BACKGROUND
the conserved dna damage response  pathway proceeds as a highly coordinated cascade of cellular events under the control of the phosphatidyl inositol 3′ kinase-related kinases , most notably tel <dig> and mec <dig> in saccharomyces cerevisiae and their homologs atm and atr, respectively, in human  <cit> .
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  <cit> .
in s. cerevisiae under genotoxic stress, tel <dig> and mec <dig> activate the ddr by phosphorylating key mediators chk <dig>  rad <dig>  mrc <dig> and rad <dig>  and others, resulting in the halt of dna replication and cell cycle progression at g <dig> and s phases or at g2/m transition  <cit> .
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 hop <dig> as a downstream effector of tel1/mec <dig> signaling and defective telomerase recruitment as a result of a tel <dig> deficiency illustrate additional roles for tel1/mec <dig> in meiosis and telomere maintenance, respectively  <cit> .
recently, a series of large-scale studies suggest that the number of tel1/mec <dig> targets is much higher than initially estimated.
a high throughput analysis in yeast treated with dna damaging reagents identified  <dig> proteins phosphorylated at s/t-q sites  <cit> .
a similar approach in human cell lines treated with uv radiation, led to the identification of  <dig> phosphosites  <cit> .
an additional search for peptides phosphorylated at atm/atr consensus sites in response to ionizing radiation yielded more than  <dig> putative protein targets, of which many lacked functional characterization of their s/t-q phosphorylation sites  <cit> .
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/mec <dig> substrates might delineate additional functions of these kinases beyond the ddr.
tel1/mec <dig> 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  <dig> s/t-q within  <dig> amino acids, the so-called s/t-q cluster domain   <cit> .
to date, an scd has been confirmed in just  <dig> tel1/mec <dig> targets with many of these having more than one scd .
not all s/t-q sites within a given scd are phosphorylated, although all  <dig> of these scd proteins possess at least  <dig> tel1/mec1-phosphorylated s/t-q within an scd.
although the functions of scd domains are not completely understood, they often mediate protein-protein interactions during signaling cascades  <cit> .
for instance, a single phosphorylation of the rad <dig> scd promotes dimerization whereas a double phosphorylation triggers rad <dig> binding to the fha domain of dun <dig>  <cit> .
similarly, sustaining the dna damage signal requires oligomerization of rad <dig> proteins at dna breaks through the interaction of its brct domain and phosphorylated scd  <cit> .
these examples suggest that scds are biologically relevant domains with important roles during the ddr.
based on the original scd definition , 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/mec <dig> targets.
the final set of targets contained  <dig> proteins including the  <dig> known scd-containing tel1/mec <dig> 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/mec <dig>  similar results were obtained when the new scd definition was applied to generate a human scd census.
RESULTS
obtaining a census of scd proteins
the scd in s. cerevisiae was previously defined as a region with at least  <dig> s/t-q within  <dig> residues.
examination of the  <dig> known scd proteins revealed the scd could be defined as having  <dig> s/t-q within just  <dig> amino acids .
to refine and ease the stringency of our census, we used scanprosite to search the uniprot database for s. cerevisiae proteins containing at least  <dig> s/t-q within a stretch of  <dig> residues or less.
we found a total of  <dig> proteins, each having at least one scd region .
this number was significantly higher than the  <dig> scd proteins expected to be present in the yeast proteome by chance  suggesting scds are indeed biologically relevant units rather than stochastic events.
since the probability of seeing an s/tq by chance alone increases as the protein length increases, we determined the distribution of the scd-containing protein lengths by performing a goodness of fit test using person’s chi-square test and we found that the distribution of protein lengths in our census is not statistically different from a log-normal distribution  .
empirical support for our scd definition could be found in several ways.
first,  <dig> of the identified proteins had evidence of phosphorylation at s/t-q sites in mass spectrometry phosphoproteomic studies, with  <dig> of those occurring within an scd   <cit> .
second, of the  <dig> mec1/tel1-dependent and rad53-independent phosphoproteins that were induced after exposure of wildtype and rad53Δ yeast to methyl methanosulfonate,  <dig> were present in our list   <cit> .
third, our list also contained  <dig> of the  <dig> proteins that were found in as tel1/mec <dig> targets in a quantitative mass spectrometry analysis   <cit> .
fourth, additional similarities with other reports were uncovered in the amino acids flanking those scds that contained phosphorylated s/t-q  motifs .
serine residues were frequently found upstream of ps/t-q, whereas glutamic acid residues were enriched at the + <dig> position.
these features corresponded to sites of dna damage-induced ps/t-q sites in human proteins  <cit> .
therefore, our scd definition of  <dig> s/t-q within  <dig> amino acids identified proteins with empirical data supporting ddr-related functions.
analysis of the go-slim ontology terms associated with the  <dig> yeast scd proteins showed a non-random distribution and a concentration in specific functions, processes and components .
as anticipated, we found significant enrichment in ontology terms that are usually associated with ddr proteins.
like known tel1/mec <dig> targets, the identified scd-containing proteins tended to be nuclear, responsive to stress signals, involved in phosphorylation and signal transduction, and have roles in ddr-related pathways such as cell cycle progression and transcriptional regulation .
when the analysis was extended to all go terms, those related to ddr pathways remained significantly over-represented , validating our approach.
as expected for a list enriched for ddr genes, we found a significant enrichment in proteins involved in cell cycle progression, transcription, dna replication and dna repair .
in addition, our yeast scd census contained a significant enrichment of proteins associated with protein localization and endocytosis, suggesting a broader and more direct role for tel1/mec <dig> in these pathways than previously thought   <cit> .
the yeast census was also enriched for proteins involved in cytokinesis and cell division, pathways where a role for tel1/mec <dig> kinases was previously hinted .
these included components of the actin cortical cytoskeleton, which control later stages of mitosis to ensure both proper nuclear migration to the cellular bud and cell division during cytokinesis   <cit> .
this may also explain the enrichment of scd proteins from the cellular bud and cell cortex in the census .
in addition, several proteins involved in the spindle assembly checkpoint  and spindle position and orientation checkpoint as well as other networks controlling mitotic exit were found to contain an scd   <cit> .
consistent with a putative role for tel <dig> and mec <dig> kinases monitoring key transition steps in mitosis, we found a concentration of scd proteins localized to the kinetochore and spindle pole body, two key subcellular compartments central to the sac and completion of mitosis .
our yeast scd census also uncovered proteins not known to be tel1/mec <dig> targets, but with characterized roles in pathways well-known to be regulated by tel1/mec <dig> kinases.
the pathways included dna repair, dna replication, gene expression, meiosis, and telomere homeostasis .
for example, there was an over-representation of proteins influencing rna polii-dependent transcription , such as components of the pre-initiation complex and mediator as well as members of the saga and compass complexes .
furthermore, several ddr transcription factors such as rfx <dig> were also found to contain an scd, raising the possibility they may be under direct control of tel1/mec <dig> kinases  <cit> .
many of these scd-containing proteins belong to groups of proteins influencing more than one known ddr-related pathway.
for instance, most of the nucleases and helicases involved in dna double strand break repair containing an scd have also been associated with replication and telomere homeostasis .
similarly, several scd proteins are involved in sumoylation, ubiquitination, chromatin remodeling and the establishment of sister chromatid cohesion, which are activities known to influence several ddr pathways such as dna replication, transcription regulation, dna repair, and cell cycle progression .
another example of crosstalk among ddr related pathways by scd proteins in our census is a subset of transcription factors that ensure proper transitions within phases of the cell cycle .
this suggests these potential novel tel1/mec <dig> targets may serve as a link between cell cycle progression and global transcription changes, two key components during ddr.
census of human scd proteins overlaps with the yeast census
for a given scd-containing protein, the presence of an scd in orthologues increases the probability that it is a biological entity rather than generated randomly.
for this reason, we searched the human proteome for proteins containing this newly defined scd.
this census identified  <dig>  proteins, including  <dig> of the  <dig> proteins used to formulate the original scd definition   <cit> , and  <dig> of the  <dig> proteins found to contain ps/t-q sites following dna damage  <cit> .
the extremely low probability of coincidence between our human scd census and these experimental data  indicates that human scd proteins are significantly enriched for proteins known to be phosphorylated following dna damage  <cit> .
furthermore, the ontology terms over-represented in this list overlapped largely with those found experimentally in human ddr proteins and with our yeast scd census.
these included terms such as dna repair, cell cycle progression, gene expression, dna replication, and response to dna damage    <cit> .
as with the yeast scd census, we found an unexpected abundance of proteins belonging to the microtubule organizing center and spindle as well as the actin cytoskeleton .
also similar to yeast, the human scd census was enriched in mediator and key ddr transcription factors such as the human homologue of rfx <dig> .
network analysis of the human scd census revealed connections between scd proteins involved in cell signaling pathways including jnk, erk, ras, akt, calmodulin signaling, and nf-ĸb, a pathway in which atm plays important roles   <cit> .
remarkably, these networks included insulin signaling proteins which are heavily phosphorylated at s/t-q sites after dna damage, suggesting these phosphorylations may occur in the context of an scd   <cit> .
finally, as found in the network analysis of the yeast scd census, one of the human scd networks included several components of the nuclear pore complex as well as proteins involved in nucleocytoplasmic transport like importin-beta and exportin  <dig>  .
* phosphorylated in s/t-q sites in mass spectrometry searches.
** phosphorylated in s/t-q sites within an scd in mass spectrometry searches.
discussion
the scd is neither a motif nor a true protein domain in that a consensus alignment cannot completely define the region and there is variable spacing between each s/t-q.
this has made its identification in proteins difficult, relying on loose definitions extending to include 25% of the yeast proteome.
using a more stringent scd definition of a sequence containing at least  <dig> s/t-q in a stretch of  <dig> amino acids, we arrived at a refined census of  <dig> proteins in the yeast proteome, still a much larger number than expected at random.
the validity of this approach is supported by the enrichment of proteins phosphorylated at s/t-q sites in mass spectrometry studies and the presence of all well-characterized scd-containing proteins phosphorylated by tel1/mec <dig>  in addition, ontology terms related to the ddr are significantly over-represented in this census.
we propose that this newly defined scd can be used to predict new roles for tel1/mec <dig> during the ddr and to identify novel putative targets for these kinases.
while the presence of an scd in a protein may have arisen stochastically, the existence of several scd proteins in the same pathway is much more unlikely.
therefore, the definition has a higher predictive value when assigning new processes regulated by tel1/mec <dig>  similarly, for a given scd-containing protein, the presence of an scd in homologues in other organisms increases the probability that the scd is a biological entity and not randomly generated.
for this reason, we searched the human proteome for proteins matching this newly defined scd to look for similarities and differences.
table  <dig> shows a list of interesting yeast proteins in our census whose human orthologue either contains ps/t-q sites, possess an scd in their sequence, or both.
these genes are likely to be tel1/mec <dig> targets in yeast and, in fact, several of them were phosphorylated in s/t-q sites in high throughput mass spectrometry approaches  <cit> .
mitosis
as hinted by previous reports, the presence of scds in several sac proteins such as bub <dig>  mad <dig>  and cdc <dig> indicates tel <dig> and/or mec <dig> may control cell cycle progression at the metaphase-anaphase transition in addition to their well-known roles in the g2/m, g <dig> and s checkpoints  <cit> .
consistent with this, bub <dig>  mad <dig> and cdc <dig> have phosphorylated s/t-q sites after dna damage and exit from mitosis was recently shown to be regulated by tel1/mec <dig> in yeast and by atm/atr in humans  <cit> .
the significant enrichment of scd-containing proteins involved in later stages of mitosis and cell division, including these and other putative novel scd targets in the sac, the spindle orientation checkpoint and cytokinesis, seems to emphasize the notion that tel1/mec <dig> is very active during these processes.
while the presence of scd proteins in the kinetochore relates to its functional role as a reservoir for sac proteins, the presence of scd proteins in the spindle pole body suggests an unknown role for tel1/mec <dig> in monitoring spindle formation and orientation during mitosis.
consistent with this, the human tel <dig> homologue atm resides in the centrosome, which we found was significantly enriched with scd proteins  <cit> .
interestingly, several members of the yeast spindle orientation checkpoint such as bub <dig> are scd proteins.
bub <dig> resides in the spindle pole body and activates the mitotic exit network once the spindle has been correctly positioned providing a link between spindle orientation and mitotic progression into cytokinesis  <cit> .
the presence of an scd in bub <dig> suggests that this surveillance mechanism may also be under tel1/mec <dig> control.
in addition to microtubules, both the yeast and human proteomes have a significant concentration of scd proteins in the actin cytoskeleton.
in yeast, several of these localize to the cellular bud and cell cortex to direct nuclear migration, spindle orientation, nuclear division and cell division during cytokinesis.
for instance bni <dig>  an scd protein in both yeast and human, is a formin protein that organizes actin filaments and is involved in mitotic spindle orientation  <cit> .
deletion of rad <dig> or chk <dig> in yeast causes aberrant mitotic movements of the nucleus into the bud neck without triggering anaphase, suggesting the ddr machinery also controls nuclear migration in mitosis  <cit> .
in addition, several yeast scd proteins form the contractile ring during cytokinesis  <cit> .
examples of such proteins are cla <dig>  a protein involved in ring assembly, and cdc <dig>  a septin which is a component of the contractile ring and whose human homologue also contains an scd and has ps/t-q sites upon dna damage.
having functional scds in these processes would strengthen the notion that crosstalk occurs among the actin cytoskeleton governing nuclear migration, cytokinesis and the ddr.
to complete mitosis in yeast, the mitotic exit network  must be inactivated and the daughter cell completely separated from the mother cell.
two transcription factors, amn <dig> and ace <dig>  play key roles in these steps and contain sequences that meet our scd definition.
amn <dig> acts by downregulating men, whereas ace <dig> is restricted to the daughter cell where it activates several chitinases and glucanases that sever remaining links between bud and mother cell  <cit> .
moreover, mob <dig> is another scd-containing protein belonging to the ram  pathway, whose function is essential for daughter cell-specific transcription required for cell separation  <cit> .
thus, scd proteins are enriched for roles revolving around the end of mitosis, from the mitotic networks that control entry into anaphase and telophase to the regulation and formation of the contractile ring during cytokinesis to pathways that control cytokinesis and telophase completion.
we also identified scds in yeast proteins controlling other aspects of cell cycle progression, especially those regulating other cell cycle boundaries.
examples are: mih <dig>  which is involved in g2/m transition, and whi <dig> and whi <dig>  which coordinate start entry with cell size  <cit> .
genes whose expression is tightly linked to cell cycle progression often contain specific promoter sequences that allow their concerted and timely expression.
several transcription factors that recognize these sequences contain an scd, suggesting that tel1/ mec <dig> may also control cell cycle progression by influencing the expression of cell cycle regulated genes.
in addition, two of the major e <dig> ubiquitin ligase complexes controlling cell cycle progression, apc/c and scf, have members in the yeast scd census   <cit> .
for example, cdc <dig> is an scd-containing protein belonging to the apc/c complex, which regulates the metaphase-anaphase transition.
similarly, cdc <dig> is an scd protein forming part of the scf ubiquitin ligase, a complex that regulates entry into s-phase.
moreover, the cdc <dig> human orthologue, fbxw <dig>  is phosphorylated at s/t-q sites after dna damage  <cit> .
cdc <dig> also contains a so-called f-box that is the substrate recognition component of scf complexes.
related to this,  <dig> of the  <dig> known f-box proteins in yeast were found in our census   <cit> .
while several of these f-box proteins play cell cycle-related roles, others are involved in cell morphology and cell growth.
furthermore, mec <dig> is known to activate the scf/ufo <dig> complex to degrade ho, an endonuclease involved in mating type switching.
the presence of proteins involved in protein ubiquitination in the yeast scd census supports the fact that in human cells several e <dig> ligases such as brca <dig>  mdm <dig>  rnf <dig> and rnf <dig> are well-known mediators and effectors of ddr  <cit> .
dna replication
the yeast scd census also contains several proteins performing critical roles in dna replication, such as pre-replication complex members mcm <dig> and mcm <dig>  helicase dna <dig>  licensing factor cdt <dig> and polymerases pol <dig> and pol <dig>  this correlates with the observations that human mcm members and the human homologue pol <dig> are known atm/atr targets  <cit> .
in yeast, these pre-replication complex and replication fork proteins may be targets of the mec1-dependent dna replication checkpoint  triggered by replication fork stalling, which is mediated by founding scd members mrc <dig>  sgs <dig> and rad <dig>  <cit> .
interestingly, the binding of mrc <dig> to pol <dig> is required to stabilize pol <dig> at stalled replication forks  <cit> .
moreover, the drc is dependent on ctf <dig>  an scd protein whose human homologue contains an scd and is phosphorylated at s/t-q sites following dna damage.
along with scd proteins chl <dig> and pds <dig>  ctf <dig> is required for chromatid cohesion, a process regulated by the ddr in human cells through the phosphorylation of smc <dig> cohesion subunit by atr  <cit> .
smc proteins constitute a family of atpases forming the condensin and cohesion complexes as well as the smc5-smc <dig> complex in yeast.
in addition to cohesion, several other scd proteins belong to these complexes.
for instance, smc <dig> and ycs <dig> are two scd proteins belonging to the condensin complex whereas mms <dig>  an e <dig> sumo ligase, and nse <dig>  belong to the smc5-smc <dig> complex, which is involved in dna repair, cohesion and recovery of stalled replication forks  <cit> .
dna repair
during the ddr, tel <dig> and mec <dig> coordinate the halt of cell cycle progression with the activation of dna repair mechanisms.
consistent with this, four of the known tel1/mec <dig> targets with characterized scds are directly involved in dna repair: esc <dig>  slx <dig>  sgs <dig> and sae <dig>  <cit> .
in human cells, atm and atr kinases directly target homologous recombination factors nbs <dig> and rad <dig> and mismatch repair factor msh <dig>  <cit> .
as anticipated, our yeast scd census contained a significant enrichment of proteins associated with all types of dna repair pathways  <cit> .
homologous recombination was the most over-represented dna repair pathway with scd proteins involved in every step, including processing and resection , homologous pairing and strand exchange , dna synthesis , holliday junction resolution  and dissolution of homologous recombination intermediates   <cit> .
the mrx complex is a known sensor of dna damage that recruits tel1/mec <dig> to double strand breaks during the ddr.
our data indicate the mrx component rad <dig> contains an scd both in yeast and human, which is known to be phosphorylated at s/t-q sites following dna damage  <cit> .
furthermore, xrs <dig> and the human orthologue nbs <dig> are known targets of the tel1/mec <dig> and atm/atr kinases during the ddr  <cit> .
since the majority of known factors involved in end processing during double strand break repair contain scds, this process may be under tight control of tel1/mec <dig>  perhaps regulating the pathway of double strand break repair, homologous recombination versus nonhomologous end joining, undertaken, an outcome dependent upon the level of resection present at the double strand breaks.
many of these proteins also impact telomere homeostasis and, therefore, the presence of scds in this particular group of proteins may reflect tel1/mec <dig> regulation of their telomeric functions or simply the degree of telomere end resection as recently proposed  <cit> .
in addition to homologous recombination, proteins impacting other dna repair pathways were identified in the yeast scd census.
for example, mismatch repair proteins msh <dig> and mlh <dig> were identified as possible tel1/mec <dig> targets, which correspond with the known phosphorylations of the msh <dig> and mlh <dig> human homologues at s/t-q sites after dna damage  <cit> .
other dna repair proteins found in our scd census are nej <dig>  required during nhej, and mms <dig>  an e <dig> ubiquitin ligase that acts with scd-containing tel1/mec <dig> targets esc <dig> and slx <dig> to promote replication and recovery from replication fork arrest on damaged dna  <cit> .
furthermore, the abundance of chromatin modification proteins mentioned below may be related to the roles they play during dna repair in addition to transcription regulation.
overall, the high enrichment of dna repair proteins in our census, along with the concordance between the yeast and human data, suggests that tel1/mec <dig> may have a more significant role in directly phosphorylating proteins involved in dna repair pathways during the ddr than currently recognized.
transcription regulation
another profound effect of inflicting dna damage is a global change in transcription, which affects 5% of the yeast genome  <cit> .
not surprisingly, we found gene expression as one of the most over-represented ontology terms in our census, which corresponded to several transcription factors that regulate the expression of cell cycle, dna repair and dna replication genes.
one of the major gene expression changes during the ddr involves upregulation of the rnr genes, which results in a 6- <dig> fold increase in dntp levels in cells  <cit> .
rfx <dig>  a transcription factor that binds and regulates rnr gene promoters, was found both in our yeast and human scd censuses.
while dun1-dependent phosphorylation of rfx <dig> during the ddr is well established, our data suggest a more direct role of tel1/mec <dig> in rfx <dig> regulation.
perhaps more surprisingly, we found a significantly greater number of proteins in the rna polii pre-initiation and mediator complexes in both the yeast and human scd censuses than expected.
this suggests that, in addition to gene specific transcription factors, the basal transcription machinery may be part of the ddr.
protein subunits of other complexes known to influence gene expression were also found to contain scds.
for instance, we found scds in components of the histone methylation compass complex , the saga complex , the histone acetyl-transferase sas complex , the nua <dig> complex  and the swi/snf and rsc remodeling complexes .
scds were also identified in several yeast proteins involved in heterochromatin formation such as sir <dig>  sir <dig>  rif <dig> and tbf <dig>  <cit> .
the abundance of chromatin modification proteins correlates with the way human tip <dig>  and nua <dig> bind to mdc <dig> and participates in the ddr  <cit> .
additionally, transcription factors matα <dig> and matα <dig>  the yeast mating type loci, contain an scd and bind scd-containing mcm <dig>  further suggesting additional targets for tel1/mec <dig> during mating type switching.
rna metabolism
our yeast scd census was also significantly enriched for proteins involved in a panoply of processes required for mrna processing and protein synthesis such as mrna capping , mrna cleavage and polyadenylation , splicing , translation initiation , translation regulation , translation termination  and ribosome synthesis .
this correlates well with studies in human cells which show a concentration of proteins involved in splicing, translation and protein synthesis among those phosphorylated at s/t-q sites following dna damage  <cit> .
while tel1/mec <dig> effectors like dun <dig> are known to influence rna processing, our findings suggest that tel <dig> and mec <dig> are capable of directly regulating this process.
meiosis
during meiosis, mec <dig> phosphorylates scd-containing proteins sae <dig> and hop <dig>  <cit> .
similar to sae <dig>  other proteins involved in homologous recombination also play roles during normal meiotic progression and thus, the presence of an scd in their sequence may identify them as possible tel1/mec <dig> targets in meiosis.
consistent with this, the mrx complex, sgs <dig> and exo <dig> are all scd-containing and are proposed targets of mec <dig> during normal meiotic progression.
it is also possible that mlh <dig>  a mismatch repair scd protein involved in meiotic recombination, may be also a mec <dig> target during meiosis.
moreover, our yeast scd census identified, in addition to hop <dig>  other meiotic–specific proteins.
examples include ime <dig>  a transcription factor that serves as a master regulator of meiosis and triggers entry into meiosis in the presence of starvation conditions; msh <dig> and dmc <dig>  proteins involved in processing programmed dna double strand breaks during meiotic recombination; and csm <dig>  a kinetochore-localized protein required for accurate segregation of homologous chromosomes in anaphase i  <cit> .
nuclear pore
the significant enrichment of scd proteins that localize to the nuclear pore was surprising.
while rad <dig> phosphorylates several nuclear pore components, evidence for phosphorylation of these by tel1/mec <dig>  as proposed by this census, is lacking.
the functional role of nuclear pore phosphorylation during the ddr is not fully understood, but it is known nuclear pore components influence dna repair, gene expression and telomere homeostasis which are all pathways directly targeted by tel1/mec <dig>  alternatively, the presence of importins and other transport proteins in our census may indicate a direct role of tel1/mec <dig> in regulating transport across the nuclear membrane during the ddr.
consistent with this, los <dig>  an scd protein which is the primary exon-containing trna exporter in yeast, is phosphorylated in a mec1- and rad53-dependent manner during the ddr and induces the rapid accumulation of trna in the nucleus and arrest at g <dig> before start  <cit> .
therefore, the tel1/mec <dig> kinases couple nucleocytoplasmic trafficking with cell cycle progression in the presence of dna damage.
our census may have unveiled additional novel tel1/mec <dig> targets that also coordinate protein transport across the nuclear pore with other ddr pathways.
for instance, toa <dig> a tfiia subunit contains an scd and is transported into the nucleus by an scd-containing importin  while nup <dig> and nup <dig> bind mex <dig>  the major mrna exporter in yeast, suggesting tel1/mec <dig> may also couple nuclear transport with gene expression  <cit> .
furthermore kap <dig>  an scd protein, imports histones h <dig> and h <dig> into the nucleus, which suggests another possible mechanism by which the tel1/mec <dig> kinases regulate dna replication and cell cycle progression  <cit> .
finally, kap <dig>  the major importin of nls-containing cargo proteins in yeast, has an scd which may provide a mechanism for tel1/mec <dig> to regulate several nuclear pathways by regulating the ability of kap <dig> to transport its components  <cit> .
telomeres
tel <dig> promotes the elongation of short telomeres  <cit> .
although telomeric cdc <dig> protein can be phosphorylated by tel <dig> in vitro, it appears not to be a tel <dig> target in vivo  <cit> .
tel1′s influence on telomeres may be due to its effects on dna end processing by proteins that function not only at double strand breaks but also at telomeres as previously proposed  <cit> .
consistent with this, our yeast census identified several such scd containing proteins .
interestingly, our yeast scd census also identified two additional proteins with roles in telomere homeostasis, tbf <dig> and rif <dig>  tbf <dig> functions in parallel with tel <dig> to promote preferential elongation of shorter telomeres  <cit> .
one of the s/t-q sites in rif <dig> is phosphorylated in vivo  <cit>  and it has been proposed that tel <dig> phosphorylation of rif <dig> may serve to relieve rif <dig> negative inhibition of telomerase, downstream of telomerase recruitment  <cit> .
thus, tel1′s role in telomere length homeostasis is likely complex.
moreover, several scd proteins are required for establishing heterochromatin at subtelomeric regions  further expanding putative roles of tel <dig> at telomeres.
cell signaling
while phosphoproteome analysis revealed several putative tel1/mec <dig> targets that localize exclusively to the cytoplasm, the presence of tel1/mec <dig> in cellular compartments other than the nucleus remains to be demonstrated.
in contrast, human atm/atr localize, in part, in the cytoplasm where they function in endocytosis and several cell signaling pathways.
for instance, atm plays roles in nf-ĸb signaling where, upon dna damage, atm binds and phosphorylates nemo and translocates to the cytoplasm  <cit> .
consistent with this, our human scd census identified protein networks involved in nf-ĸb and other cell signaling pathways like the erk, insulin, jnk, ras and akt.
also in humans, atm is known to induce autophagy in the presence of reactive oxygen species by repressing the torc pathway.
in yeast, the torc pathway elicits a response to nutrient deprivation and metabolic stress, by inducing transcriptional activation of metabolic genes, repressing protein synthesis and inducting autophagy  <cit> .
our yeast scd census revealed a number of proteins involved in the torc pathway and other nutrient signaling mechanisms, suggesting in yeast, as in human cells, tel1/mec <dig> may regulate the torc pathway .
similarly, we found the signal transduction pathway controlling glycerol production in response to hyperosmotic stress is highly enriched in scd-containing proteins suggesting tel1/mec <dig> may contribute to the response to this type of stress as well   <cit> .
endocytosis
although atm has been found in endocytic vesicles, its precise role in endocytosis remains to be determined.
surprisingly, our yeast scd census was significantly enriched for proteins involved in endocytosis, indicating that tel1/mec <dig> may also be involved in endocytosis in yeast.
moreover, it is known that the actin cytoskeleton and several motor proteins are involved in transporting of endocytic vesicles across the cytoplasm  <cit> .
therefore, the presence of scds in proteins involved in cortical cytoskeleton may reflect their role in endocytosis in addition to their involvement in telophase.
CONCLUSIONS
overall, we have shown that our newly defined scd definition can be used to predict pathways under control of tel1/mec <dig> and to identify novel putative targets for these kinases.
a census of scd-containing proteins in yeast has revealed a wide network of proteins involved in cytokinesis, mrna processing, protein transport, mating type switching and endocytosis suggesting that tel1/mec <dig> roles in yeast are broader than previously recognized and contain extensive parallels to pathways and targets under control of atm/atr in mammalian cells.

 the conserved dna damage response pathway proceeds as a highly coordinated cascade of cellular events under the control of the phosphatidyl inositol 3′ kinase-related kinases, most notably tel and mec in saccharomyces cerevisiae and their homologs atm and atr, respectively, in human. 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. in s. cerevisiae under genotoxic stress, tel and mec activate the ddr by phosphorylating key mediators chk rad mrc and rad and others, resulting in the halt of dna replication and cell cycle progression at g and s phases or at g2/m transition. 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 hop as a downstream effector of tel1/mec signaling and defective telomerase recruitment as a result of a tel deficiency illustrate additional roles for tel1/mec in meiosis and telomere maintenance, respectively. recently, a series of large-scale studies suggest that the number of tel1/mec targets is much higher than initially estimated. a high throughput analysis in yeast treated with dna damaging reagents identified proteins phosphorylated at s/t-q sites. a similar approach in human cell lines treated with uv radiation, led to the identification of phosphosites. an additional search for peptides phosphorylated at atm/atr consensus sites in response to ionizing radiation yielded more than putative protein targets, of which many lacked functional characterization of their s/t-q phosphorylation sites. 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/mec substrates might delineate additional functions of these kinases beyond the ddr. tel1/mec 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 s/t-q within amino acids, the so-called s/t-q cluster domain. to date, an scd has been confirmed in just tel1/mec targets with many of these having more than one scd. not all s/t-q sites within a given scd are phosphorylated, although all of these scd proteins possess at least tel1/mec1-phosphorylated s/t-q within an scd. although the functions of scd domains are not completely understood, they often mediate protein-protein interactions during signaling cascades. for instance, a single phosphorylation of the rad scd promotes dimerization whereas a double phosphorylation triggers rad binding to the fha domain of dun. similarly, sustaining the dna damage signal requires oligomerization of rad proteins at dna breaks through the interaction of its brct domain and phosphorylated scd. these examples suggest that scds are biologically relevant domains with important roles during the ddr. based on the original scd definition, 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/mec targets. the final set of targets contained proteins including the known scd-containing tel1/mec 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/mec similar results were obtained when the new scd definition was applied to generate a human scd census.  obtaining a census of scd proteins the scd in s. cerevisiae was previously defined as a region with at least s/t-q within residues. examination of the known scd proteins revealed the scd could be defined as having s/t-q within just amino acids. to refine and ease the stringency of our census, we used scanprosite to search the uniprot database for s. cerevisiae proteins containing at least s/t-q within a stretch of residues or less. we found a total of proteins, each having at least one scd region. this number was significantly higher than the scd proteins expected to be present in the yeast proteome by chance suggesting scds are indeed biologically relevant units rather than stochastic events. since the probability of seeing an s/tq by chance alone increases as the protein length increases, we determined the distribution of the scd-containing protein lengths by performing a goodness of fit test using person’s chi-square test and we found that the distribution of protein lengths in our census is not statistically different from a log-normal distribution. empirical support for our scd definition could be found in several ways. first, of the identified proteins had evidence of phosphorylation at s/t-q sites in mass spectrometry phosphoproteomic studies, with of those occurring within an scd. second, of the mec1/tel1-dependent and rad53-independent phosphoproteins that were induced after exposure of wildtype and rad53Δ yeast to methyl methanosulfonate, were present in our list. third, our list also contained of the proteins that were found in as tel1/mec targets in a quantitative mass spectrometry analysis. fourth, additional similarities with other reports were uncovered in the amino acids flanking those scds that contained phosphorylated s/t-q motifs. serine residues were frequently found upstream of ps/t-q, whereas glutamic acid residues were enriched at the + position. these features corresponded to sites of dna damage-induced ps/t-q sites in human proteins. therefore, our scd definition of s/t-q within amino acids identified proteins with empirical data supporting ddr-related functions. analysis of the go-slim ontology terms associated with the yeast scd proteins showed a non-random distribution and a concentration in specific functions, processes and components. as anticipated, we found significant enrichment in ontology terms that are usually associated with ddr proteins. like known tel1/mec targets, the identified scd-containing proteins tended to be nuclear, responsive to stress signals, involved in phosphorylation and signal transduction, and have roles in ddr-related pathways such as cell cycle progression and transcriptional regulation. when the analysis was extended to all go terms, those related to ddr pathways remained significantly over-represented, validating our approach. as expected for a list enriched for ddr genes, we found a significant enrichment in proteins involved in cell cycle progression, transcription, dna replication and dna repair. in addition, our yeast scd census contained a significant enrichment of proteins associated with protein localization and endocytosis, suggesting a broader and more direct role for tel1/mec in these pathways than previously thought. the yeast census was also enriched for proteins involved in cytokinesis and cell division, pathways where a role for tel1/mec kinases was previously hinted. these included components of the actin cortical cytoskeleton, which control later stages of mitosis to ensure both proper nuclear migration to the cellular bud and cell division during cytokinesis. this may also explain the enrichment of scd proteins from the cellular bud and cell cortex in the census. in addition, several proteins involved in the spindle assembly checkpoint and spindle position and orientation checkpoint as well as other networks controlling mitotic exit were found to contain an scd. consistent with a putative role for tel and mec kinases monitoring key transition steps in mitosis, we found a concentration of scd proteins localized to the kinetochore and spindle pole body, two key subcellular compartments central to the sac and completion of mitosis. our yeast scd census also uncovered proteins not known to be tel1/mec targets, but with characterized roles in pathways well-known to be regulated by tel1/mec kinases. the pathways included dna repair, dna replication, gene expression, meiosis, and telomere homeostasis. for example, there was an over-representation of proteins influencing rna polii-dependent transcription, such as components of the pre-initiation complex and mediator as well as members of the saga and compass complexes. furthermore, several ddr transcription factors such as rfx were also found to contain an scd, raising the possibility they may be under direct control of tel1/mec kinases. many of these scd-containing proteins belong to groups of proteins influencing more than one known ddr-related pathway. for instance, most of the nucleases and helicases involved in dna double strand break repair containing an scd have also been associated with replication and telomere homeostasis. similarly, several scd proteins are involved in sumoylation, ubiquitination, chromatin remodeling and the establishment of sister chromatid cohesion, which are activities known to influence several ddr pathways such as dna replication, transcription regulation, dna repair, and cell cycle progression. another example of crosstalk among ddr related pathways by scd proteins in our census is a subset of transcription factors that ensure proper transitions within phases of the cell cycle. this suggests these potential novel tel1/mec targets may serve as a link between cell cycle progression and global transcription changes, two key components during ddr. census of human scd proteins overlaps with the yeast census for a given scd-containing protein, the presence of an scd in orthologues increases the probability that it is a biological entity rather than generated randomly. for this reason, we searched the human proteome for proteins containing this newly defined scd. this census identified proteins, including of the proteins used to formulate the original scd definition, and of the proteins found to contain ps/t-q sites following dna damage. the extremely low probability of coincidence between our human scd census and these experimental data indicates that human scd proteins are significantly enriched for proteins known to be phosphorylated following dna damage. furthermore, the ontology terms over-represented in this list overlapped largely with those found experimentally in human ddr proteins and with our yeast scd census. these included terms such as dna repair, cell cycle progression, gene expression, dna replication, and response to dna damage. as with the yeast scd census, we found an unexpected abundance of proteins belonging to the microtubule organizing center and spindle as well as the actin cytoskeleton. also similar to yeast, the human scd census was enriched in mediator and key ddr transcription factors such as the human homologue of rfx. network analysis of the human scd census revealed connections between scd proteins involved in cell signaling pathways including jnk, erk, ras, akt, calmodulin signaling, and nf-ĸb, a pathway in which atm plays important roles. remarkably, these networks included insulin signaling proteins which are heavily phosphorylated at s/t-q sites after dna damage, suggesting these phosphorylations may occur in the context of an scd. finally, as found in the network analysis of the yeast scd census, one of the human scd networks included several components of the nuclear pore complex as well as proteins involved in nucleocytoplasmic transport like importin-beta and exportin. * phosphorylated in s/t-q sites in mass spectrometry searches. ** phosphorylated in s/t-q sites within an scd in mass spectrometry searches. discussion the scd is neither a motif nor a true protein domain in that a consensus alignment cannot completely define the region and there is variable spacing between each s/t-q. this has made its identification in proteins difficult, relying on loose definitions extending to include 25% of the yeast proteome. using a more stringent scd definition of a sequence containing at least s/t-q in a stretch of amino acids, we arrived at a refined census of proteins in the yeast proteome, still a much larger number than expected at random. the validity of this approach is supported by the enrichment of proteins phosphorylated at s/t-q sites in mass spectrometry studies and the presence of all well-characterized scd-containing proteins phosphorylated by tel1/mec in addition, ontology terms related to the ddr are significantly over-represented in this census. we propose that this newly defined scd can be used to predict new roles for tel1/mec during the ddr and to identify novel putative targets for these kinases. while the presence of an scd in a protein may have arisen stochastically, the existence of several scd proteins in the same pathway is much more unlikely. therefore, the definition has a higher predictive value when assigning new processes regulated by tel1/mec similarly, for a given scd-containing protein, the presence of an scd in homologues in other organisms increases the probability that the scd is a biological entity and not randomly generated. for this reason, we searched the human proteome for proteins matching this newly defined scd to look for similarities and differences. table shows a list of interesting yeast proteins in our census whose human orthologue either contains ps/t-q sites, possess an scd in their sequence, or both. these genes are likely to be tel1/mec targets in yeast and, in fact, several of them were phosphorylated in s/t-q sites in high throughput mass spectrometry approaches. mitosis as hinted by previous reports, the presence of scds in several sac proteins such as bub mad and cdc indicates tel and/or mec may control cell cycle progression at the metaphase-anaphase transition in addition to their well-known roles in the g2/m, g and s checkpoints. consistent with this, bub mad and cdc have phosphorylated s/t-q sites after dna damage and exit from mitosis was recently shown to be regulated by tel1/mec in yeast and by atm/atr in humans. the significant enrichment of scd-containing proteins involved in later stages of mitosis and cell division, including these and other putative novel scd targets in the sac, the spindle orientation checkpoint and cytokinesis, seems to emphasize the notion that tel1/mec is very active during these processes. while the presence of scd proteins in the kinetochore relates to its functional role as a reservoir for sac proteins, the presence of scd proteins in the spindle pole body suggests an unknown role for tel1/mec in monitoring spindle formation and orientation during mitosis. consistent with this, the human tel homologue atm resides in the centrosome, which we found was significantly enriched with scd proteins. interestingly, several members of the yeast spindle orientation checkpoint such as bub are scd proteins. bub resides in the spindle pole body and activates the mitotic exit network once the spindle has been correctly positioned providing a link between spindle orientation and mitotic progression into cytokinesis. the presence of an scd in bub suggests that this surveillance mechanism may also be under tel1/mec control. in addition to microtubules, both the yeast and human proteomes have a significant concentration of scd proteins in the actin cytoskeleton. in yeast, several of these localize to the cellular bud and cell cortex to direct nuclear migration, spindle orientation, nuclear division and cell division during cytokinesis. for instance bni an scd protein in both yeast and human, is a formin protein that organizes actin filaments and is involved in mitotic spindle orientation. deletion of rad or chk in yeast causes aberrant mitotic movements of the nucleus into the bud neck without triggering anaphase, suggesting the ddr machinery also controls nuclear migration in mitosis. in addition, several yeast scd proteins form the contractile ring during cytokinesis. examples of such proteins are cla a protein involved in ring assembly, and cdc a septin which is a component of the contractile ring and whose human homologue also contains an scd and has ps/t-q sites upon dna damage. having functional scds in these processes would strengthen the notion that crosstalk occurs among the actin cytoskeleton governing nuclear migration, cytokinesis and the ddr. to complete mitosis in yeast, the mitotic exit network must be inactivated and the daughter cell completely separated from the mother cell. two transcription factors, amn and ace play key roles in these steps and contain sequences that meet our scd definition. amn acts by downregulating men, whereas ace is restricted to the daughter cell where it activates several chitinases and glucanases that sever remaining links between bud and mother cell. moreover, mob is another scd-containing protein belonging to the ram pathway, whose function is essential for daughter cell-specific transcription required for cell separation. thus, scd proteins are enriched for roles revolving around the end of mitosis, from the mitotic networks that control entry into anaphase and telophase to the regulation and formation of the contractile ring during cytokinesis to pathways that control cytokinesis and telophase completion. we also identified scds in yeast proteins controlling other aspects of cell cycle progression, especially those regulating other cell cycle boundaries. examples are: mih which is involved in g2/m transition, and whi and whi which coordinate start entry with cell size. genes whose expression is tightly linked to cell cycle progression often contain specific promoter sequences that allow their concerted and timely expression. several transcription factors that recognize these sequences contain an scd, suggesting that tel1/ mec may also control cell cycle progression by influencing the expression of cell cycle regulated genes. in addition, two of the major e ubiquitin ligase complexes controlling cell cycle progression, apc/c and scf, have members in the yeast scd census. for example, cdc is an scd-containing protein belonging to the apc/c complex, which regulates the metaphase-anaphase transition. similarly, cdc is an scd protein forming part of the scf ubiquitin ligase, a complex that regulates entry into s-phase. moreover, the cdc human orthologue, fbxw is phosphorylated at s/t-q sites after dna damage. cdc also contains a so-called f-box that is the substrate recognition component of scf complexes. related to this, of the known f-box proteins in yeast were found in our census. while several of these f-box proteins play cell cycle-related roles, others are involved in cell morphology and cell growth. furthermore, mec is known to activate the scf/ufo complex to degrade ho, an endonuclease involved in mating type switching. the presence of proteins involved in protein ubiquitination in the yeast scd census supports the fact that in human cells several e ligases such as brca mdm rnf and rnf are well-known mediators and effectors of ddr. dna replication the yeast scd census also contains several proteins performing critical roles in dna replication, such as pre-replication complex members mcm and mcm helicase dna licensing factor cdt and polymerases pol and pol this correlates with the observations that human mcm members and the human homologue pol are known atm/atr targets. in yeast, these pre-replication complex and replication fork proteins may be targets of the mec1-dependent dna replication checkpoint triggered by replication fork stalling, which is mediated by founding scd members mrc sgs and rad. interestingly, the binding of mrc to pol is required to stabilize pol at stalled replication forks. moreover, the drc is dependent on ctf an scd protein whose human homologue contains an scd and is phosphorylated at s/t-q sites following dna damage. along with scd proteins chl and pds ctf is required for chromatid cohesion, a process regulated by the ddr in human cells through the phosphorylation of smc cohesion subunit by atr. smc proteins constitute a family of atpases forming the condensin and cohesion complexes as well as the smc5-smc complex in yeast. in addition to cohesion, several other scd proteins belong to these complexes. for instance, smc and ycs are two scd proteins belonging to the condensin complex whereas mms an e sumo ligase, and nse belong to the smc5-smc complex, which is involved in dna repair, cohesion and recovery of stalled replication forks. dna repair during the ddr, tel and mec coordinate the halt of cell cycle progression with the activation of dna repair mechanisms. consistent with this, four of the known tel1/mec targets with characterized scds are directly involved in dna repair: esc slx sgs and sae. in human cells, atm and atr kinases directly target homologous recombination factors nbs and rad and mismatch repair factor msh. as anticipated, our yeast scd census contained a significant enrichment of proteins associated with all types of dna repair pathways. homologous recombination was the most over-represented dna repair pathway with scd proteins involved in every step, including processing and resection, homologous pairing and strand exchange, dna synthesis, holliday junction resolution and dissolution of homologous recombination intermediates. the mrx complex is a known sensor of dna damage that recruits tel1/mec to double strand breaks during the ddr. our data indicate the mrx component rad contains an scd both in yeast and human, which is known to be phosphorylated at s/t-q sites following dna damage. furthermore, xrs and the human orthologue nbs are known targets of the tel1/mec and atm/atr kinases during the ddr. since the majority of known factors involved in end processing during double strand break repair contain scds, this process may be under tight control of tel1/mec perhaps regulating the pathway of double strand break repair, homologous recombination versus nonhomologous end joining, undertaken, an outcome dependent upon the level of resection present at the double strand breaks. many of these proteins also impact telomere homeostasis and, therefore, the presence of scds in this particular group of proteins may reflect tel1/mec regulation of their telomeric functions or simply the degree of telomere end resection as recently proposed. in addition to homologous recombination, proteins impacting other dna repair pathways were identified in the yeast scd census. for example, mismatch repair proteins msh and mlh were identified as possible tel1/mec targets, which correspond with the known phosphorylations of the msh and mlh human homologues at s/t-q sites after dna damage. other dna repair proteins found in our scd census are nej required during nhej, and mms an e ubiquitin ligase that acts with scd-containing tel1/mec targets esc and slx to promote replication and recovery from replication fork arrest on damaged dna. furthermore, the abundance of chromatin modification proteins mentioned below may be related to the roles they play during dna repair in addition to transcription regulation. overall, the high enrichment of dna repair proteins in our census, along with the concordance between the yeast and human data, suggests that tel1/mec may have a more significant role in directly phosphorylating proteins involved in dna repair pathways during the ddr than currently recognized. transcription regulation another profound effect of inflicting dna damage is a global change in transcription, which affects 5% of the yeast genome. not surprisingly, we found gene expression as one of the most over-represented ontology terms in our census, which corresponded to several transcription factors that regulate the expression of cell cycle, dna repair and dna replication genes. one of the major gene expression changes during the ddr involves upregulation of the rnr genes, which results in a 6- fold increase in dntp levels in cells. rfx a transcription factor that binds and regulates rnr gene promoters, was found both in our yeast and human scd censuses. while dun1-dependent phosphorylation of rfx during the ddr is well established, our data suggest a more direct role of tel1/mec in rfx regulation. perhaps more surprisingly, we found a significantly greater number of proteins in the rna polii pre-initiation and mediator complexes in both the yeast and human scd censuses than expected. this suggests that, in addition to gene specific transcription factors, the basal transcription machinery may be part of the ddr. protein subunits of other complexes known to influence gene expression were also found to contain scds. for instance, we found scds in components of the histone methylation compass complex, the saga complex, the histone acetyl-transferase sas complex, the nua complex and the swi/snf and rsc remodeling complexes. scds were also identified in several yeast proteins involved in heterochromatin formation such as sir sir rif and tbf. the abundance of chromatin modification proteins correlates with the way human tip and nua bind to mdc and participates in the ddr. additionally, transcription factors matα and matα the yeast mating type loci, contain an scd and bind scd-containing mcm further suggesting additional targets for tel1/mec during mating type switching. rna metabolism our yeast scd census was also significantly enriched for proteins involved in a panoply of processes required for mrna processing and protein synthesis such as mrna capping, mrna cleavage and polyadenylation, splicing, translation initiation, translation regulation, translation termination and ribosome synthesis. this correlates well with studies in human cells which show a concentration of proteins involved in splicing, translation and protein synthesis among those phosphorylated at s/t-q sites following dna damage. while tel1/mec effectors like dun are known to influence rna processing, our findings suggest that tel and mec are capable of directly regulating this process. meiosis during meiosis, mec phosphorylates scd-containing proteins sae and hop. similar to sae other proteins involved in homologous recombination also play roles during normal meiotic progression and thus, the presence of an scd in their sequence may identify them as possible tel1/mec targets in meiosis. consistent with this, the mrx complex, sgs and exo are all scd-containing and are proposed targets of mec during normal meiotic progression. it is also possible that mlh a mismatch repair scd protein involved in meiotic recombination, may be also a mec target during meiosis. moreover, our yeast scd census identified, in addition to hop other meiotic–specific proteins. examples include ime a transcription factor that serves as a master regulator of meiosis and triggers entry into meiosis in the presence of starvation conditions; msh and dmc proteins involved in processing programmed dna double strand breaks during meiotic recombination; and csm a kinetochore-localized protein required for accurate segregation of homologous chromosomes in anaphase i. nuclear pore the significant enrichment of scd proteins that localize to the nuclear pore was surprising. while rad phosphorylates several nuclear pore components, evidence for phosphorylation of these by tel1/mec as proposed by this census, is lacking. the functional role of nuclear pore phosphorylation during the ddr is not fully understood, but it is known nuclear pore components influence dna repair, gene expression and telomere homeostasis which are all pathways directly targeted by tel1/mec alternatively, the presence of importins and other transport proteins in our census may indicate a direct role of tel1/mec in regulating transport across the nuclear membrane during the ddr. consistent with this, los an scd protein which is the primary exon-containing trna exporter in yeast, is phosphorylated in a mec1- and rad53-dependent manner during the ddr and induces the rapid accumulation of trna in the nucleus and arrest at g before start. therefore, the tel1/mec kinases couple nucleocytoplasmic trafficking with cell cycle progression in the presence of dna damage. our census may have unveiled additional novel tel1/mec targets that also coordinate protein transport across the nuclear pore with other ddr pathways. for instance, toa a tfiia subunit contains an scd and is transported into the nucleus by an scd-containing importin while nup and nup bind mex the major mrna exporter in yeast, suggesting tel1/mec may also couple nuclear transport with gene expression. furthermore kap an scd protein, imports histones h and h into the nucleus, which suggests another possible mechanism by which the tel1/mec kinases regulate dna replication and cell cycle progression. finally, kap the major importin of nls-containing cargo proteins in yeast, has an scd which may provide a mechanism for tel1/mec to regulate several nuclear pathways by regulating the ability of kap to transport its components. telomeres tel promotes the elongation of short telomeres. although telomeric cdc protein can be phosphorylated by tel in vitro, it appears not to be a tel target in vivo. tel1′s influence on telomeres may be due to its effects on dna end processing by proteins that function not only at double strand breaks but also at telomeres as previously proposed. consistent with this, our yeast census identified several such scd containing proteins. interestingly, our yeast scd census also identified two additional proteins with roles in telomere homeostasis, tbf and rif tbf functions in parallel with tel to promote preferential elongation of shorter telomeres. one of the s/t-q sites in rif is phosphorylated in vivo and it has been proposed that tel phosphorylation of rif may serve to relieve rif negative inhibition of telomerase, downstream of telomerase recruitment. thus, tel1′s role in telomere length homeostasis is likely complex. moreover, several scd proteins are required for establishing heterochromatin at subtelomeric regions further expanding putative roles of tel at telomeres. cell signaling while phosphoproteome analysis revealed several putative tel1/mec targets that localize exclusively to the cytoplasm, the presence of tel1/mec in cellular compartments other than the nucleus remains to be demonstrated. in contrast, human atm/atr localize, in part, in the cytoplasm where they function in endocytosis and several cell signaling pathways. for instance, atm plays roles in nf-ĸb signaling where, upon dna damage, atm binds and phosphorylates nemo and translocates to the cytoplasm. consistent with this, our human scd census identified protein networks involved in nf-ĸb and other cell signaling pathways like the erk, insulin, jnk, ras and akt. also in humans, atm is known to induce autophagy in the presence of reactive oxygen species by repressing the torc pathway. in yeast, the torc pathway elicits a response to nutrient deprivation and metabolic stress, by inducing transcriptional activation of metabolic genes, repressing protein synthesis and inducting autophagy. our yeast scd census revealed a number of proteins involved in the torc pathway and other nutrient signaling mechanisms, suggesting in yeast, as in human cells, tel1/mec may regulate the torc pathway. similarly, we found the signal transduction pathway controlling glycerol production in response to hyperosmotic stress is highly enriched in scd-containing proteins suggesting tel1/mec may contribute to the response to this type of stress as well. endocytosis although atm has been found in endocytic vesicles, its precise role in endocytosis remains to be determined. surprisingly, our yeast scd census was significantly enriched for proteins involved in endocytosis, indicating that tel1/mec may also be involved in endocytosis in yeast. moreover, it is known that the actin cytoskeleton and several motor proteins are involved in transporting of endocytic vesicles across the cytoplasm. therefore, the presence of scds in proteins involved in cortical cytoskeleton may reflect their role in endocytosis in addition to their involvement in telophase.  overall, we have shown that our newly defined scd definition can be used to predict pathways under control of tel1/mec and to identify novel putative targets for these kinases. a census of scd-containing proteins in yeast has revealed a wide network of proteins involved in cytokinesis, mrna processing, protein transport, mating type switching and endocytosis suggesting that tel1/mec roles in yeast are broader than previously recognized and contain extensive parallels to pathways and targets under control of atm/atr in mammalian cells. 
the cellular response to dna damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. during the dna damage response, the sensor kinases tel and mec in saccharomyces cerevisiae and atm and atr in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and dna repair. this new scd definition was used in a custom bioinformatics pipeline to generate a census of scd-containing proteins in both yeast and human. to identify all putative scd-containing proteins, we analyzed the distribution of s/t-q motifs within verified tel1/mec targets and arrived at a unifying scd definition of at least s/t-q within a stretch of residues. we also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel tel1/mec targets in these pathways. in the human proteome, a wide range of similar proteins were identified, including homologs of some scd-containing proteins found in yeast. using a bioinformatic approach, we have generated a census of scd-containing proteins that are involved not only in known ddr pathways but several other pathways under tel1/mec control suggesting new putative targets for these kinases.
 the cellular response to dna damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. during the dna damage response, the sensor kinases tel and mec in saccharomyces cerevisiae and atm and atr in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and dna repair. a subset of kinase substrates are recognized by the s/t-q cluster domain, which contains motifs of serine or threonine followed by a glutamine. however, the full repertoire of proteins and pathways controlled by tel and mec is unknown.  to identify all putative scd-containing proteins, we analyzed the distribution of s/t-q motifs within verified tel1/mec targets and arrived at a unifying scd definition of at least s/t-q within a stretch of residues. this new scd definition was used in a custom bioinformatics pipeline to generate a census of scd-containing proteins in both yeast and human. in yeast, proteins were identified, a significantly larger number of hits than were expected by chance. these scd-containing proteins did not distribute equally across go-ontology terms, but were significantly enriched for those involved in processes related to the ddr. we also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel tel1/mec targets in these pathways. in the human proteome, a wide range of similar proteins were identified, including homologs of some scd-containing proteins found in yeast. this list also included high concentrations of proteins in the mediator, spindle pole body/centrosome and actin cytoskeleton complexes.  using a bioinformatic approach, we have generated a census of scd-containing proteins that are involved not only in known ddr pathways but several other pathways under tel1/mec control suggesting new putative targets for these kinases. dna damage responsephosphorylationproteometel1/mec1atmatr 