Antonia Lopez-Girona, Junko Kanoh, Paul Russell Current Biology

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Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast Antonia Lopez-Girona, Junko Kanoh, Paul Russell Current Biology Volume 11, Issue 1, Pages 50-54 (January 2001) DOI: 10.1016/S0960-9822(00)00026-9

Figure 1 Elimination of the Cdc25 nuclear localization signal (NLS) causes nuclear exclusion of Cdc25. (a) Immunolocalization of “12-myc”-tagged Cdc25 proteins expressed from the cdc25 genomic locus is shown. Cdc25-nls has a mutant NLS and is excluded from the nucleus, whereas Cdc25-nls/NLS has the SV40 T-antigen NLS and appears exclusively nuclear. Nuclei were visualized with DAPI. Cdc25 nuclear staining was decreased in binucleate cells, as previously described [9]. (b) A cdc25–22 strain (1; PR1483) is inviable at 35.5°C, whereas cdc25–22 strains having integrated plasmids that express Cdc25, Cdc25-nls, or Cdc25-nls/NLS (2–4; AL2660, AL2749, and AL2750, respectively) are viable at 35.5°C. (c) An anti-myc immunoblot of 12-myc-tagged cdc25+, cdc25-nls, and cdc25-nls/NLS strains mock-irradiated (lanes 1, 3, and 5, respectively) or exposed to 100 Gy IR (lanes 2, 4, and 6) is shown. (d) Inactivation of the exportin Crm1 does not change Cdc25-nls nuclear exclusion. JK2421 (cdc25-nls:12myc) and JK2425 (cdc25-nls:12myc crm1–809) strains were grown at restrictive temperature (25°C) for 4 hr prior to immunolocalization analysis Current Biology 2001 11, 50-54DOI: (10.1016/S0960-9822(00)00026-9)

Figure 2 Forced nuclear inclusion or exclusion of Cdc25 does not abrogate damage checkpoint responses. (a) Immunolocalization of Cdc25-nls and Cdc25 nls/NLS are unaffected by DNA damage induced by bleomycin. Bleomycin treatment (45 min) caused cells of both genotypes to undergo cell cycle arrest and become elongated. (b) Ultraviolet (UV) survival is unimpaired in cdc25-nls and cdc25 nls/NLS strains. (c) Checkpoint delay in cdc25+, cdc25-nls, and cdc25-nls/NLS strains but not Δchk1 cells is shown. Cells were synchronized in G2 phase by elutriation and exposed to 5 mU ml-1 bleomycin (+BL) for the duration of the experiment or mock-treated Current Biology 2001 11, 50-54DOI: (10.1016/S0960-9822(00)00026-9)

Figure 3 Checkpoint regulation of Cdc25 is retained in cdc25-nls and cdc25 nls/NLS strains. Strains that contained wild-type or mutant cdc25 in a wee1–50 Δmik1 background were grown at 25°C and synchronized in G2 phase by centrifugal elutriation. Cells were irradiated (100 Gy) or mock-irradiated and then shifted to 35.5°C. The rate of mitotic induction is determined by Cdc25 activity. Irradiation caused a ∼40 min mitotic delay in all strains. Thus, the in vivo function of Cdc25, Cdc25-nls, and Cdc25-nls/NLS was equally inhibited by DNA damage Current Biology 2001 11, 50-54DOI: (10.1016/S0960-9822(00)00026-9)

Figure 4 14-3-3 proteins are important for checkpoint response irrespective of effects on Cdc25 localization. (a) Immunolocalization of Cdc25-nls and Cdc25 nls/NLS in 14-3-3-deficient (Δrad24) strains is shown. (b) The checkpoint response to ionizing radiation is abrogated in Δrad24 cells exposed to different doses of ionizing radiation. (c) Checkpoint delay is abolished in Δrad24 strains synchronized in G2 phase and exposed to bleomycin (+BL). (d) Immunolocalization of Cdc13 B-type cyclin in cells exposed to 100 Gy ionizing radiation or mock-treated is shown. Cells were photographed 30 min after irradiation Current Biology 2001 11, 50-54DOI: (10.1016/S0960-9822(00)00026-9)