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. 2014 Nov 6;42(21):13174–13185. doi: 10.1093/nar/gku1095

Figure 6.

Figure 6.

Deletion of HUG1 specifically alleviates the growth defects of some R2 mutants. (A) Invalidation of HUG1 abrogates the growth defects of epitope-tagged Rnr2 strains in the presence of HU. Five-fold serial dilutions of wild-type (WT) or epitope-tagged Rnr2 strains invalidated or not for HUG1 were spotted onto YPD plates complemented with hydroxyurea (HU) at various concentrations (15, 50 or 100 mM) and incubated at 30°C for 2 days. (B) Invalidation of HUG1 but not DIF1 abrogates the growth defects of epitope-tagged Rnr2 strains. Five-fold serial dilutions of exponentially growing cells were spotted onto YPD plates complemented with hydroxyurea (25, 50 or 100 mM) and incubated at 30°C for 2 days. Relevant genotypes are indicated on the left. (C) HUG1 deletion improves the cell-cycle progression of the RNR2–3FLAG strain after release from HU block. Wild-type (WT) or RNR2–3FLAG strains invalidated or not for HUG1 were treated for 2 h with HU (80 mM) before release. Cells were fixed with ethanol at indicated time points after release and cell-cycle profiles were analysed by flow cytometry after propidium iodide staining. 1C, 2C: DNA content; 1C corresponds to cells in G1 phase, 2C to cells in G2/M phases. (D) Invalidation of HUG1 decreases the level of Rad53 phosphorylation of the RNR2–3FLAG strain after HU treatment. Wild-type (WT) or RNR2–3FLAG strains invalidated or not for HUG1 were treated or not with HU (80 mM) for 2 h. After cell extraction, equal amounts of total protein extracts were loaded and analysed by SDS-PAGE followed by western blotting using polyclonal anti-Rad53 antibodies. (E) The suppression of the resistance of RNR2–3FLAG hug1Δ cells to HU by reintroducing HUG1 depends on Rnr2 interaction. Strains whose relevant genotype is indicated on the left were transformed with pGAL-HUG1 WT, mutant (M1, M2 or M3) or the corresponding empty vector. Serial dilutions of transformants were plated on minimal medium containing 2% galactose (SGal) complemented with various concentrations of hydroxyurea (HU) as indicated and grown for 5 days at 30°C.