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First published online 25 November 2008
doi: 10.1242/jcs.035428

Journal of Cell Science 121, 4047-4054 (2008)
Published by The Company of Biologists 2008
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The G1-S checkpoint in fission yeast is not a general DNA damage checkpoint

Marit Krohn*, Henriette C. Skjølberg*, Héla Soltani, Beáta Grallert and Erik Boye{ddagger}

Department of Cell Biology, Institute for Cancer Research, Rikshospitalet Medical Centre, Montebello, 0310 Oslo, Norway


Figure 1
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  		Fig. 1. UVC-induced phosphorylation of eIF2{alpha}. Immunoblots of total cell extracts probed with antibody against eIF2{alpha}-P and against Cdc2 as loading control, and the quantification (bottom). (A) G1-synchronised cdc10-M17 cells exposed to increasing doses of UVC irradiation. (B) Asynchronous cells auxotrophic for leucine, which were starved of leucine for 2 hours (–Leu) and asynchronous wild-type cells exposed to 0 or 1100 J/m2 of UVC. (C) Synchronous cdc10-M17 cells in G1, S and G2 phase exposed to 0 and 1100 J/m2 of UVC. The corresponding DNA histograms are shown below. (D) G1-synchronised cdc10-M17 cells irradiated with 0 (control) or 1100 J/m2 of UVC and incubated for different times after irradiation, as indicated.

 

Figure 2
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  		Fig. 2. H2O2-induced eIF2{alpha} phosphorylation and cell cycle delay. (A) Immunoblot (see Fig. 1 for details) of asynchronous cells exposed to increasing concentrations of H2O2. G1-synchronised wild-type (B) and gcn2 mutant (C) cells exposed to 0 (control) or 5 mM H2O2 and incubated for different times, as indicated. The quantified relative value for H2O2 at time 0, was arbitrarily set to 1. (D) DNA histograms of G1-synchronised wild-type (left) and gcn2 (right) cells that were untreated (control; shaded histogram) or treated with 5 mM of H2O2 (unshaded) and incubated for the times indicated (in minutes) after H2O2 treatment. (E) Pre-RC assays of G1-synchronised wild-type (left) or gcn2 mutant (right) cells after exposure to 0 (control) or 5 mM of H2O2 and incubated for different periods of time, as indicated.

 

Figure 3
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  		Fig. 3. MMS-induced eIF2{alpha} phosphorylation. For details, see Fig. 1. (A) Asynchronous cells exposed to increasing concentrations of MMS. (B) Cell survival after MMS. (C,D) G1-synchronised wild-type (C) and gcn2 mutant (D) cells were exposed to 0 or 0.15% MMS and incubated for different periods of time after MMS inactivation and removal, as indicated.

 

Figure 4
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  		Fig. 4. MMS-induced effects on cell-cycle progression. (A) Flow cytometry histograms of G1-synchronised wild-type (left) and gcn2 (right) control cells (shaded) and cells treated with 0.15% MMS (unshaded) incubated for the times indicated after MMS treatment. (B-E) Immunoblots (for details, see Fig. 1) of wild-type (B) and gcn2 (D) cells probed with antibody against Rum1. Immunoblots of wild-type (C) and gcn2 (E) cells probed with antibody against phosphorylated Cdc2. Antibody against tubulin was used as loading control. Quantifications are shown as white columns for the control and shaded columns for MMS-treated cells.

 

Figure 5
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  		Fig. 5. The effect of PUVA on eIF2{alpha} phosphorylation and cell-cycle progression. (A) Immunoblot of G1-synchronised wild-type cells exposed to psoralen and 0 or 10 kJ/m2 of UVA. (B) Flow cytometry histograms of G1-synchronised wild-type cells irradiated (unshaded) and not irradiated (shaded) and incubated for the times indicated (in minutes) after exposure. (C) Pre-RC assay of G1-synchronised cells at different time points after exposure to 0 (control) or 10 kJ/m2 of UVA.

 

Figure 6
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  		Fig. 6. The effect of ionizing irradiation on eIF2{alpha} phosphorylation. (A) Immonoblots of asynchronous cells exposed to increasing doses of IR and a comparison with leucine-starved cells (see Fig. 1). (B) Cell survival after ionizing radiation.

 

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© The Company of Biologists Ltd 2008