Strategy to identify targets of Mec1 and Tel1 in yeast. (A) WT and mec1Δ tel1Δ cells were treated with MMS to induce accumulation of Mec1/Tel1-dependent phosphorylation (black circles), specifically in WT cells. Phosphopeptides were purified and then labeled by the N-isotag reagent (d0-Leu-NHS for WT and d10-Leu-NHS for mec1Δ tel1Δ cells). Labeled phosphopeptides were then combined and analyzed by 2D liquid chromatography–tandem MS. Based on their relative abundance, phosphopeptides (and the corresponding phosphorylation sites) were then determined to be either independent of (case 1) or dependent on Mec1 and Tel1 (case 2). (B) Comparison of the distribution of proteins in each abundance range for the identified phosphoproteins (gray bars) and the total yeast proteome (black bars). Information on protein abundance was retrieved from Ghaemmaghami et al. (29).

Identification and analysis of Mec1/Tel1-dependent phosphorylation. (A) Relative ion abundances of selected phosphopeptides (with the indicated phosphorylation site) in WT (filled circles) and mec1Δ tel1Δ (open circles) cells. (Left) Example of phosphopeptides present at similar abundance in both cells, thereby containing a Mec1/Tel1-independent phosphorylation. (Right) Example of phosphopeptides specifically present in wild-type cells, thereby containing a Mec1/Tel1-dependent phosphorylation. For these Mec1/Tel-dependent phosphorylations, the open circles indicate the expected position of the phosphopeptide ions from mec1Δ tel1Δ cells (labeled with d10-Nisotag). (B) Number and percentage of Mec1/Tel1-dependent phosphorylation identified relative to the total number of phosphorylation sites identified (2,689 phosphorylation sites and 1,109 proteins; see Methods for detailed description of criteria used). (C) Comparison of the frequency of amino acids at the +1 position of phosphorylated serine or threonine for all identified phosphorylation (gray bars) and Mec1/Tel1-dependent phosphorylation (black bars).

Identification and analysis of Rad53-dependent phosphorylation. (A) Number and percentage (related to total number of Mec1/Tel1-dependent phosphorylation identified) of Rad53-dependent phosphorylation identified. All Rad53-dependent phosphorylation was also Mec1/Tel1-dependent (n = 62). (B) Frequency of amino acids at the +1 position of phosphorylated serine or threonine for Rad53-independent and Mec1/Tel1-dependent phosphorylation (black bars) and Rad53-dependent phosphorylation (gray bars). (C) Simplified representation of the Mec1, Tel1, and Rad53 kinases and their targets in the DNA damage response.

Cbf1 and Trm8 are phosphorylated in an S/T-Q motif, in an MMS-induced manner. (A and D) Relative ion abundance of the Cbf1 and Trm8 phosphopeptides, with phosphorylation at S45 and S7, respectively, in WT (filled circles) and the indicated kinase-null (open circles) cells. (B and E) Western blot analysis of Cbf1 and Trm8 immunoprecipitated (IP) from control or MMS-treated cells and eluted by using TEV protease. Phosphorylated S/T-Q motifs were detected with an antiphospho-S/T-Q antibody. For loading control, the blot was later detected with an anti-CBP antibody. (C and F) Western blot was performed as described in B and E, and proteins were purified from MMS-treated cells lacking the indicated kinases.

The nuclear pore proteins Nup1, Nup2, and Nup60 are directly phosphorylated by Rad53. Shown are in vitro kinase reactions using recombinant Rad53 and either Nup1, Nup2, or Nup60 complexes purified from untreated cells. Purified comoplexes were stained by Coomassie blue, and indicated bands were indentified by MS. Anti-FLAG Western blot is shown as loading control. Asterisks indicate partially proteolyzed fragments of indicated proteins.