DNA damage response and proliferation of <i>ddc2-S4</i> mutants.

<p>(A, B) DNA damage sensitivity of <i>ddc2-S4</i> 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), <i>ddc2Δ</i> (KSC1234) and <i>ddc2-S4</i> (KSC3153). (C) Effect of <i>ddc2-S4</i> on DNA damage checkpoint signaling. Wild-type (KSC1178), <i>ddc2Δ</i> (KSC1234), and <i>ddc2-S4</i> (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 <i>ddc2-S4</i> mutants. Wild-type (KSC1178), <i>ddc2Δ</i> (KSC1234) or <i>ddc2-S4</i> (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 <i>ddc2-S4</i> mutation on cell proliferation in the presence of <i>SML1</i>. <i>ddc2Δ</i> mutants carrying the <i>URA3</i>-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 <i>URA3</i>-marker plasmid can proliferate in the presence of 5-FOA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004136#pgen.1004136-Boeke1" target="_blank">[62]</a>.