A Novel Checkpoint and RPA Inhibitory Pathway Regulated by Rif1
Figure 4
Rif1 inhibits the checkpoint responses to telomere uncapping in cdc13-1 cells.
(a) Growth of serial dilution of cdc13-1 cells with or without additional mutations (indicated at the right of each row) at different temperatures, indicated above each plate. (b–e) Over-night cultures at 20°C were incubated with nocodazole for 2 h and then shifted to 25°C or to 27°C for 3 h. DNA was digested and hybridized with a CA-rich probe. (b) TG signals at native (non-denatured) telomeres. Numbers at the top of each lane correspond to different mutants, revealed in c. (c) Legend for b, d, and e indicating relevant mutations. (d) TG signals at denatured telomeres. (e) The percentage of ssDNA represents the fraction of native TG-ssDNA normalized to the total of TG sequences in each denatured sample. Error bars represent the standard deviation between measurements from two independent experiments. (f–g) The ssDNA was quantified by QAOS and the G2/M fraction counted by microscopy in cdc13-1 RIF1+ cells incubated for 240 min at 25°C (white circles) or 27°C (black circles). (h–i) As in f–g, except that cells were cdc13-1 rif1Δ at 25°C (white triangles) or 27°C (black triangles). (j) Rad53 activation detected by western blotting in cdc13-1 cells with wild type RIF1 (left) or with a rif1Δ mutation (right), incubated at 25°C, (except for the time 0 when the temperature was 21°C). (k) As in (j), except that the Ddc2 activation was detected.