FIG. 5.

5′ to 3′ resection at the MAT locus is unaffected by mre11-D56N in the presence of additional DSBs. (A) Physical map of the MATa- MATα switching process and illustration of the DNA intermediates as observed by alkaline gel electrophoresis after a StyI-BamHI double digestion. The probe used reveals the 0.9- and 1.9-kb StyI fragments from the uncut MATa and MATα loci, respectively. When expressed, HO cuts the MAT locus to produce a smaller 0.7-kb HO-StyI cut fragment. As a result of the 5′ to 3′ resection of the right end of the HO break, some StyI and BamHI cut sites become single stranded and resistant to cleavage, generating high-molecular-weight ssDNA fragments when electrophoresed under alkaline conditions (ssDNA1 to -5). The sizes of the different DNA intermediates are indicated in parentheses. S, StyI; B, BamHI; HO, HO cut site. (B) A 60-min HO induction was performed in MRE11 (LSY1477) and mre11-D56N (LSY1483) strains, and ssDNA formation at MAT was analyzed by alkaline gel electrophoresis (see Materials and Methods). The sizes of the ssDNA species (ssDNA1 to -5) are indicated in parentheses. LEU2, hybridization control; t, time in minutes between the beginning of HO induction and the collection of cells for DNA analysis. (Bottom) Quantitation of the ssDNA intensities, normalized with the LEU2 signal and expressed as the fraction of the maximum intensity of ssDNA1 for each strain. Error bars correspond to the range of the data from two independent experiments.