Figure 2.

The DSB-dependent decreases in 3C interaction frequencies requires RAD51, RAD52, and MPS3. (A) 3C interactions in G1-arrested cells. CY1276 cells (MATa “donorless,” isogenic to JKM179) were arrested in the G1 phase of the cell cycle by treatment with 4 μM α-factor prior to DSB induction with galactose. 3C analysis was carried out as in Figure 1B using a primer on the DSB-containing fragment and various primers specific for the neighboring fragments. G1 arrest was monitored by flow cytometry, and DSB formation was confirmed by Southern blotting (Supplemental Fig. S3). (B,C) Recombination proteins are required for the decrease in interactions of the DSB seen by 3C. 3C interactions in asynchronous rad51Δ (B) and rad52Δ (C) strains were analyzed as in Figure 1B. (D) The Mps3p N-terminal domain is required for DSB-induced changes in 3C interaction frequencies. 3C interactions in asynchronous wild-type or mps3Δ75-150 cells were analyzed as in Figure 1B. For each panel, representative experiments of at least three independent data sets are shown. Error bars represent one standard deviation from the mean for three separate PCRs. DSB formation was shown to occur normally in each of these mutants (Supplemental Fig. S8).