The conserved DNA damage response (DDR) pathway proceeds as a highly coordinated cascade of cellular events under the control of the phosphatidyl inositol 3′
kinase-related kinases (PIKKs), most notably Tel1 and Mec1 in Saccharomyces cerevisiae
and their homologs ATM and ATR, respectively, in human [1
]. During the DDR, sensor proteins detect DNA damage and then recruit and activate multiple proteins that mediate and transduce signals to elicit, among others, transcriptional programs, cell cycle arrest, DNA repair activity and, in the setting of irreparable damage, apoptosis or senescence [1
]. In S. cerevisiae
under genotoxic stress, Tel1 and Mec1 activate the DDR by phosphorylating key mediators Chk1, Rad53, Mrc1 and Rad9, and others, resulting in the halt of DNA replication and cell cycle progression at G1 and S phases or at G2/M transition [5
]. These events are coordinated with global changes in transcriptional patterns and DNA repair activation to ensure that the cell cycle progresses and DNA replication resumes once the damage is repaired. In addition, the discoveries of Hop1 as a downstream effector of Tel1/Mec1 signaling and defective telomerase recruitment as a result of a Tel1 deficiency illustrate additional roles for Tel1/Mec1 in meiosis and telomere maintenance, respectively [3
Recently, a series of large-scale studies suggest that the number of Tel1/Mec1 targets is much higher than initially estimated. A high throughput analysis in yeast treated with DNA damaging reagents identified 355 proteins phosphorylated at S/T-Q sites [7
]. A similar approach in human cell lines treated with UV radiation, led to the identification of 570 phosphosites [8
]. An additional search for peptides phosphorylated at ATM/ATR consensus sites in response to ionizing radiation yielded more than 700 putative protein targets, of which many lacked functional characterization of their S/T-Q phosphorylation sites [9
]. While many of these phospho-targets function in DDR pathways, others belong to pathways that were not known to be under ATM/ATR control. Therefore, alternative methods to obtain a full census of Tel1/Mec1 substrates might delineate additional functions of these kinases beyond the DDR.
Tel1/Mec1 kinases phosphorylate well-known DDR proteins at S/T-Q consensus sites. In some targets, these sites appear to be concentrated within a relatively short stretch of sequence previously defined as at least 3 S/T-Q within 100 amino acids, the so-called S/T-Q cluster domain (SCD) [4
]. To date, an SCD has been confirmed in just 11 Tel1/Mec1 targets with many of these having more than one SCD (Figure and Additional file 1
: Table S1). Not all S/T-Q sites within a given SCD are phosphorylated, although all 11 of these SCD proteins possess at least 1 Tel1/Mec1-phosphorylated S/T-Q within an SCD.
Figure 1 SCD-containing proteins in S. cerevisiae. (A) List of eleven reported SCD domains . For each protein, the number of S/T-Q sites within the SCD(s) is indicated with the amino acid positions in brackets. Graphical representations of the approximate location (more ...)
Although the functions of SCD domains are not completely understood, they often mediate protein-protein interactions during signaling cascades [4
]. For instance, a single phosphorylation of the Rad53 SCD promotes dimerization whereas a double phosphorylation triggers Rad53 binding to the FHA domain of Dun1 [10
]. Similarly, sustaining the DNA damage signal requires oligomerization of Rad9 proteins at DNA breaks through the interaction of its BRCT domain and phosphorylated SCD [11
]. These examples suggest that SCDs are biologically relevant domains with important roles during the DDR.
Based on the original SCD definition (at least 3 S/T-Q motifs within 100 amino acids), more than 25% of the proteins in the S. cerevisiae proteome contain an SCD. To better discriminate against false positives, we used a more stringent definition of the SCD to identify potential Tel1/Mec1 targets. The final set of targets contained 436 proteins including the 11 known SCD-containing Tel1/Mec1 targets. This SCD census was enriched for proteins in DDR-related pathways such as cell cycle progression and checkpoints, DNA repair and transcriptional regulation. In addition, we observed an over-representation of proteins with roles in several pathways previously only weakly linked to Tel1/Mec1. Similar results were obtained when the new SCD definition was applied to generate a human SCD census.