Fig. 7.

rsc mutants are defective in DSB repair in G₂ but not in G₁. (A and B) Haploid BY4741 cells were grown to mid-log phase, arrested at G₁ (10 μg/ml α-factor for 3 h) (A) or G₂ (10 μg/ml nocodazole) (B), and then incubated with MMS (0.2% for 20 min). Cells were washed and resuspended in YEPD supplemented with 10 μg/ml nocodazole and allowed to recover for up to 8 h with samples for PFGE taken at the indicated time points. DNA separated by PFGE as shown in panels A to F was subjected to Southern blot analysis to determine the percentage of repair using the PEX10 fragment in chromosome IV as a probe. The percentage of the repair of broken chromosome IV was calculated by measuring the amount of chromosome IV after MMS damage at each time point and then normalized to the mock-treated samples at the same time point. The repair efficiency after 8 h of recovery in wild-type (wt) cells was set to 1. The averages of three independent experiments ± the standard deviation are shown. (C and D) Diploid yeast cells with a MATα deletion (and thus of the MATa type instead of the typical MATa/α type) were arrested at G₁ (C) or G₂ (D), treated with MMS, and allowed to recover in nocodazole-containing but MMS-free medium. Shown are FACS profiles of diploid wild-type and rsc7 and rad52 mutant cells arrested at G₁(C) or G₂ (D), treated with MMS, and allowed to recover in nocodazole-containing but MMS-free medium. (E and F) Quantification of the repair efficiency of haploid yeast cells with MATa (BY4741) (E) and diploid cells (BY4743) (F) at G₂ using Southern blotting with rad59Δ and rad51Δ mutant combinations. Repair efficiency at G₂ was determined as described for panel A and plotted. The averages of three independent experiments ± the standard deviation are shown.