DNA damage checkpoint maintenance through sustained Chk1 activity

doi:10.1242/jcs.01204

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First published online 22 June 2004
doi: 10.1242/jcs.01204

Journal of Cell Science 117, 3489-3498 (2004)
Published by The Company of Biologists 2004

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DNA damage checkpoint maintenance through sustained Chk1 activity

Christine Latif¹^,2, Nicole R. den Elzen¹ and Matthew J. O'Connell¹^,2^,3^,*

¹ Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, VIC 8006, Australia
² Department of Genetics, University of Melbourne, Parkville, VIC 3010, Australia
³ Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA

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Fig. 1. Chk1 kinase activity is rapidly induced following UV irradiation and remains elevated for the duration of the checkpoint. (A) Checkpoint response of wild-type (chk1:ep) G2 cells to 150 J m^–2 UV-C. Cells were synchronized by centrifugal elutriation and then irradiated ( ${blacksquare}$ ) or left untreated ( ${square}$ ) (t=0). The proportion of cells either passing mitosis or with septa and cell number was determined. Chk1 kinase activity was assayed at the time points indicated. Data shown is representative of three independent elutriations. (B) Phosphorylated Chk1 (Chk1

) was detected by western blotting. (C) Asynchronous wild-type cells (chk1:ep) were irradiated with 0 J m^–2, 25 J m^–2, 50 J m^–2, 75 J m^–2 or 150 J m^–2 UV-C, and extracts were prepared 30 minutes after irradiation. The average Chk1 kinase activity from three concurrent assays is shown and error bars represent standard error. Chk1 and hyperphosphorylated Chk1 (Chk1

) was detected by immunoblotting with anti-HA antibodies

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Fig. 2. chk1-ts1 is a conditional allele of chk1 that is active at 25°C but not at 36°C. (A) UV-C and ionizing radiation survival curve for wild-type (chk1:ep) ( ${square}$ ), chk1 ${Delta}$ ( ${blacksquare}$ ) and chk1-ts1 ( ${circ}$ ) at 25°C and 36°C. Strains were grown at the temperature shown and irradiated with the indicated dose of UV-C (J m^–2). Data are normalized against unirradiated controls, and error bars represent s.e.m. (n=3-9). (B) MMS sensitivity assay for chk1:ep, chk1 ${Delta}$ and chk1-ts1. Strains were grown at the temperatures indicated then serial dilutions of culture inoculated onto YES agar plates containing 0.01% MMS. Plates were incubated at the specified temperature to allow colony formation.

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Fig. 3. chk1-ts1 is conditionally checkpoint defective. (A) Checkpoint assays for asynchronously growing wild-type (chk1:ep) ( ${square}$ ), chk1D ( ${blacksquare}$ ) and chk1-ts1 ( ${circ}$ ) at 25°C and 36°C. Cells were irradiated with 150 J m^–2 UV-C and septation indices counted at each time point. Data are normalized against the time of irradiation (t=0). (D) DAPI-stained chk1-ts1 cells from both the 25°C and 36°C asynchronous time courses, with or without UV-C irradiation. Cells showing aberrant mitotic figures are indicated by the arrows. Scale bar, 10 µm.

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Fig. 4. chk1-ts1 kinase activity is induced following irradiation at 25°C but not at 36°C. (A) Comparison of kinase activity of asynchronous wild-type (chk1:ep) and chk1-ts1 cells grown at 25°C or 36°C and irradiated with 150 J m^–2 UV-C or 150 Gy ionizing radiation. Samples were taken 10 minutes, 20 minutes and 30 minutes after irradiation. Error bars represent standard errors. (B) Chk1 and hyperphosphorylated Chk1 (top) was detected by immunoblotting with anti-HA antibodies.

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Fig. 5. Inactivation of chk1-ts1 following the accumulation of DNA damage results in a rapid increase in aberrant mitoses. (A) Asynchronous wild-type (chk1:ep), chk1 ${Delta}$ and chk1-ts1 cells were treated with either 0 Gy or 250 Gy ionizing radiation (0.7 Gy minute^–1, 6 hour exposure) or 0.0075% MMS (16 hour exposure) at 25°C. Cultures were then shifted to 36°C to inactivate the chk1-ts1 allele. For each strain, the number of aberrant mitoses was counted every 15 minutes for 1 hour after temperature shift for irradiated ( ${bullet}$ ), MMS-treated ( ${square}$ ) and untreated ( ${blacksquare}$ ) cells. (B) DAPI-stained wild-type (chk1:ep), chk1 ${Delta}$ and chk1-ts1 cells are shown at 25°C, at 25°C following 250 Gy irradiation and following a temperature shift after irradiation to 36°C for 60 minutes. Cells showing aberrant mitotic figures are indicated by the arrows.

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Fig. 6. Chk1 is required for ionizing-radiation-induced checkpoint maintenance. (A) Asynchronously growing wild-type (chk1:ep) ( ${square}$ ), chk1 ${Delta}$ ( ${blacksquare}$ ) and chk1-ts1 ( ${circ}$ ) cells were incubated at 25°C for the duration of the experiment or shifted to 36°C at 15 minutes after irradiation. The proportion of binucleate cells was counted at the time points indicated by DAPI and calcafluor staining, and normalized to that of t₀ (binucleate index). In each case, shifting the culture to 36°C led to a chk1-independent decrease in the number of binucleate cells as a result the temperature-shift-induced heat shock. (B) The checkpoint response of asynchronously growing wild type (chk1:ep) ( ${square}$ ), chk1 ${Delta}$ ( ${blacksquare}$ ) and chk1-ts1 ( ${circ}$ ) to 150 Gy ionizing radiation. Cultures were incubated at 25°C for the duration of the experiment or shifted to 36°C at 15 minutes after irradiation. The percentage of binucleate cells was counted at the indicated timepoints. In the absence of Chk1 activity (chk1-ts1 and chk1 ${Delta}$ ), the percentage of binucleate cells increased more rapidly than wildtype (chk1:ep) controls. (C) Checkpoint proficiency of asynchronously growing wild-type (chk1:ep) ( ${square}$ ), chk1 ( ${blacksquare}$ ) and chk1-ts1 ( ${circ}$ ) to 150 Gy ionising radiation. Cultures were incubated at 25°C for the duration of the experiment, or shifted to 36°C at 15 minutes post irradiation. The proportion of aberrant mitoses was determined at the indicated time points. The proportion of aberrant mitoses is higher in the absence of Chk1 activity (chk1-ts1 and chk1 ${Delta}$ ).

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Fig. 7. Chk1 is required for UV-C-induced checkpoint maintenance. (A) Wild-type (cdc10-M17chk1:ep) cells were grown to mid-logarithmic phase at 25°C and then shifted to 36°C for 4.75 hours, after which the cultures were shifted to 25°C. Half of the culture was irradiated with 50 J m^–2 UV-C ( ${blacksquare}$ ) and the other half mock irradiated ( ${square}$ ). After an additional 20 minutes at 25°C, one irradiated and one mock-irradiated culture were shifted to 36°C, with duplicate cultures left at 25°C. Samples were taken at the indicated times and cells passing mitosis assayed by DAPI staining. (B) Repeat of the experiment in (A) but using cdc10-M17chk1 ${Delta}$ cells. (C) Repeat of the experiment in (A) but using cdc10-M17chk1-ts1 cells.