Fig. 3. Ddc2-YFP foci disappear rapidly upon repair when donor loci are present for HR. (A) Ddc2-YFP fusion allows visualisation of the same Mec1-Ddc2 foci detected by IF after DSB induction (see Figs 1 and 2). We monitored the presence of Ddc2-YFP foci in two isogenic strains either bearing wild-type (WT) HML and HMR donor loci (KD-131; light-grey bars) or bearing hml and hmr deletions (GA-2358; dark-grey bars) after induction of the HO endonuclease on galactose for the indicated times. (B) Focus disappearance requires donor loci and Rad51. To show whether loss of foci coincides with repair by HR, we scored for Ddc2-YFP foci both in the strains used in panel A and in KD-132, in which wild-type HML and HMR are combined with a full rad51 deletion. Cells were grown on YPLGg overnight and GAL::HO was induced by 2% galactose for 1 hour. Thereafter, glucose was added to repress the endonuclease. The fluorescence image (bottom) shows a panel of typical cells bearing Ddc2-YFP (yellow) on a background of Nup49-CFP signal (red) after 1 hour of growth on galactose with 2 hours of glucose recovery. The corresponding phase image is also shown (top). (Graph) The percentage of cells with Ddc2-YFP foci were monitored in all strains after 1 hour of growth on galactose (gal), and then again after 2, 4 and 6 hours on glucose (glu). Note that the processing of DSBs continues even after the switch to glucose. Bar, 2 µm.