Table 1. Strains used in this work. Name K699 YAN21/8d YFP20 YFP62/1d YMIC5A3 YFP125/6d YFP167/1a YFP206/1a Y799 YFP218/1a YFP74 YFP161/5C YFP328 YFP330 YMIC6C3 YFP223 YFP225 YFP230 Relevant Genotype MATa ade2-1 trp1-1 leu2-3,112 his3-11,15 ura3-1, can1-100 (K699) ddc1D::kanMX6 (K699) dpb11-1 (K699) ddc1D::kanMX6 dpb11-1 (K699) mec1-1 sml1 (K699) mrc1D::HIS3 (K699) dpb4D::HIS3 (K699) ddc1D::KanMX6 dpb4D::HIS3 (K699) drc1-1 (K699) ddc1D::KanMX6 drc1-1 (K699) rad9D::HIS3 (K699) rad9D::HIS3 dpb11-1 (K699) crt1D::HIS3 (K699) ddc1D::KanMX6 crt1D::HIS3 dpb11-1 (K699) tel1D::KanMX6 (K699) ddc1::KanMX6 tel1D::HIS3 (K699) dpb11-1 tel1D::HIS3 (K699) ddc1::KanMX6 dpb11-1 tel1D::HIS3 Sensing Replication Stress Reference K.Nasmyth A. Nespoli Puddu, 2008 Puddu, 2008 M. Giannattasio Wang & Elledge, 1999 This Work This Work This Work This Work This Work This Work This Work This Work This Work This Work This Work M. Giannattasio doi:10.1371/journal.pgen.1002022.t001 One software (BioRad) and normalizing it, first on the loading controls and then on the level of phospho-H2A in the untreated/ G1-arrested sample of each strain. Immunofluorescence 1 ml of a 56106 cells/ml culture were fixed overnight at 4uC with fixation buffer (3,7% formaldehyde, 0,1 M K-phosphate pH 6,4, 0,5 mM MgCl2). Cells were then washed three times with wash buffer (0,1 M K-phosphate pH 6,4, 0,5 mM MgCl2), one time with spheroplasting solution (1,4 M sorbitol, 0,1 M K- phosphate pH 6,4, 0,5 mM MgCl2) and resuspended in 200 ml of the same solution. Spheroplasts were prepared using 5 ml of 10 mg/ml Zymolyase at 37uC. Spheroplasts were washed with the same solution and used to prepare multi-well immunofluorescence slides which were incubated overnight with a-tubulin antibody (YOL1/34, Seralab) diluted 1:100 in PBS-5%BSA. Sensitivity assay HU plates were prepared by serial dilutions of the 2 M stock solution. Plates containing 25 mM, 50 mM and 100 mM HU were prepared. Overnight grown cultures were diluted to 16106 cell/ml, then 10-fold serial dilutions were prepared and 10 ml of the suspensions were spotted on HU plates, which were incubated at 25uC. Images were taken 2 to 7 days later. Pulsed field gel electrophoresis Agarose plugs containing yeast chromosomes were prepared as described previously [57]. These were incubated overnight at 37uC in 0.5 ml/plug TE containing 1 mg/ml RNAseA. After extensive washes with Wash Buffer (10 mM Tris-HCl pH 7.5 50 mM EDTA), plugs were loaded on 1% agarose gel and sealed in the wells with a solution of 1% LMP agarose in TBE 0.5X. Gels were run at 4uC for 24 h at 165 V, with 60 seconds pulses for 12 h and 90 second pulses for 12 h, using an Amersham Gene Navigator system. Supporting Information Figure S1 H2A is phosphorylated during the cell cycle. (A) Strains K699 (WT) and YMIC5A3 (mec1-1 sml1) were arrested in G1 with a-factor and UV irradiated. At the indicated time-points Mec1 activation was assayed by western blotting monitoring cH2A, using actin as loading control. (B) Strains K699 (WT) and YMIC6C3 (tel1D) were arrested in M phase with nocodazole and UV irradiated. At the indicated time-points Mec1 activation was assayed by western blotting monitoring cH2A and using actin as loading control. (C) K699 (WT) was synchronized in G1 with a- factor and released into fresh medium. Every 10 min samples of the culture were withdrawn and progression into the cell cycle was monitored by FACS analysis. H2A phosphorylation was moni- tored by western blotting. (D) Strains K699 (WT), YMIC6C3 (tel1D), YFP20 (dpb11-1) and YFP225 (tel1Ddpb11-1) were pro- cessed as in B. (TIF) Figure S2 Tel1 contributes minimally to Rad53 phosphoryla- tion in the absence of either Ddc1 or Dpb11-AAD. (A) Strains K699 (WT), YAN21/8d (ddc1D), YFP20 (dpb11-1), YMIC6C3 (tel1D), YFP223 (tel1Dddc1D), YFP225 (tel1Ddpb11-1) were syn- chronized in G1 with a-factor and released in 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 strains K699 (WT), YMIC6C3 (tel1D), YFP62/1d (ddc1Ddpb11-1), YMIC5A3 (mec1-1) and YFP230 (ddc1Ddpb11-1tel1D) were spotted on YPD plates supplemented with HU at the indicated concentration. Survival was assayed by monitoring cell growth after 6 days. (TIF) Figure S3 Low levels of Mec1 activity are sufficient to delay mitosis following S-phase arrest. (A) K699 (WT), YFP20 (dpb11-1), PLoS Genetics | www.plosgenetics.org 9 March 2011 | Volume 7 | Issue 3 | e1002022