Yeast two-hybrid assay

Matchmaker GAL4 Two-Hybrid System 3 (Clontech) was used for the Epe1-Swi6 interaction analysis. pGBKT7 (containing TRP1) was used for bait expression. pGADT7 (containing LEU2) was used for prey expression. pGBKT7 and pGADT7 plasmids were introduced into the AH109 host strain by a polyethylene glycol/lithium acetate (PEG/LiAc)-mediated method. AH109 harbors a HIS3 reporter gene. Yeast strains were cultured on proper minimal synthetic dropout (SD) media according to the user manual (Clontech). 3-AT (15 mM; 3-amino-1,2,4-triazole), an inhibitor of the HIS3 product, was added for precise analysis of the Epe1-Swi6 interaction. Glucose was applied to SD media as the carbon source. The experimental procedure for serial dilution assays of S. cerevisiae strains was the same as for S. pombe strains.