Intra-G1 arrest in response to UV irradiation in fission yeast

Abstract

G1 is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in G1 poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early G1 phase. We find that irradiation results in delayed progression through G1, as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3- and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-G1- and intra-S-phase delays. We suggest that intra-G1 delay may be widely conserved and discuss significance and possible mechanisms.

Fig. 1.

Demonstration of a UV-inducible delay is shown. (A and B Upper) Synchronized cdc10-M17 (A) and cdc10-M17 rad3 (B) cells were UV-irradiated, and samples were removed for flow cytometry at the times indicated (in min). DNA histograms are shown for unirradiated control cells (filled profile) and irradiated cells (open profile with bold outline). (A and B Lower) Quantification of the fraction of cells with a 1C complement of DNA in the respective DNA histograms. ○, Control cells; •, irradiated cells. (C) Quantification of histograms from cdc10 cells irradiated and incubated in the presence of caffeine.

Fig. 2.

Arrested cells have not replicated the early origin ars3001. (A) Mutant cdc10^ts cells were incubated at 36°C for 4 h before release at 25°C. Samples were taken before release and at the times indicated for control (Left) and UV-irradiated (Right) cells. DNA was purified from each sample and subjected to 2D electrophoresis, blotting, and probing with an ars3001-specific probe. All panels are from the same experiment and the same blot. (B) Synchronized cdc10^ts mutant cells carrying the Mcm4-GFP allele were treated as described in Fig. 1, and samples were removed at the time of irradiation and after1hof incubation at 25°C. The cells were permeabilized, and unbound Mcm4 was extracted with detergent. GFP fluorescence was detected, and the cells were classified as either negative (Left) or positive (Right). (C) Percentage of positive cells, after 0 min and after 60 min, with or without UV. Standard errors are indicated.

Fig. 3.

Cds1, but not Chk1, is involved in the delay. (A–C) Synchronized cdc10^ts cells carrying chk1 (A), cds1 (B), or cds1 chk1 (C) mutations were treated as described in Fig. 1. Quantification of the fraction of 1C cells is shown, as described in the legend to Fig. 1. (D) Synchronized cdc10^ts mutant cells were UV-irradiated, and samples were taken after 0, 60, and 90 min and analyzed for Cds1 kinase activity. (Upper) The relative amounts of Cds1 protein used in the kinase assay were estimated by immunoblotting. (Lower) The kinase activity was measured by phosphorylation of myelin basic protein (MBP). The hydroxyurea (HU)-treated cells gave such a strong kinase signal that we reduced the intensity of the exposure of this lane to visualize the MBP bands in all lanes. Quantification showed that the signal from HU-treated cells was 8.5-fold higher than that from the UV90 sample.

Fig. 4.

The Cdc2 protein is not phosphorylated in the initial phases of the delay. Synchronized cdc10^ts mutant cells were UV-irradiated, and samples were taken for flow cytometry and for immunoblotting at different time points after irradiation. (A) The immunoblots were probed with antibodies to phosphorylated Cdc2 (Upper) and to total Cdc2 protein (Lower). (B) Quantification of the immunoblot, with correction for loading and using total Cdc2 as the loading control. (C) Percentage of 1C cells, as determined by flow cytometry. ○, Unirradiated control cells; •, irradiated cells. (D) A blot of total RNA probed with a cig2-specific probe (Top). Quantification is shown (Middle), as is the gel after ethidium bromide staining (Bottom), to demonstrate even loading.