Fig. 6
Mrc1 is phosphorylated by Snf1 upon low glucose availability. a Mrc1 is phosphorylated by Snf1 in vivo. This experiment was carried out as in Fig. 1a. b Quantification of five independent phosphorylation experiments with the indicated strains. Data represent the mean and standard deviation. The graph indicates the relative phosphorylation of Mrc1 (assessed as in a) in the indicated strains normalized to the total amount of precipitated Mrc1–TAP protein and then referenced to control conditions. The data represent the mean and standard deviation of five independent experiments. Values that are significantly different (respect to the control conditions) from Student's t test are indicated (***p < 0.005). c Phosphorylation of Mrc1 by Snf1 delays DNA replication when the cells are subjected to nutrient deprivation (low glucose), whereas mrc13A or snf1 mutant cells bypass this delay. Cells were synchronized and released in low glucose (LG), and DNA replication over time was followed using FACS. d Low glucose delays DNA replication. Replication forks in WT and mrc13A cells in control conditions or in LG stress were analyzed using DNA combing. The graph indicates the distribution of BrdU track length (kb) during 1 h of labeling. Box, 25–75 percentile range; Whiskers, 5–95 percentile range. *Values are significantly different (**p < 0.01, ***p < 0.0001). e Snf1 is phosphorylated in vivo upon low glucose. Cells were synchronized and released into S phase in low glucose (LG). Snf1 phosphorylation over time was followed using western blotting with specific antibodies. f Mrc1 and Snf1 interact in vivo. HA–Snf1 was immunoprecipitated (IP) from cells and coprecipitated Mrc1–TAP was assayed using western blotting. g snf1 and mrc13A cells show a higher percentage of Rad52-YFP foci upon low glucose. Data represent the mean and standard deviation of three independent experiments. Asterisks indicate statistically significant differences by Student's t test (*p < 0.05, **p < 0.01, ***p < 0.005) of stress versus control conditions
