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Resection, recombinants, and HR–dependent restitution of MMS–derived DSBs.
Logarithmically growing apn1/2 cells as well as triple mutants containing a deletion of an HR gene rad52 or rad51 were arrested in G2/M with nocodazole, treated with MMS (0.1%, 15 min) in PBS and returned to the YPDA+nocodazole medium. Cells were collected at the indicated times and processed for PFGE analysis. Chromosomes were visualized by ethidium bromide staining (A) and by Southern blotting (B) with a CHA1 probe that identifies Chr III. The SMD was estimated in the ethidium bromide stained gel (A) by comparing the amount of DNA material in the SMD region to DNA in the small chromosomes that experienced little breakage, “SMD / Chr (I+VI)”. The MMS-induced breakage of the circular Chr III is indicated on the Southern blot (B). Resection, identified by the upward “PFGE-shift” of linear Chr III in (B), is seen in all three strains. The rad51- and rad52-dependent generation of dimer-size linear chromsome III is also indicated.