DSB Repair by NHEJ and SSA

DSB repair by NHEJ and SSA in YMV45 strains were detected by Southern blot analysis using an Asp718-SalI fragment containing part of the LEU2 gene as a probe, as previously described [71,72]. To determine the efficiency of NHEJ, the intensity of the uncut band at 30 min after HO induction (maximum efficiency of DSB formation), normalized respect to a loading control, was subtracted to the normalized values of the same band at the subsequent time points after glucose addition. The obtained values were divided by the normalized intensity of the uncut band in raffinose (100%). To determine the efficiency of DSB repair by SSA, the normalized intensity of the SSA product band at different time points after HO induction was divided by the normalized intensity of the uncut band at time zero before HO induction (100%). The loading control was obtained by hybridization of the filters with a probe that anneals to the RAD52 gene. DSB repair by NHEJ in JKM139 strains was detected by Southern blot analysis of SspI-digested genomic DNA with a MATa probe (200870–201587 coordinates of chromosome III).