Tetracycline

Drugs were used at the following concentrations. Auxin (Merck Cat# I5148): 1 mM. Tetracycline (nzytech Cat# MB02202): 0.6 mM in 2% glucose, 0.3 mM in 0.02% glucose. Nicotinamide (Merck Cat# N0636): 4 mM. Hydroxyurea (Merck Cat# H8627): as indicated. Rapamycin (Merck Cat# R0395): as indicated. Chlorpromazine (Merck Cat# C8138): as indicated.

Yeast strains were grown in YPD medium containing 2% glucose as carbon source at 25 °C. For cell cycle synchronization, exponentially grown cells at 25 °C were synchronized either in G1 phase by adding alpha factor (Genscript) to a final concentration of 3–5 mg/ml for 2 h or in G2/M phase with Nocodazole to a final concentration of 10 µg/ml together with DMSO to a final concentration of 1% for 2 h at 25 C. The cells were released from the respective arrests by washing them with YP medium (YP + 1%DMSO in case of Nocadazole arrest) followed by its release into fresh YPD medium containing 0.033% MMS at 28 °C. To inhibit de-novo Sgs1 translation in cells expressing pADH1-Tc-3HA, tetracycline (NZYTech) was added to a final concentration of 1 mM at the indicated times. For drug sensitivity assays, cells from overnight cultures were counted and diluted before being spotted on YPD plates containing the indicated concentrations of drugs and incubated at 28 °C for 2–3 days.