FIG. 5.

The pot1Δ rqh1-hd double mutant and rqh1-hd single mutant released from S-phase arrest have RPA foci during the M phase. (A) Visualization of RPA foci during the M phase in asynchronous living cells. Rad11-GFP-expressing living rqh1-hd cells and pot1Δ rqh1-hd cells were observed in a series of time-lapse images taken at 1-min intervals at 30°C. Bar = 5 μm. Time zero represents the time just before cells enter anaphase. (B) Percentages of cells in which the RPA foci (shown in panel A) appeared on the chromosome bridges are shown. The data from the analysis of the Rad11-mRFP-expressing living pot1Δ rqh1-hd rad51Δ were also added. The total number of M phase cells that were observed in this experiment (N) is shown at the top. (C) Visualization of RPA foci during the M phase in rqh1-hd cells released from S-phase arrest. Rad11-GFP-expressing living rqh1-hd cells released from S-phase arrest were observed at 30°C. We added 10 mM HU to an asynchronous culture of Rad11-GFP-expressing rqh1-hd cells in YEA medium. After 4 h in HU, the cells were washed and transferred to YEA medium without HU and cultured for a further 2 h. The resulting cells were observed. Two different rqh1-hd cells are shown. Bar = 5 μm. (D) Percentages of rqh1-hd cells in which the RPA foci, shown in panel C, appeared on chromosome bridges are shown. The total number of M-phase cells that were observed in this experiment (N) is shown at the top. (E) RPA foci in the pot1Δ rqh1-hd double mutant do not colocalize with Gar2 (rDNA). Merged images of fluorescence micrographs showing Rad11-mRFP (red) and Gar2-GFP (green) in the two different pot1Δ rqh1-hd double mutant cells during the M phase at 30°C.