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DNA Repair 12 (2013) 791– 799

Contents lists available at ScienceDirect

DNA Repair

jo ur nal home p age: www.elsev ier .com/ locate /dnarepai r

ini review

nterplays between ATM/Tel1 and ATR/Mec1 in sensing and signalingNA double-strand breaks

lisa Gobbini, Daniele Cesena, Alessandro Galbiati, Arianna Lockhart, Maria Pia Longhese ∗

ipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy

r t i c l e i n f o

rticle history:eceived 22 July 2013ccepted 23 July 2013vailable online 13 August 2013

a b s t r a c t

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the poten-tial to cause mutations, chromosomal rearrangements and genomic instability. The cellular response toDSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which areconserved from yeasts to humans. These pathways can sense DNA damage and transduce this informationto specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian

eywords:NA double-strand breaksRN/MRX

TM/Tel1TR/Mec1heckpoint

protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1,act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSBprocessing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly andreciprocal manner.

© 2013 Elsevier B.V. All rights reserved.

esection

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7912. Resection of DNA ends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792

2.1. Positive regulators of DSB resection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7922.2. Negative regulators of DSB resection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

3. The ATR/Mec1 and ATM/Tel1 checkpoint kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7944. Activation of ATM/Tel1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7945. Activation of ATR/Mec1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7966. Interplays between ATM/Tel1 and ATR/Mec1 signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

Conflict of interest statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

. Introduction

DNA double-strand breaks (DSBs) are among the most cytotoxicorms of DNA damage because failure to repair them can lead

obligate intermediates during physiological cellular processes,such as meiotic recombination and lymphoid differentiation.Moreover, the ends of eukaryotic chromosomes, i.e. the telomeres,are structurally related to DSBs.

Two major pathways take care of repairing DSBs: non-

o loss of genetic information and chromosome rearrangements,hich are hallmarks of cancer cells. DSBs can occur either acciden-

ally during normal cell metabolism or can be caused by exposureo exogenous agents, such as certain types of chemotherapeu-ic drugs or ionizing radiation (IR). Nevertheless, they are also

∗ Corresponding author. Tel.: +39 0264483425; fax: +39 0264483565.E-mail address: mariapia.longhese@unimib.it (M.P. Longhese).

568-7864/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.dnarep.2013.07.009

homologous end-joining (NHEJ) and homologous recombination(HR). NHEJ directly ligates together the two broken ends with lit-tle or no processing [1] and is highly efficient, but it can lead tomutations at the joining sites, as well as inversions and transloca-tions. HR is more accurate, because it uses undamaged homologous

DNA sequences (sister chromatids or homologous chromosomes)as a template for repair in an error-free manner [2]. Making theright choice between NHEJ and HR is important to ensure genomestability.

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Generation of DNA DSBs elicits the activation of sophisticatedurveillance mechanisms, the DNA damage checkpoints, which ini-iate a coordinated cellular response [3]. Activation of the DNAamage checkpoint results in cell cycle arrest and DNA repair orrogrammed cell death. Key players in the checkpoint responsere phosphatidylinositol 3-kinase related protein kinases, such asammalian ATM (ataxia-telangiectasia-mutated) and ATR (ATM-

nd Rad3-related), Saccharomyces cerevisiae Tel1 and Mec1, andchizosaccharomyces pombe Tel1 and Rad3. In humans, ATM con-enital deficiency results in ataxia-telangiectasia [4], which is aare, autosomal recessive disorder characterized by progressiveerebellar ataxia, neuro-degeneration, radiosensitivity, checkpointefects, genome instability and predisposition to cancer. Simi-

arly, mutations in ATR are associated with Seckel syndrome, alinically distinct disorder characterized by proportionate growthetardation and severe microcephaly [5]. Here we will focus onhe work done in S. cerevisiae and mammals to review the earlyteps in DSB processing and signaling, as well as the regulation ofTM/Tel1 and ATR/Mec1 signaling activities in responding to DNASBs.

. Resection of DNA ends

The highly conserved MRN/MRX complex (Mre11-Rad50-Nbs1n metazoan; Mre11-Rad50-Xrs2 in yeast) and the Ku70/Ku80eterodimer (hereafter referred to as Ku) are the first protein com-lexes to be recruited at DSBs [6]. The presence of Ku and MRN/MRXediates the recruitment of proteins that religate the broken DNA

nds by NHEJ [7–9]. NHEJ is active only on blunt or minimallyrocessed DNA ends, and therefore is inhibited by the nucleolyticegradation of the 5′ strands. The latter process, referred to as 5′–3′

esection, generates 3′ single-stranded DNA (ssDNA) tails at the DSBnds and commits DSB repair to HR [10]. The Replication Protein ARPA) complex binds to the ssDNA tails and recruits the ATR/Mec1heckpoint kinase. Thus the decision to resect a DSB is fundamen-al not only to initiate DSB repair by HR, but also to activate theTR/Mec1-mediated checkpoint response.

.1. Positive regulators of DSB resection

In S. cerevisiae, the MRX complex initiates DSB resection togetherith the Sae2 protein [11,12]. The Mre11 component of MRX

xhibits 3′–5′ double-strand DNA (dsDNA) DNA exonuclease activ-ty and ssDNA endonuclease activity [13–15]. It has been proposedhat MRX together with Sae2 can remove oligonucleotides fromhe 5′ ends of the break, giving rise to short 3′-ended ssDNA tailshat are then subjected to extensive resection [16,17] (Fig. 1). Sae2hows endonuclease activity in vitro that is stimulated by the MRXomplex [18]. Whether Sae2 promotes DSB resection by regulatingRX nuclease activity or by acting as a nuclease itself remains to

e determined. Sae2 involvement in DSB processing is conservedmong eukaryotes, as also its putative orthologs in humans and S.ombe (CtIP and Ctp1, respectively) have critical functions in DSBesection [19,20].

The function of S. cerevisiae Sae2 in end resection requires itshosphorylation on Ser267 by cyclin-dependent kinases (CDK)21]. In fact, a sae2-S267A mutant exhibits defective generation of′-ended ssDNA and reduced HR-mediated DSB repair. On the con-rary, the phospho-mimicking mutant sae2-S267E does not causehese phenotypes and partially bypasses the requirement for CDKctivity in DSB ends processing. This CDK-dependent control of

ae2 function in DSB resection is conserved also in the Sae2 ver-ebrate ortholog CtIP, two sites of which are phosphorylated byDK and control the efficiency of resection [22–24]. How CDK phos-horylation promotes Sae2/CtIP activity in DSB resection is still

ir 12 (2013) 791– 799

unknown, but the use of CDK activity to promote Sae2 functionin resection is one of the mechanisms that cells use to suppress HRduring the G1 phase of the cell cycle (when sister chromatids arenot yet present for HR and CDK activity is low) to avoid genomicrearrangements [25,26].

The requirement for MRX and Sae2 in end resection dependsupon the nature of DNA ends. The initial endonucleolytic cleav-age of the 5′ strands catalyzed by MRX and Sae2 is crucial forthe processing of “dirty” DNA ends such as those created afterexposure to IR, camptothecin, bleomycin and methylating agents,where protein-DNA adducts or altered DSB ends structures must beremoved to allow further processing [27–32]. Conversely, resectionof “clean” DSB ends, such as those generated by endonucleases,can occur also in the absence of MRX and Sae2. In fact, initia-tion of resection at an endonuclease-induced DSB is impaired incells lacking MRX or Sae2, but once resection is initiated its rateis similar to that of wild-type cells [11,12,28,29,31]. It is worthnoting that the defect in initiating resection is more severe inmre11� cells than in sae2� cells or mre11 nuclease defectivemutants, and this difference is likely due to reduced recruitmentat DSBs of other proteins involved in resection (Sgs1, Dna2 andExo1) rather than to a specific requirement for MRX to initiateresection.

More extensive DSB resection is catalyzed by the 5′-3′ exonu-clease Exo1 and the 3′-5′ RecQ helicase Sgs1, which control twopartially redundant pathways [16,17] (Fig. 1). The ssDNA formedby Sgs1-mediated DNA unwinding is degraded by the bipolar 5′

flap endonuclease Dna2, which is a CDK target in DSB resection[33]. Sgs1 interacts with the type I topoisomerase Top3 and theoligonucleotide/oligosaccharide-binding (OB)-fold containing pro-tein Rmi1 to form the STR complex [34,35]. Recruitment of Sgs1,Dna2 and Exo1 to DSBs requires the MRX complex [36], andthis can explain why mre11� cells have more severe resectiondefects than sae2� and mre11 nuclease defective mutants. Bycontrast, Sgs1 and Dna2 are still recruited in sae2� and mre11nuclease defective mutants, indicating that these proteins cancompensate for MRX-Sae2 nuclease function in initiation ofresection.

In vitro data indicate that resection in humans occurs via twopathways, which are similar to those described for S. cerevisiae. Inone of them, the human counterpart of Sgs1, BLM and DNA2 phys-ically interact and collaborate in 5′–3′ resection of DNA ends [37],while MRN promotes resection by recruiting BLM to DNA ends [37].In the second pathway, MRN, RPA and BLM stimulate resectionby promoting the action of human EXO1 to DNA ends, with BLMenhancing EXO1 affinity for DSB ends and MRN increasing EXO1processivity [38].

Interestingly, these reconstitution in vitro experiments of theresection machinery has revealed an essential role for the RPAcomplex in promoting the unwinding activity of Sgs1/BLM andenforcing the 5′–3′ resection polarity of Dna2 [34,35,38,39]. Thesein vitro findings have been recently confirmed in vivo, as depletionof S. cerevisiae RPA eliminates both the Sgs1-Dna2 and Exo1-dependent resection pathways [40]. Furthermore, RPA shields the3′-ended ssDNA overhangs from nucleolytic attack and inappropri-ate annealing that could lead to genetic rearrangements [40].

DSB resection is also influenced by histone modifications andATP-dependent chromatin remodeling reactions [41]. Interest-ingly, recent data indicate that Exo1- and Sgs1/Dna2-mediated DSBprocessing require distinct chromatin remodeling events [42]. Infact, either removal of H2A-H2B dimers or incorporation of thehistone variant H2A.Z markedly enhances Exo1 activity, suggest-

ing that ATP-dependent chromatin-remodeling enzymes regulateExo1-mediated resection. By contrast, resection by the Sgs1-Dna2machinery remains efficient when chromatin fibers are subsatu-rated with nucleosomes, suggesting that initiation of resection by

E. Gobbini et al. / DNA Repair 12 (2013) 791– 799 793

Fig. 1. Model for DNA-end resection. MRX/MRN, Ku and Sae2/CtIP rapidly bind DNA ends. Upon phosphorylation of Sae2/CtIP by CDK, MRX/MRN and Sae2/CtIP catalyze theinitial processing of the 5′ strand. This clipping removes Ku or creates substrates that are no longer bound by Ku. The 5′ strand is then extensively processed through twoparallel pathways governed by Exo1 and the STR complex in concert with Dna2. MRX facilitates the extensive resection by promoting the recruitment of Exo1 and STR-Dna2.Extensive DSB resection is inhibited by the checkpoint adaptor protein Rad9/53BP1, which is bound to methylated histone H3 at lysine 79 (H379me) and histone H2A thath omotea legen

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as been phosphorylated at serine 129 (�H2A). The chromatin remodeler Fun30 prre indicated as red dots. (For interpretation of the references to color in this figure

his pathway might simply require a nucleosome-free gap nexto the DSB. Consistent with this hypothesis, the helicase activityf yeast Sgs1 is reduced on nucleosomal substrates, and effi-ient resection by the Sgs1-Dna2-dependent machinery requires

nucleosome-free gap adjacent to the DSB [42]. In this context,t has been recently shown that Tbf1 protein and its interactorid22 promote DSB resection by facilitating the persistence ofRX at the DSB ends [43]. Furthermore, these proteins appear to

acilitate nucleosome eviction around the DSB [43]. As Tbf1 haseen shown to decrease nucleosome occupancy at some promo-ers [44,45] and to prevent the propagation of silent chromatint telomeres [46,47], Tbf1 and Vid22 might promote the action

f DSB processing enzymes by maintaining a nucleosome-freeegion around the DSB. Whether these two proteins specificallyupport the Sgs1-Dna2-dependent machinery remains to be deter-ined.

s DSB resection by removing Rad9 from the DSB ends. The phosphorylation eventsd, the reader is referred to the web version of the article.)

2.2. Negative regulators of DSB resection

DNA end resection is also negatively regulated. In particular,deletion of YKU70 or YKU80 allows DSB resection in S. cerevisiae G1cells [48,49]. Ku and the MRX complex have been shown to bindindependently and simultaneously the DSB ends [51]. Moreover, G1cells lacking Ku show an increased recruitment of Mre11 at the DSBends, whereas loss of MRX increases Ku binding [8,48,51]. Theseresults suggest that Ku and MRX compete for binding to DSBs andthat DSB-bound Ku limits the formation of ssDNA by impairing theloading and/or the activity of resection factors. Notably, resection ofa single DSB in Ku-deficient G1 cells occurs independently of CDK

activity, although it is limited to DNA regions close to the breaksite [48]. This finding suggests that Ku is the principal rate-limitingfactor for initiation of resection in G1, and its action is preventedin G2 by CDK-dependent phosphorylation events. Interestingly, the

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ack of Ku suppresses the DNA damage sensitivity of Sae2-deficientells, and this suppression requires both Exo1 and Sgs1 [50]. Theseesults suggest that CDK-mediated Sae2 activation promotes Exo1nd Dna2 action by removing Ku from DNA ends (Fig. 1).

DSB resection is also restricted by the checkpoint protein Rad9,hich acts as barrier toward end processing enzymes [52,53]

Fig. 1). It has been recently shown that the ATP-dependent chro-atin remodeler Fun30 is capable to overcome the Rad9 barrier to

esection by promoting both Exo1 and Sgs1/Dna2-dependent path-ays [54–56] (Fig. 1). This finding suggests that the role for Fun30uring DSB resection is not to disrupt nucleosomes per se, butather to antagonize the resection inhibitor Rad9. Also the humanounterpart of Fun30, SMARCAD1, participates in end resection,s its knockdown causes a reduction in ssDNA generation at IR-nduced DSBs [55].

The human structural and functional ortholog of Rad9 is 53BP1, protein that interacts with histones and histone-binding pro-eins. Similarly to S. cerevisiae Rad9, mammalian 53BP1 inhibits DSBesection promoted by CtIP in G1 [57]. DSB resection is allowedy the removal of 53BP1 from DSB ends, which is promoted byhe BRCA1 protein [58]. Moreover, loss of 53BP1 partially res-ues the HR defects of BRCA1 mutant cells, confirming that BRCA1vercomes 53BP1 function at DSBs [58–60]. The mechanism ofRCA1 inhibition by 53BP1 involves RIF1, a protein that accumu-

ates at the DSB sites in an ATM- and 53BP1-dependent manner61]. Similarly to 53BP1-deficiency, RIF1 ablation enhances 5′ DNA-nd resection at IR-induced DSBs and dysfunctional telomeres61–63]. Furthermore, depletion of 53BP1 or RIF1 restores resectionn BRCA1-depleted cells, but it fails to do so in CtIP-deficient cells64]. Interestingly, RIF1 accumulation at DSB sites inhibits resectiony CtIP and BRCA1 during the G1 phase of the cell cycle, whereashe phosphorylated BRCA1-CtIP complex prevails after S phasentry and activation of CDK complexes, thus leading to initiation ofesection [64]. Altogether, these data support a model where 53BP1nd RIF1 protect DSB ends from BRCA1-CtIP-dependent resectionn G1 cells, while phosphorylated BRCA1-CtIP displaces 53BP1-RIF1rom DSBs in S/G2, facilitating the action of the MRN complex.hus, a regulatory circuit involving 53BP1-RIF1 and BRCA1-CtIPnfluences DSB repair pathway choice in mammals.

S. cerevisiae Rif1 is involved in telomere metabolism and phys-cally interacts with the telomeric proteins Rif2 and Rap1 [65].he lack of Rif1 enhances generation of ssDNA at native telomeresnd assists the function of the CST (Cdc13-Stn1-Ten1) complex inelomere protection [66,67]. Furthermore, it counteracts the bind-ng of RPA at telomeric ends [68,69]. Whether the role of buddingeast Rif1 in regulating resection is restricted to telomeres or can bextended also to accidentally occurring DSBs remains to be deter-ined.

. The ATR/Mec1 and ATM/Tel1 checkpoint kinases

Generation of DNA DSBs triggers the activation of the DNA dam-ge checkpoint signal transduction pathways, which coordinatehe DNA damage response [3]. Checkpoint signaling comprises arotein kinase cascade initiated by the two apical protein kinases,hich are called ATM and ATR in mammals or Tel1 and Mec1,

espectively, in S. cerevisiae (Fig. 2). In collaboration with accessoryroteins, these two kinases respond to DNA damage by phosphory-

ating downstream effectors that coordinate cell cycle progressionith DNA repair.

ATM and ATR are members of the phosphoinositide 3-kinase-

elated protein kinase (PIKK) family. The consensus motif for phos-horylation by ATM/ATR is hydrophobic-X-hydrophobic-[S/T]-Q.ther members of the PIKK family include the DNA-dependent pro-

ein kinase (DNA-PK), the homolog of Caenorhabditis elegans SMG-1

ir 12 (2013) 791– 799

(SMG1) and the mammalian target of rapamycin (mTOR). The PIKKenzymes are large proteins (270–450 kDa) that have analogousstructures, characterized by N-terminal HEAT repeat domains fol-lowed by relatively small kinase domains [70]. The kinase domainis located near the C-terminus and is flanked by two regions ofsequence similarity called FAT (FRAP, ATM, TRRAP) and FATC (FATC-terminus) domains, which might interact and participate in theregulation of the kinase activity [71]. The remaining part of eachprotein consists of multiple �-helical HEAT repeats [72], a singleHEAT unit being a pair of interacting anti-parallel helices linkedby a flexible “intra-unit” loop [73]. These regions are predictedto adopt large superhelical conformations creating extended sur-faces that mediate protein and DNA interactions. The presence ofa common evolutionarily conserved structure among all PIKK-likeproteins raises the possibility that, despite their different biologicalroles, all these proteins share common underlying properties.

Both ATM/Tel1 and ATR/Mec1 are activated by DNA damage,but their DNA damage specificities are distinct. ATM/Tel1 is acti-vated by DSBs, whereas ATR/Mec1 responds to a broad spectrum ofDNA lesions that induces the generation of ssDNA [3]. DNA damage-activated ATR/Mec1 and/or ATM/Tel1 promote the activation of thedownstream effector kinases Rad53 and Chk1 (vertebrate CHK2 andCHK1, respectively) [74]. In S. cerevisiae, Mec1 activates both Rad53and Chk1, while human ATM and ATR primarily activate CHK2 andCHK1, respectively.

Activation of the effector kinases requires mediator proteins,among which are the BRCT-domain-containing protein Rad9 andits metazoan ortholog 53BP1 (Fig. 2). In particular, Rad9 is phos-phorylated in a Mec1- and/or Tel1-dependent manner upon DNAdamage, and these phosphorylation events create a binding sitefor Rad53, which then undergoes in-trans autophosphorylationevents required for its full activation as a kinase [75–77]. Mec1-dependent phosphorylation of Rad53 allows further autoactivation[78,79]. Moreover, Rad9 oligomerization is required to maintaincheckpoint signaling through a feedback loop involving Rad53-dependent phosphorylation of the Rad9 BRCT domain [80]. Fullyactivated Rad53 is then released from the hyperphosphorylatedRad9 complex [81]. Vertebrate CHK2 is also known to dimerize andto undergo ATM-dependent autophosphorylation in-trans, but therole for the DNA damage mediators in this activation remains to beinvestigated.

Recruitment of the Rad9/53BP1 mediator to chromatin involvesmultiple pathways (Fig. 2). In unperturbed conditions, Rad9 isalready bound to chromatin via interaction between its Tudordomain and methylated histone H3 at lysine 79 [82–85]. This con-stitutive Rad9 recruitment to chromatin is thought to facilitate theefficiency of the Rad9-dependent response to DNA damage, whichrequires additional histone modifications. In fact, Rad9 bindingto the sites of damage is further strengthened by the interactionbetween its BRCT domain with histone H2A that has been phos-phorylated at serine 129 (�H2A) [82,84,86–88]. Similarly, 53BP1binding to DSBs is facilitated by phosphorylation of the histonevariant H2AX at serine 139 [89,90].

4. Activation of ATM/Tel1

It is known that ATM is activated primarily by DSBs, but thespecific signals that activate ATM following DSB induction are stillunclear. It has been suggested that the initial trigger of ATM acti-vation might be a modification in chromatin structure surroundingthe DSB rather than direct contact of ATM with broken DNA [91].

Full activation of human ATM is dependent on autophosphoryla-tion on Ser1981 and interaction with the MRN complex at the DSBsites. ATM exists as an inactive dimer in unperturbed cells, but itundergoes intermolecular autophosphorylation on Ser1981 after

E. Gobbini et al. / DNA Repair 12 (2013) 791– 799 795

Fig. 2. Tel1/ATM and Mec1/ATR activation by DSBs. Recognition of the DSB by MRX/MRN (MRX/N) leads to recruitment of Tel1/ATM, which phosphorylates Sae2/CtIP andhistone H2A (�H2A). MRX/MRN, Sae2/CtIP and other nucleases resect the DSB ends to generate 3′-ended ssDNA tails that, once coated by RPA, allow the loading of the Mec1-Ddc2/ATR-ATRIP complex. Tel1/ATM, possibly by acting on the MRX/MRN complex, promotes DSB resection, which activates Mec1/ATR and concomitantly inhibits Tel1/ATMsignaling. Mec1/ATR activation requires Dpb11/TopBP1, the 9-1-1 complex and possibly the MRX/MRN complex itself. Once recruited to the DSB, Mec1/ATR regulates theg activR d53/Cf Exo1

Dt[S[rfi

eneration of 3′-ended ssDNA by phosphorylating Sae2/CtIP and histone H2A. Mec1ad53/Chk2 itself. Moreover, phosphorylated Rad9/53BP1 promotes activation of Ra

rom DNA and can regulate both DSB processing by phosphorylating and inhibiting

SB formation or treatment with agents that alter chromatin struc-ure, resulting in dissociation of the dimer into active monomers91]. Besides Ser1981, other autophosphorylation sites (Ser367,er1893 and Ser2996) play a role in the ATM activation process

92,93]. Although the functional significance of ATM autophospho-ylation is still unclear, it has been recently shown to be requiredor the stabilization of activated ATM at DSB sites, albeit not for thenitial ATM recruitment [94,95].

ates the downstream checkpoint kinase Rad53/Chk2 by phosphorylating Rad9 andhk2 by allowing its in-trans autophosphorylation. Activated Rad53 is then releasedand its specific targets in the checkpoint cascade.

ATM/Tel1 activation requires also the MRN/MRX complex.Mre11 contains a C-terminal DNA binding domain as well as a phos-phoesterase domain that provides ssDNA endonuclease and 3′–5′

dsDNA exonuclease activities [13–15]. Both Rad50 and Xrs2/Nbs1

enhance the nuclease activity of Mre11 in vitro, and Rad50 hasin vitro ATP-binding and hydrolysis activities that are critical forMRN/MRX function [96]. Notably, cells defective in any componentof the MRN/MRX complex are defective in ATM/Tel1 activation,

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ndicating that this complex is crucial for ATM/Tel1 function.hich is the exact molecular mechanism of ATM/Tel1 activation

y MRN/MRX remains to be elucidated, although it is clear thatTM/Tel1 is recruited to sites of DNA DSBs through its interactionith the C-terminal domain of Nbs1/Xrs2 [97–100]. In mammals,irect tethering of a large number of Mre11, Nbs1 or ATM moleculeso a specific chromosome locus activates ATM even in the absencef DSBs [101], suggesting that one of the functions of the ATM-MRNnteraction is to accumulate ATM/Tel1 at the damage sites. Inter-stingly, the action of Mre11 at the DSB ends has been reportedo produce small DNA fragments that can stimulate ATM activ-ty [102], raising the possibility that the MRN/MRX complex hasther functions besides the recruitment of ATM/Tel1 at the DSBs.urthermore, Tel1 kinase activity is stimulated by MRX binding toNA-protein complexes at DSBs [103], suggesting that the MRXomplex might control Tel1 catalytic activity by monitoring proteininding at DNA ends.

. Activation of ATR/Mec1

Although ATR is primarily activated by replication stresses, itsctivation can be promoted also by DNA DSBs. However, in mam-als ATR activation is slower than ATM activation and occurs

redominantly in the S/G2 phase of the cell cycle [104]. In bothammals and yeast, recruitment of ATR/Mec1 at the DSB sites

equires the presence of RPA-coated ssDNA 3′ overhangs [105],hich are generated by nuclease-mediated DSB resection (Fig. 2).ecognition of RPA-coated ssDNA by ATR/Mec1 depends on anTR/Mec1 interacting protein, called ATRIP in mammals and Ddc2

n S. cerevisiae. Biochemical studies indicate that ATRIP binds thePA complex directly and this interaction involves an ATRIP acidic-helix that binds to the basic cleft of the N-terminal oligonu-leotide/oligosaccharide binding (OB)-fold domain of the largePA1 subunit [106]. Loss of ATRIP/Ddc2 causes the same pheno-ypes as loss of ATR/Mec1 in both mammals and yeast, indicatinghat ATRIP/Ddc2 is required for all known ATR/Mec1 functions107,108].

The interaction of ATR-ATRIP with RPA-coated ssDNA is suf-cient for ATR-ATRIP recruitment to DNA lesions, but it is notufficient to fully activate ATR. In response to DNA damage, theeterotrimeric ring-shaped complex 9-1-1 (RAD9-RAD1-HUS1 inumans; Ddc1-Rad17-Mec3 in S. cerevisiae) is loaded at the junc-ions between ssDNA and dsDNA by the RFC-like clamp loaderAD17-RFC2-5 in humans or Rad24-Rfc2-5 in S. cerevisiae. In bud-ing yeast, co-localisation of Mec1-Ddc2 and 9-1-1 at damageites directly stimulates Mec1 kinase activity [109,110]. Evi-ence demonstrating that the 9-1-1 clamp can similarly stimulateTR/Rad3 kinase activity in vertebrates and S. pombe is lacking.ather, the 9-1-1 complex has been proposed to stimulate ATRinase activity by recruiting TopBP1 via an interaction betweenhosphorylated RAD9 and TopBP1 [111–114]. This interactionacilitates the association of TopBP1 with ATRIP, which in turntimulates ATR kinase activity.

TopBP1-mediated activation of ATR is conserved in S. cere-isiae, where the Ddc1 subunit of the 9-1-1 complex recruits theopBP1 ortholog Dpb11 to DNA damage sites [115–118]. Phos-horylation of Ddc1 by Mec1 is critical for Dpb11 function inheckpoint signaling, suggesting that RPA-recruited Mec1-Ddc2ay have sufficient activity to phosphorylate Ddc1 before Ddc1-bp11 interaction takes place [118].

Interestingly, some data in both yeast and mammals suggest

hat the MRN/MRX complex is involved in ATR/Mec1 activation119,120]. Because MRN/MRX has a role in DSB resection, it haseen proposed that MRN/MRX effects on ATR/Mec1 may be dueo DSB processing. However, by using defined ATR-activating DNA

ir 12 (2013) 791– 799

structures in Xenopus egg extracts, MRN has been shown to rec-ognize the ss/dsDNA junctions and to recruit TopBP1 to DNA[121]. Furthermore, Nbs1 is important for ATR-dependent phos-phorylation of RPA. This function does not require 9-1-1 andcan be separated from ATM activation and DSB resection [122].Taken together, these findings lead to a working model where thess/dsDNA junctions at the DSBs have two roles in ATR activation:activating TopBP1 by facilitating its interaction with the 9-1-1 com-plex, and recruiting TopBP1 by promoting its association to theMRN complex.

6. Interplays between ATM/Tel1 and ATR/Mec1 signaling

A challenging question is how ATM and ATR actions are coor-dinated at DSBs. Interestingly, while activation of both ATM andATR depends on the ss/dsDNA junctions, they are oppositely regu-lated by the lengthening of single-stranded overhangs [123]. Bluntdouble-strand ends, as well as ends with short single-strandedtails, are the preferred substrates for ATM activation. As the single-stranded tail increases in length, it simultaneously potentiates ATRactivation and attenuates ATM activation [123]. A similar mecha-nism has been proposed for budding yeast Tel1, whose signalingactivity is disrupted when the DSB ends are subjected to 5′–3′

exonucleolytic degradation [124]. These data suggest that thenature of the DSB ends is important to dictate the engagement ofATM/Tel1 or ATR/Mec1.

In both humans and yeast, ATM/Tel1 activation promotes theaccumulation of ssDNA at DSB ends and therefore is critical for thesubsequent activation of ATR/Mec1 [104,120,123–125]. As genera-tion of ssDNA ultimately leads to ATM inactivation, this mechanismensures an efficient switch from ATM/Tel1 to ATR/Mec1. HowATM/Tel1 promotes DSB resection is unknown. One possibility isthat ATM/Tel1 influences the function of the MRN/MRX complex ina positive feedback loop. Consistent with this hypothesis, Tel1 wasrecently shown to regulate the generation of ssDNA at telomeresby promoting MRX function [126].

Although MRN/MRX is required for ATM/Tel1 recruitment in allthe species analyzed so far, different organisms apparently exhibitdifferences in the roles of ATM and ATR orthologs in checkpointactivation. For example, Tel1-deficient S. cerevisiae cells do notshow obvious hypersensitivity to DNA damaging agents and arenot defective in checkpoint activation in response to a single DSB[124]. This might be due to differences between ATM and Tel1 intheir intrinsic kinase activity and/or in their ability to interact withspecific targets and/or DNA–protein complexes at DNA ends. On theother hand, the ability of ATM/Tel1 to signal at a DSB is attenuatedwhen the DSB ends are resected [124]. This finding suggests that theapparent minor role of Tel1 in DSB signaling may be explained bythe ability of yeast cells to rapidly convert the DSB ends into ssDNAsubstrates that preferentially stimulate Mec1 kinase activity. Thus,although Tel1 contribution to the checkpoint can be masked by theprevailing activity of Mec1, the mechanism governing ATM- andATR-dependent checkpoint activation in humans operates also inS. cerevisiae.

The importance of the ATM/Tel1 to ATR/Mec1 switch inthe response to DSBs remains to be determined. Noteworthy,resection-defective S. cerevisiae cells, such as sae2�, exo1�, fun30�or sgs1� mutants, fail to turn off the checkpoint in response toan unrepaired DSB [56,127,128]. Moreover, the sae2� mutationenhances Tel1-mediated Rad53 activation after DNA damage. This

enhancement requires the function of MRX, whose persistenceat DSBs is increased in sae2� cells, thus promoting sustainedTel1 signaling via interaction with Xrs2 [127,128]. In this context,resection would have two distinct consequences: (i) generation

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f ssDNA that recruits/activates ATR/Mec1; (ii) displacement ofRN/MRX from the DSB site to limit ATM/Tel1 signaling activity.Altogether, these data are consistent with a working model

here, after DSB formation, binding of MRN/MRX to DNA endsromotes the recruitment of ATM/Tel1 to the DSB and subse-uent ATM/Tel1-dependent checkpoint activation (Fig. 2). Then,TM/Tel1 promotes the generation of ssDNA, which in turn acti-ates ATR/Mec1 and concomitantly inhibits ATM/Tel1 signaling.nterestingly, ATR/Mec1 itself might regulate the generation of 3′-nded ssDNA at DNA ends, as Mec1-dependent phosphorylation ofae2 is important for Sae2 function in DSB resection during bothitosis and meiosis [129,130]. Furthermore, Mec1 phosphorylates

istone H2A on Ser129, and this phosphorylation event seems toegulate the resection rate at DSBs [56]. Finally, Mec1 activateshe Rad53 checkpoint kinase that phosphorylates Exo1, and thishosphorylation appears to negatively regulate Exo1 activity inesection [131]. Thus, it is possible that ATR/Mec1 might regulate itswn activation by acting on the resection machinery, and this cane part of a negative feedback loop to prevent excessive resection.

onflict of interest statement

All the authors declare that there are no conflicts of interest.

cknowledgements

We thank Giovanna Lucchini and Michela Clerici for criticaleading of the manuscript. The work in MPL laboratory is supportedy grants from Associazione Italiana per la Ricerca sul Cancro (AIRC)grant IG11407) and Cofinanziamento 2010–2011 MIUR/Universitài Milano-Bicocca. We apologize to all authors whose publicationsave not been cited because of space limitation.

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