FIG. 6.

Kinetics of repair of HO endonuclease-induced DSBs. Haploid strains were transformed with pHT51 carrying the HO gene under the control of the galactose promoter. HO endonuclease expression was induced by suspending the cells in a galactose-containing medium for 1 h and was then repressed by suspending the cells in a glucose-containing medium, and the DSB repair kinetics were observed as described in Materials and Methods. (A) Physical maps used for DSB repair detection. (i) The MATα locus. The indicated probe (a 1.0-kb NdeI-HindIII fragment) was used to detect the 2.2-kb MAT distal, 1.8-kb MATα, and 0.7-kb HO-cut fragments after StyI digestion. HO endonuclease produces a 0.7-kb HO-cut fragment from the 1.8-kb MATα fragment, and this 0.7-kb fragment is replaced by a 0.9-kb MATa fragment when the mating type switches from MATα to MATa. (ii) pHT51. The indicated probe (a 2.4-kb PvuII-PvuII fragment obtained from pJH283) was used to detect 5.7-kb parental and 3.3-kb HO-cut fragments after HindIII digestion. Stippled and solid rectangles, the homologous regions of pHT51 and the genome and the 35-mer HO recognition sequence, respectively. (B) Kinetics of DSB repair at the MATα locus. (C) Kinetics of DSB repair on pHT51. Strains: HTY925 (rad⁺), HTY927 (mre11Δ), HTY929 (rad52Δ), HTY931 (mre11-58), and HTY933 (rad50S).