DSB-stimulated ectopic gene conversion (EGC) frequency determination

The frequency of repair of a HO endonuclease catalyzed DSB at the HIS3 locus on chromosome XV (his3-Δ3'-HOcs) by conservative homologous recombination with a his3-′MscI allele proximal to the LEU2 locus on chromosome III was determined as described previously [29]. A minimum of 10 one milliliter YPGL cultures were inoculated with single colonies of selected strains and grown at 30°C overnight to a density of 1 – 2 x 10⁷ cells/ml before addition of 20% galactose to a final concentration of 2% followed by an additional four hour incubation at 30°C. Dilutions were plated onto YPD medium to assess viability and onto synthetic complete medium lacking histidine (SC-His) to select for recombinants. Plates were incubated at 30°C for three days and the number of colonies counted. Frequencies of EGC were determined by dividing the number of His⁺ recombinants by the number of viable cells plated. Mean EGC frequencies, 95% confidence intervals and t-test values were calculated with Prism. Gene conversion events at the HIS3 locus in select His⁺ recombinants were verified by Southern blot analysis.