6576 Nucleic Acids Research, 2011, Vol. 39, No. 15 Figure 4. Detection of DSBs caused by the lacO-LacI array. (A) Schematic of chromosome 3 showing the position of the ade6 locus and probes used for the Southern blot analysis of the CHEF gel in (B). (B) Detection of DSBs by whole chromosome CHEF analysis. The DNA in lanes a–f and i–n are from strains containing the lacO array with and without LacI (++) expression. The DNA in lanes g, h, o and p are from a strain that contains a MATa cleavage site in place of the lacO array and either pREP81 (HO ) or pACYCREP81-HO (HO +). Cultures were grown in EMMG lacking leucine, histidine and thiamine at 30 C for 20 h (lacO array strains) or 24 h (MATa strains) before isolating genomic DNA in agarose plugs. (C) Schematic showing the position of the lacO array relative to AvrII sites ﬂanking the ade6 locus and the probe used for the Southern blot analysis of the CHEF gel in (D). (D) CHEF analysis of AvrII digested genomic DNA from strains with and without LacI (++) expression. (E) Viability of strains relative to minus LacI control after 24 h of LacI (++) induction. (F) The percentage of viable colonies in E that are Ade+. In both (E) and (F) the error bars are the standard deviations about the mean. repair. Indeed a rad22D mutant containing the lacO array grows more slowly upon LacI (++) induction than when LacI is repressed, whereas a wild-type strain grows simi- larly whether LacI (++) is induced or not (Supplementary Figure S2). Intriguingly the growth of a rad51D mutant containing the lacO array is largely unaffected by LacI (++) induction indicating that Rad51 is not essential for the repair of lacO-LacI-induced DSBs (Supplementary Figure S2), which suggests that like recombinant forma- tion viability might depend on SSA. If true then mutants deﬁcient in SSA that contain the lacO array should show a reduction in viability when LacI (++) is induced. To test this we compared the viability of wild-type, rqh1D exo1D, rad22D and rad22D swi10D strains with and without the lacO array after 24 h of LacI (++) induction (Figure 4E). Each of the mutant strains containing the lacO array exhibit a reduction in viability after LacI (++) induction compared to strains without the lacO array (Figure 4E). Moreover this reduced viability correlates with a reduc- tion in Ade+ recombinants suggesting that SSA between the ade6 genes that ﬂank the lacO array is a major path- way for repairing the DSBs that are induced within it (Figure 4F). Based on the data in Figure 4D, at least 80% of rqh1D exo1D mutant cells containing the lacO array experience a DSB after 24 h of LacI (++) in- duction. In contrast viability is reduced only by 25% in