corresponding lanes using Quantity PLoS Genetics | www.plosgenetics.org 8 March 2011 | Volume 7 | Issue 3 | e1002022