DSB Induction and DNA Resection Assay

Yeast cells were grown overnight in the pre-induction YEP–raffinose medium (1% yeast extract, 2% peptone, and 2% raffinose) to a density of ~1 × 10⁷ cells/ml. DSB induction was initiated by adding 2% galactose. For testing the resection under replication stress, 200 mM HU (final concentration) was added to each sample when starting the galactose induction. Samples were collected at different time points following break induction. DNA isolated by glass bead disruption using a standard phenol extraction method was digested with EcoRI and separated on 0.8% agarose gels. The resolved DNA was transferred onto a Nylon hybridization transfer membrane (Perkin Elmer). Radiolabeling of DNA probes was carried out according to the manufacturer's instructions (Takara). Southern blotting and hybridization were carried out as described previously (Chen et al., 2012 (link)). The signal on the phosphor screen was captured by scanning with an OptiQuant Cyclone Plus machine (Perkin Elmer). Quantities of DNA loaded on gels for each time point were normalized using the TRA1 DNA probe. The resulting values were further normalized to that of the control sample (uncut). Three independent experiments were performed for each strain.