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Supplementary Figure 1 (A) Flow cytometry analysis of DNA content in exponentially growing wild type cells exposed to HU, MMS or CPT as described in Fig. 3. Cells are collected before induction of Gal-HO. (B) Viability of cells exposed to Zeocin and HU as described in Fig. 2C. 100 cells were washed and plated on YPD. Viable colonies were counted after 48h at 30°C. Supplementary Figure 2 (A) PCR analysis of DSB repair at the MAT locus after induction of HO in wild type cells (PP723) exposed or not to HU, MMS and CPT. Cell growth and arrest was performed as described in figure 3. *: ARG5,6 DNA. (B) Position of PCR primers is indicated with arrows. (C) Quantitation of the intensity of the DNA repair band in untreated (Ctrl) cells or in cells exposed to HU, MMS and CPT. Band intensity was normalized to ARG5,6. Supplementary Figure 3 Analysis of DSB resection at the MAT locus by alkaline gel electrophoresis in wild type cells exposed to HU, MMS and CPT. (A) Wild type cells (PP648) were exposed to genotoxic drugs as indicated in Fig. 3 and were harvested at the indicated times after induction of the HO endonuclease. Genomic DNA was digested with StyI and was analyzed by alkaline gel electrophoresis. Resection generates ssDNA fragments of increasing length (fragments 1, 2 and 3) due to the progressive loss of StyI restriction sites. C: HO cut fragment. *: StyI fragments detected by the probe. (B) Southern blot analysis of HO-induced resection in untreated cells or in cells exposed to HU, MMS or CPT. (C) Quantitation of the intensity of resection bands 1, 2 and 3 normalized to a control fragment on chromosome III. Supplementary Figure 4 (A) Schematic representation of the MAT locus. The progressive loss of StyI restriction fragments upon induction of the HO endonuclease with galactose and resection of 5’-ends the break is shown. See figure 4C for the corresponding Sothern blot. (B) Quantitation of unequal sister-chromatid exchange (uSCE) in wild type (PP915) and rad51 (PP985) cells exposed for 45 minutes to 0.02% MMS. The experiment was performed as described previously (Fasullo et al., 2001).