Figure 3. DNA damage response and proliferation of ddc2-S4 mutants.

(A, B) DNA damage sensitivity of ddc2-S4 mutants. Serial dilutions of cultures were spotted on YEPD medium without or with MMS or HU (A). Cultures spotted on YEPD medium were irradiated with UV light (B). Plates were incubated at 30°C for two-three days. Strains used were wild-type (KSC1178), ddc2Δ (KSC1234) and ddc2-S4 (KSC3153). (C) Effect of ddc2-S4 on DNA damage checkpoint signaling. Wild-type (KSC1178), ddc2Δ (KSC1234), and ddc2-S4 (KSC3153) cells expressing Rad53-HA were grown to log-phase and exposed to MMS (0.05%) at 30°C for the indicated length of time. Cells were harvested and subjected to immunoblotting analysis with anti-HA antibodies. (D) G2/M-phase DNA damage checkpoint in ddc2-S4 mutants. Wild-type (KSC1178), ddc2Δ (KSC1234) or ddc2-S4 (KSC3153) cells were arrested with nocodazole and irradiated or not irradiated with UV (50 J/m²). At the indicated times after release of UV-irradiated (+UV) and unirradiated (−UV) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining. (E) Effect of ddc2-S4 mutation on cell proliferation in the presence of SML1. ddc2Δ mutants carrying the URA3-marked YCp-DDC2 plasmid (KSC3308) were transformed with YCpT-DDC2, YCpT-DDC2-S4 or the control vector. Transformants were streaked and grown on plates containing medium with or without 5-fluoroorotic acid (5-FOA) at 30°C. Only cells that have lost the URA3-marker plasmid can proliferate in the presence of 5-FOA [62].