(A) Diagram of the CRASH (Cre-Reported Altered States of Heterochromatin) assay used to measure transcriptional silencing stability at HML. (B) Schematic for directing homologous recombination between HML::cre and pseudo-MAT in strain JRY10817. The region between the dashed lines represents the 2.4 kb sequence of HML::cre that was inserted onto the left arm of chromosome VIII. The first 844 bp of the cre open reading frame were included, omitting the protein’s active-site sequences. The sequence on chromosome VIII in green indicates that it is transcribed whereas the homologous sequences in black at HML::cre sequence are silenced. The open triangles represent three SNPs that distinguish the homologous regions: two SNPs on chromosome VIII on either side of the site of the HO-induced double-strand break, SNP-L and SNP-R, and one SNP within HML that destroys the HO recognition sequence, SNP-INC. SNP-L is located 45 bp into the cre open reading frame, 721 bp from the site of HO cleavage, and SNP-R is at the 20th base pair of the Z region, ∼16 bp away from the site of the double-strand break. The arrow indicates the site of HO cleavage. (C) DNA hybridization blot of double-strand-break kinetics in JRY10817. A blot evaluating HML::cre and pseudo-MAT showed double-strand-break induction at pseudo-MAT after HO induction for the times shown. Here and in subsequent figures, the ratio of cut/total pseudo-MAT band intensities were calculated as described in Materials and Methods.