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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02703

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Production of reactive oxygen species in response to replication stress and inappropriate mitosis in fission yeast

Maria A. Marchetti^1,*, Martin Weinberger², Yota Murakami³, William C. Burhans^2, and Joel A. Huberman^1,

¹ Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
² Department of Cell Stress Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
³ Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shogoinkawahara-machi, Sakyo-ku, Kyoto 606-8507, Japan

View larger version (68K): [in a new window]   Fig. 1. Detection of ROS and death in fission yeast cells. Wild-type and dfp113-240 mutant cells were incubated for 80 minutes at 25°C in the presence of DCDHFDA to detect ROS. The cells were then harvested, washed and resuspended in buffer containing PI to detect dead cells.

View larger version (24K): [in a new window]   Fig. 2. Time- and temperature-dependence study of ROS production and PI staining in cells bearing temperature-sensitive mutations in orp2 or cdc18. The indicated strains, in log-phase, were shifted to the indicated temperatures for the indicated numbers of hours. Then DCDHFDA was added, and incubation was continued at the same temperatures for an additional 80 minutes. Other conditions were as described in Fig. 1 and supplementary material Fig. S1.

View larger version (18K): [in a new window]   Fig. 3. Temperature-dependence study of ROS production and PI staining in cells bearing temperature-sensitive mutations in orp5. This experiment was carried out as described in Fig. 2, except that the time of incubation was 4 hours in all cases.

View larger version (11K): [in a new window]   Fig. 4. N- and C-terminal deletions in dfp1 lead to ROS production and PI staining. The indicated fission yeast strains were incubated for 80 minutes at 25°C in the presence of DCDHFDA and stained with PI.

View larger version (14K): [in a new window]   Fig. 5. Mutant cells defective in pathways affecting non-homologous end-joining, the DNA-damage checkpoint, replication-fork stability and the MRN complex appear to produce less ROS than mutant cells defective in replication proteins. The indicated strains were incubated at 25°C for 80 minutes in the presence of DCDHFDA. Other conditions were as described in Figs 1 and 4.

View larger version (22K): [in a new window]   Fig. 6. ROS production by dfp1 N-terminal deletion mutants requires a functional Rad3p- and Cds1p-dependent checkpoint pathway, but ROS production by the orp5-H19 strain does not. Double-mutant strains bearing orp5-H19, dfp113-193 or dfp1183-191 together with rad3 or cds1 were generated by crossing. Results are shown for two independently derived isolates of each type. Strains lacking a rad3 or cds1 mutation are indicated by +. Cells in early log-phase were incubated at 30°C for 6 hours. (A) Levels of ROS production. (B) Levels of PI staining.

View larger version (17K): [in a new window]   Fig. 7. Deletion of any of the checkpoint-Rad genes leads, in the presence of HU, to elevated levels of ROS and PI staining. The indicated strains were incubated at 25°C for 4 hours with (+) or without (-) 12 mM HU. Then DCDHFDA was added, and incubation was continued for 80 minutes. The cells were stained with PI and analyzed by flow cytometry.

View larger version (15K): [in a new window]   Fig. 8. Stimulation of ROS production and PI staining by HU requires abrogation of both the Cds1p- and Chk1p-dependent checkpoint pathways or dysregulation of Cdc2p. The indicated strains were incubated at 25°C for 4 hours with (+) or without (-) 12 mM HU. Then DCDHFDA was added, and incubation was continued for 80 minutes. The cells were stained with PI and analyzed by flow cytometry.

View larger version (12K): [in a new window]   Fig. 9. Checkpoint-inhibited, checkpoint-stimulated and checkpoint-independent pathways leading to production of ROS and cell death in fission yeast. This figure provides a simplified summary of the pathways leading from mutations in genes that encode replication-initiation proteins, or from HU treatment, to elevated production of ROS and cell death.