Fluorescence microscopy was performed on a Nikon Eclipse Ti inverted microscope and a DeltaVision microscope (Applied Precision) using 100× objective lens with immersion oil. For sample fixation, 1 × 107 cells were harvested and treated with 1 ml of paraformaldehyde solution (4% paraformaldehyde, 3.4% sucrose in distilled water) for 15 min at room temperature. Fixed cells were collected by centrifugation and washed twice with phosphate-buffered saline (PBS). Washed cells were resuspended in 0.5 ml of PBS. Fixed samples were analyzed immediately or stored at 4°C for a few days. To visualize nuclear DNA, the fixed samples were directly treated with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg ml−1) (Invitrogen). A 96-well glass bottom plate (Metrical Bioscience) for the Nikon Eclipse Ti inverted microscope and a coverglass bottom dish (SPL Life Sciences) for the DeltaVision microscope were pretreated with concanavalin A (5 μg ml−1) (Sigma-Aldrich) to allow cells to adhere to the plates. For fluorescence quantification of the BiFC signal and RFP-tagged Rad52, the acquired images were analyzed with ImageJ software (National Institutes of Health). The fluorescence intensity was quantified by manual selection of Rad52 signals that colocalized with DAPI signals, and the background intensity was obtained from a random position that was not colocalized with DAPI signals. The relative fluorescence intensity was calculated by subtracting the background intensity from the fluorescence intensity of Rad52 BiFC or RFP signals.

Time-lapse microscopy of living cells was performed using a DeltaVision microscope. Cells were grown in SC media containing 2% raffinose to an OD600 = 1.0 at 30°C, and 2% galactose was directly added to cell cultures. Cells were adjusted to an OD600 = 0.2 with SC media containing 2% galactose, and 150 μl of cell culture was placed on concanavalin A–treated coverglass bottom dish. Fluorescence images were acquired every 5 min in an environmental chamber of the DeltaVision microscope. To maintain cell growing condition, the environmental chamber was heated to 30°C while time-lapse images were acquired.

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