Figure 3. Cohesin promotes recovery after replication fork arrest.
(A) Drop assays on exponentially growing wild type (JC-470) and scc1-73 (JC-1339) cells. 1:10 serial dilutions were grown on YPD + 0.2M HU or 0.005% MMS at 25 or 30°C.
(B-C) Scc1 is required for recovery of MMS-induced forks stalling. WT (PP-872) and scc1-73 (PP-1186) cells were grown at 25°C and synchroni zed in G1 with α-factor 30°C then released into S phase with 400 μg/ml BrdU to label newly-replicated DNA and 0.033% MMS for 60 minutes at 30°C. Cells were then washed and resuspended in fresh medium at 30°C for the indicated time after MMS release (t=0). Samples were subjected to PFGE analysis. (B) Southern blot probed for chromosomes 3, 4 and 14. (C) Quantification of chromosome mobility in wild-type (open circles) and scc1-73 (black circles). Median values and standard deviation for the three probes are shown.
(D-E) Representative DNA fibers of WT or scc1-73 cells exponentially grown at permissive temperature (25°C), arrested in G1 with α-factor at semi-permissive (30°C) and released synchronously into S phase in t he presence of 0.033% MMS for 60 minutes at 30°C. Green: BrdU, red: DNA, arro ws: unreplicated DNA. Bar: 50 kb. (E) Box plots depict the percentage of unreplicated DNA per DNA fiber and the length of unreplicated gaps. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively.
(F) 2D gel analysis of replication intermediates in sgs1Δ (PP-35) and sgs1Δ scc1-73 (PP-1688) cells shifted at restrictive temperature (37°C) upon G1 arrest and released synchronously into S phase in the presence of 0.033% MMS for 30 minutes at 37°C. Genomic DNA was extracted and dige sted with EcoRI and the replication intermediates were analyzed by southern blot with a probe recognizing the ARS305 region.
(G) Rates of interhomologous recombination were measured as described (Mozlin et al., 2008) in diploid ade2-I/ade2-n WT (JC-2596) and scc1-73 (JC-2597) cells. Error bars correspond to s.d. of 9 diploids per condition in each experiment. Frequencies were determined at least twice in separate experiments giving comparable results at 30°C after overnight i ncubation in YPD + 10 mM HU or 0.001% MMS.