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Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair

¹ Center for Molecular Oncology, University of Chicago, Chicago IL 60637, USA
² Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago IL 60637, USA

View larger version (50K): [in a new window]   Fig. 1. G1 cells respond to radiation. Wild-type cells were f arrested and irradiated or mock-treated then incubated at 22°C for 15 minutes before equilibrating on ice for processing. (A) Isolated chromosomes from f-arrested cells were probed with rabbit -Rfa1 (1:2000) then costained with DAPI and Alexa-488-conjugated -rabbit secondary (1:400). The superimposition of DAPI and Alexa fluorescence is shown. (B) 50 µg protein from f-arrested cells was run on 10% SDS-PAGE for western analysis. Blots were probed with -Rfa1 (1:1000) and visualized as described in the Materials and Methods. (C) 50 µg protein from f-arrested cells carrying RAD53-13Myc was run on 8% SDS-PAGE for western analysis.

View larger version (51K): [in a new window]   Fig. 2. G1 radiation results in dose-dependent replication delay. f-arrested wild-type (A), rad9 and rad17 (B) cells were irradiated and released to media at 30°C. 0.5 ml aliquots were fixed at indicated time points for flow cytometry. Lines drawn across graphs show 1N and 2N positions. Identical results were obtained with 0 cells (data not shown).

View larger version (18K): [in a new window]   Fig. 3. Spindle pole body duplication and bud emergence delay after G1 irradiation. SPC42-GFP (Schutz and Winey, 1998) strains were arrested in f and released following irradiation. 0.5 ml aliquots of cells were fixed in 3.7% formaldehyde to score bud emergence and SPD or fixed for flow cytometry (data not shown).

View larger version (33K): [in a new window]   Fig. 4. Rad53-13Myc shifts while G1 is extended. RAD53-13Myc cells were arrested with f and released following irradiation. Protein samples were collected for -Myc westerns, and p13suc1 associated histone H1 kinase activity (A,B) was measured at the indicated times. At the same time points, additional aliquots were fixed for flow cytometry (C) or incubated for 90 minutes in trapping media (with and without f) before fixation (D). The slight delay in rad9, rad17 progression after irradiation compared with unirradiated wild type was observed only in the RAD53-13Myc background.

View larger version (12K): [in a new window]   Fig. 5. Genetic requirements for G1 arrest. f-arrested cells were released after increasing doses of irradiation. f/nocodazole-trapping media was added at the indicated time points, and cells were incubated 90 minutes before fixation. (For wt, rad9 and rad17: , 0 Gy; , 100 Gy; , 200 Gy; , 400 Gy. For rad52 (, 0 Gy; , 50 Gy; , 100 Gy; , 200 Gy)). For comparisons, the solid line shows the percentage of 1N of the starting population and the hatched line marks 50% 1N. rad9, rad17 double mutants were indistinguishable from either single mutant in this assay (data not shown).

View larger version (21K): [in a new window]   Fig. 6. POL is not required for the G1 checkpoint. Indicated strains were treated as in Fig. 5.

View larger version (29K): [in a new window]   Fig. 7. G1 delay is the predominant response to tolerable radiation. Log phase cells were incubated for 3 hours at 450 rad/minute. At 60 minute intervals, cells were collected for plating efficiency (A), OD600 measurements (B) or fixed for flow cytometry (C). In (A), 3 µl of 10-fold serial diluted cells were spotted to YPD plates and incubated 48 hours at 30°C.

View larger version (41K): [in a new window]   Fig. 8. Tolerable radiation maintains G1. f-arrested cells were released into fresh media. Cells were split between a 22°C control (A) and irradiated samples (B) and (C). 15 minute time points were then collected from controls for flow cytometry. 90 minutes after release, irradiated samples were removed from the 137Cs source and incubated with (C) or without (B) f, and the time course was repeated.

View larger version (25K): [in a new window]   Fig. 9. Unbudded G1 arrest can last for 18 hours after DNA damage. Log-phase strains were irradiated with 3000 Gy then diluted to 104 cells/ml before plating on YPD (A) or were plated before UV irradiation at 100 J/m2 (B). Cell numbers were counted immediately (0 Hr) and at 18 hours post irradiation. (C) Cells were plated and incubated 0 or 20 minutes before 100 J/m2 irradiation. (Error bars represent s.d. of three experiments.)

View larger version (74K): [in a new window]   Fig. 10. Unbudded cells remain sensitive to f. Wild-type cells (see Fig. 9A,B) were collected 18 hours after (A) or UV (B) radiation and incubated in f for 3.5 hours at 30°C. Cells were fixed as for flow cytometry and scored (X63, Zeiss Axioskop) for formation of mating projections (arrow). Mating projections were not observed after incubation without f.

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