Figure 6. Histone accumulation triggers a Swe1-dependent Cdc28CDK1 phosphorylation.
(a) Pds1 phosphorylation (Pds1-HA) in rad53-AID and rad53-AID lsm1∆ cells either after 4 hr treatment with NAA or bleomycin (Ble, 15 µg/ml). Pds1 appears as a doublet. Its phosphorylation can be visualised as a shifted band after bleomycin treatment. (b) Average number of Mad2 foci (Mad2-GFP) that co-localise with kinetochores (Mtw1-mCHERRY) in rad53-AID and rad53-AID lsm1∆ cells after 4 hr of treatment with either DMSO or Auxin. Two independent experiments were performed in which at least 100 cells were counted. (c) Cell-cycle progression from G2/M to the next G1 in rad53-AID lsm1∆ cells after treatment or not with Auxin. Exponentially growing cells were treated (or not) with Auxin for 2 hr and incubated for 2 additional hours with Nocodazole. Cells were then washed to eliminate Nocodazole and released into media containing Auxin and alpha-factor to arrest cells able to enter mitosis the next G1. This experiment was performed in cells carrying either a PDS1-HA tag or a SCC1-MYC tag. The presence of Clb2 and Rap1 was monitored with anti-Clb2 and anti-Rap1 antibodies. Rap1 was used in both as a loading control. A representative full example of the experiment for each strain is shown in the Figure 6—figure supplement 1a and b. This data file also includes the results obtained with the single mutant that are not shown in the this figure. Graphs below each panel represent the progression of cells to the next G1. These percentages were estimated from fixed cells that were analysed using DAPI staining. (d) Swe1 protein levels in rad53-AID and rad53-AID lsm1∆ cells treated as indicated. (e and f) Cdc28CDK1 phosphorylation levels in the indicated strains. Cdc28CDK1 phosphorylation is monitored with phospho-cdc2 (Tyr15) antibodies. Cells were treated as indicated (g) Cell-cycle progression from G2/M to the next G1 in rad53-AID lsm1∆ and rad53-AID lsm1∆ swe1∆ similar to the one described in (6d), using the strain that expresses Pds1-HA. The results obtained with the single mutant rad53-AID are shown in the Figure 6—figure supplement 1c. All the experiments observed in Figure 6 were performed twice and gave similar results.
Figure 6—figure supplement 1. (a and b) Representative full examples of the experiments described in Figure 6d.
