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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02477

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Apoptosis in budding yeast caused by defects in initiation of DNA replication

¹ Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
² Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

View larger version (47K): [in a new window]   Fig. 1. Loss of Orc2p function leads to the production of ROS. (A) Wild-type (W303) and orc2-1 (GA1410) cells stained with PI (red) and the ROS probe H2DCF-DA (green) before and after shifting to the nonpermissive temperature of 37°C for 6 hours. (B) FACS detection of ROS and PI staining of GA1410 orc2-1 cells before and after incubation at 37°C for 6 hours. Data are plotted on logarithmic scales. Signals from unstained cells correspond to autofluorescence. (C) Temperature- and time-dependent production of ROS (green) and PI (red) signals in GA1410 orc2-1 cells. Signals from autofluorescing cells were disregarded during quantitation of ROS and PI signals. (D) Suppression of ROS production in GA1410 orc2-1 cells by ectopic expression of ORC2. (E,F) Suppression of ROS in GA1410 orc2-1 cells transformed with a plasmid expressing the co-chaperone Mge1p, but not in cells transformed with an empty vector control. (G) Western blot showing that orc2-1p is destabilized in a proteasome-dependent fashion within 30 minutes of a shift to 37°C. Glucose (glu) represses transcription of orc2-1 driven by a galactose-regulated promoter in the GAL–orc2-1 cells used in this experiment. MG132 is an inhibitor of the proteasome and was used at a final concentration of 250 µM. Mcm2p was used as a loading control. (H) Effects of proteasome inhibition on lethality of high temperatures in GA1410 orc2-1 cells. (I) Effects of proteasome inhibition on production of ROS in GA1410 orc2-1 cells.

View larger version (26K): [in a new window]   Fig. 2. Loss of Orc2p function activates the budding-yeast metacaspase Yca1p. (A) Detection of signals by FACS in wild-type and GA1410 orc2-1 cells stained with propidium iodide (PI) and FITC-VAD-FMK, which stains cells containing activated caspases green. (B) The proportions of W303 wild-type and GA1410 orc2-1 cells with caspase activity before and after shifting to 35°C for 6 hours in cells from which the YCA1 metacaspase-encoding gene had (yca1) or had not been deleted. (C) Detection of caspase activity in wild-type and GA1410 orc2-1 cells with YCA1 deleted, and transformed with a plasmid expressing Yca1p or an empty vector control plasmid. The increase in caspase activation in controls for this experiment compared with the experiment presented in B is caused by culturing cells in defined medium required for plasmid maintenance instead of rich medium (data not shown). (D) Effect of yca1 on viability of GA1410 orc2-1 cells. Viability was measured as colony-forming units and was normalized to viability measured in cells maintained at 23°C compared with cells shifted to 35°C for indicated times.

View larger version (33K): [in a new window]   Fig. 3. Cell-cycle-dependent effects on apoptosis in GA1410 orc2-1 cells. (A) Survival (left) and ROS production (right) in GA1410 orc2-1 cells after synchronization in G1 or S phase for 3 hours at 23°C followed by a shift to 37°C for 3 hours. Viability measurements were normalized to viability of cells synchronized in G1 or S phase in the absence of the temperature shift to account for minor effects on viability of synchronization regimens. (B,C) FACS measurements of temperature-dependent effects on DNA replication in cdc6-1 (B) and GA1410 orc2-1 (C) cells synchronized in G1 with mating factor and subsequently released into S phase. Cells were synchronized in G1 phase for 2 hours at 23°C and maintained in G1 phase for an additional 5 hours at this temperature or at 37°C (`G1'), or were synchronized in G1 phase for 2 hours at 23°C and then maintained at this temperature or at 37°C for an additional 3 hours followed by release into S phase for 2 hours at the same temperature (`G1>S'). (D) Effects of orc2-1p depletion on DNA replication in GAL–orc2-1 cells that regulate orc2-1 expression from a galactose-inducible and glucose-repressible promoter. (E) GA1410 orc2-1 cells arrest in S phase after shifting to 37°C for 6 hours. DNA is stained with DAPI. (F) Effects of temperature on the cell cycle in exponentially proliferating GA1410 orc2-1 cells. (G-I) Effects of temperature on viability of cdc6-1 (G) and GA1410 orc2-1 (H) cells, and on ROS production in GA1410 orc2-1 cells (I) synchronized in G1 phase and then released (`G1>S') or not released (`G1') into S phase using the same regimen described for FACS analysis (B,C).

View larger version (26K): [in a new window]   Fig. 4. Apoptosis in orc2-1 cells is stimulated by the Mec1p checkpoint protein and coincides with a Rad53-dependent DNA-damage response. (A) Production of Mec1p stimulates the production of ROS in mec1-21 orc2-1 cells. (B) Rad53p phosphorylation in GA1410 orc2-1 and W303 wild-type cells. orc2-1 (1/10 ROS) are strain YB0057. (C) Induction of HUG1 expression in GA1410 orc2-1 and wild-type cells. Signals are arbitrary units of fluorescently labeled cDNA produced from mRNA isolated from cells shifted to 37°C for indicated times and hybridized to HUG1 sequences on an Affymetrix microarray chip. Values represent the average of two independent experiments without subtraction of background.

View larger version (29K): [in a new window]   Fig. 5. ROS is induced in other temperature-sensitive initiation mutants and is amplified by additional loci that also respond to DNA damage. (A) Temperature- and time-dependent induction of ROS in cdc6-1, GA1410 orc2-1 and wild-type (W303) cells. (B) ROS induction in wild-type and orc2-1 segregants of a GA1410 orc2-1 x ORC2 mating shifted to 37°C for 6 hours. (C) ROS induction by DNA-damaging agents in parental wild-type (W303) or GA1410 orc2-1 cells. (D) ROS induction by DNA-damaging agents in wild-type (ORC2) segregants of the GA1410 orc2-1 x ORC2 mating that do [ROS (+)] or do not [ROS (–)] harbor the additional determinants of ROS present in GA1410 orc2-1 cells. Cells were exposed for 6 hours at 23°C to the DNA-damaging agent adozelesin (ado) at a final concentration of 4 µM or to 0.1% methylmethane sulfonate (MMS).

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