Figure 3A. Exponentially growing S. cerevisiae WT and yeh2∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for Western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each timepoint and plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented as the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated.C. Exponentially growing S. cerevisiae WT and ste∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for inspection by fluorescence microscopy. The establishment of DNA resection factories was assessed by counting the number of Rfa1-CFP foci per cell. Scale bar is 4 µm. Left: the mean values of each timepoint of three independent experiments were used to obtain this graph (mean and SEM). Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. Right: representative images of both strains at timepoint 120 min. Scale bar is 4 µm.D. WT and yeh2∆ cells carrying a plasmid bearing the HO nuclease gene under a galactose-inducible promoter were exposed to 2% galactose to trigger the expression of the HO nuclease to cut at the MAT locus, and samples were retrieved at the indicated time points for genomic DNA extraction. Subsequent SspI digestion, in combination with the use of a probe targeted to MAT, allows defining the fate of the cut fragment by Southern blot analysis. As resection progresses on DNA, SspI restriction sites are lost, leading to progressively longer ssDNA fragments (whose sizes are indicated at the right of the gel) that can be separated on an agarose gel under denaturing conditions. Given the much stronger signal of the "cut" fragments with respect to the "resected" fragments, the gel has been split into low and high contrast halves, respectively. The ssDNA over cut yet unresected DSB molecules was calculated for each shown resection intermediate. The graph on the right plots the sum of all these values (cumulative ssDNA/DSB) for a given resected intermediate during the interval from 90 to 240 min. The error bar is the SEM of three independent experiments. * derives from applying a t-test that compares the two populations of values.E. Cells were pre-loaded with 60 µM oleate 30 µM BSA (mock) for 4 h, and then exposed to 0.005% MMS for the indicated times. Total genomic DNA was extracted (left panel) and 1500 ng of each condition were spotted onto a nylon membrane directly. The DNA, after being crosslinked to the membrane, was subjected to detection using an anti-ssDNA antibody (middle panel). The raw ssDNA signals obtained this way were quantified for three independent kinetics. The graph (right panel) shows the mean raw ssDNA signal value and the error bars are the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. As a control for the specificity of the anti-ssDNA antibody, we used the sample "MMS 3 h" for treatment with Mung Bean Nuclease (MBN), in order to digest single stranded DNA (ssDNA), or for denaturation, by addition of NaOH to a final 0.4 N concentration. These three samples are shown in the top part of the membrane.