Studying the DNA damage response using in vitro model systems

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DNA Repair 8 (2009) 1025–1037

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DNA Repair

journa l homepage: www.e lsev ier .com/ locate /dnarepai r

tudying the DNA damage response using in vitro model systems

lizabeth Garner, Vincenzo Costanzo ∗

enome Stability Unit, London Research Institute, Clare Hall Laboratories, South Mimms,erts EN6 3LD, United Kingdom

r t i c l e i n f o

rticle history:vailable online 23 May 2009

eywords:

a b s t r a c t

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order toprevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathwaysthat delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory

enopus laevisNA damage responseenome stabilityell cycleNA repair

network of proteins has been identified that participate in DNA damage checkpoint pathways. Central tothis network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis ofATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro modelsystems to understand the detailed mechanisms underlying their function. Here we describe some recentfindings obtained analysing these pathways using in vitro model systems. In particular we focus on thestudies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response

cycle.
in the context of the cell
. Introduction

In order to preserve genomic stability, eukaryotic cells har-our highly flexible and integrated signalling systems that facilitatehe sensing of DNA damage and subsequent responses to pro-

ote repair or cell death. Concomitantly, such signalling mayodulate cell cycle progression to support the process of repair.

he process of cell cycle arrest as a consequence of DNA dam-ge signalling is known as the ‘checkpoint response’. Checkpointesponses are intrinsically linked to the cell cycle and as suchome of the first experiments that supported the concept of check-oints in response to DNA damage were performed in cyclingukaryotic cell systems such as yeasts and cells derived from mam-alian hosts. The genetic malleability of yeast systems and the

ase with which cell cycle progression can be observed makeseast a powerful tool in the study of the cell cycle. Addition-lly, the virtue of the genetic similarity of various mammalianell systems combined with advances in technology of cell biol-gy applications has paved the way for furthering our knowledgef the eukaryotic DNA damage response. However, despite thenequivocal value of such systems, the study of this response hasained important insights regarding the molecular mechanisms
nderlying their function via the exploitation and developmentf several important in vitro systems. In vitro systems make usef molecular and biochemical techniques to recapitulate proteinunction, protein–protein and protein–DNA interactions that pro-
∗ Corresponding author.E-mail address: [email protected] (V. Costanzo).

568-7864/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2009.04.015

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vide a profound insight into various aspects of DNA damagesignalling.

2. In vitro systems to examine DNA damage responses

2.1. Purified protein systems

Biochemical techniques are at the core of understanding manyaspects of protein structure, activity and function and indeed thecellular response to DNA damage has been subjected to intensein vitro biochemical characterisation. The ultimate goal of recon-stituted biochemical systems is to reproduce, at least in part,complex biochemical reactions with purified components in anattempt to study the role of the single players in the overall path-way. Purified proteins are of pivotal importance, providing a basisfor understanding precise molecular interactions and frequentlyprovide unequivocal support toward models proposed followingobservations in other systems. The knowledge of several compo-nents of checkpoint pathways has allowed in the recent years thereconstitution of many important reactions with purified recombi-nant proteins especially concerning the early steps of DNA damagerecognition and initial checkpoint activation. The definition of suchsystems has led to the discovery of detailed molecular mecha-nisms behind fundamental checkpoint pathways [1–7]. However,it is clear that although powerful, this approach cannot account for
yet undefined and unknown steps in the complex processes thatit tries to reproduce, especially when some of the players are yetto be discovered. In addition, large proteins are often difficult toobtain in useful amounts (Table 1). An important complement topurified systems is the use of cell extracts capable of recapitulating
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omplex biological events in the test tube in a more physiologicalontext.

.2. Yeast, Drosophila and mammalian cell extracts

Cell extracts from yeast, Drosophila and mammalian sourcessuch as human and mouse) can be used to partially reconstituteNA replication, DNA repair and checkpoint responses. There are

everal advantages to this, in that extracts from mutant isogenicines can be compared and the ease of genetic manipulation avail-ble to yeast experimentation can be exploited in an in vitro system.ammalian cell extracts have been used quite extensively to study

spects of DNA repair. Such assays frequently combine refined pro-ein purification techniques to examine the specific activities ofurified proteins in a system that reconstitutes repair processes onynthetic DNA molecules containing specific lesions [8–12]. How-ver, it has been more difficult to define systems that recapitulateNA damage checkpoints. This is likely due to technical issues suchs the ability to obtain functional protein mixtures and the rela-ively limited protein yield that can be obtained from such extractystems (Table 1). A further problem with such purified systems ishe limited ability to recreate complex phenomenon such as the cellycle. As such, these systems have not been utilized as successfullys the Xenopus laevis egg extract to study checkpoint responses inhich cell cycle context is an important and intrinsic factor.

.3. Xenopus laevis egg extract

The Xenopus laevis egg extract provides a platform that reca-itulates chromatin formation, nuclear assembly, DNA replication,itotic spindle assembly and chromosome segregation. Xenopus

ggs contain an abundance of proteins required to drive cell cyclerogression and multiple rounds of cell division upon fertilisation in
he absence of transcription. Consequently, biological reactions areriven by protein–protein interactions, protein post-translationalodifications and enzymatic activities. Historically, many of the
nitial observations regarding aspects of early embryonic cell cyclevents were made from the study of Xenopus egg cell division, such

able 1he pros and cons of in vitro systems.

dvantages

enopus egg cell free extractEggs can be obtained in large quantities to make egg extractEgg extract provides a rich source of biologically active proteins andmembrane componentsEgg extract is capable of recapitulating the cell cycle events such asgenomic DNA replication and mitosisProteins can be depleted or added in allowing a biochemical ‘knock-out’or ‘knock-in’ approachEgg extract retains the ability for post-translational modification ofproteinsHigh degree of conservation of pathways from Xenopus to mammals

east, mammalian, Drosophilia cell free extractGenomic modification of yeast strains is relatively quick and easy

Drosophila are amenable to mutational analysisGenetic similarity of mammalian extracts and cell cultures from patientsavailableExtract systems represent powerful systems for examining DNA repairprocesses

urified protein systemsStructural studies can yield information about interacting partners,domains and candidate regions for post-translational modificationThe function of single proteins with defined stoichiometries can beanalysed

pair 8 (2009) 1025–1037

as the oscillating levels of maturation promoting factor (MPF) inmeiotic and mitotic cell cycles [13,14] or from similar systems suchas fertilized sea urchin eggs [15]. The desire for a more experimen-tally flexible system to study biochemical aspects of the cell cycleled to the development of the sensitive egg extract cell free system[16–24]. While over the years many protocols have refined the wayin which extract is prepared and detailed the production of vari-ous types of extract the fundamental process is the same for mostderivations. Essentially, eggs are crushed by centrifugation creatinga density separation that allows the extraction of egg cytoplasm.The egg cytoplasm is uniquely able to undergo rapid and sponta-neous oscillation of cyclin dependent kinase (Cdk) activity drivingthe extract into consecutive rounds of mitosis and S-phase [25].Demembranated sperm nuclei derived from the male testis, whenadded to the egg extract, undergo complete rounds of chromatinand nuclear membrane assembly, semi-conservative replication,nuclear envelope breakdown and mitotic chromosome condensa-tion [25]. Multiple passages through the cell cycle are achievableusing this system and as such, egg extracts have proven a power-ful tool for the in vitro study of both DNA replication and cell cycleprogression [17,18,26]. A more elaborate version of the Xenopus eggextract entails the processing of nuclei assembled in egg extract toproduce a highly enriched fraction of nuclear proteins (nucleoplas-mic extract or NPE) that allows the replication of double-strandedDNA (dsDNA) templates in the absence of a nuclear membrane [27].The egg extract mostly recapitulates rapid embryonic cell cycleevents taking place in the absence of transcription and as such itconstitutes an approximation of somatic cell cycle processes. How-ever, embryonic cell cycle events can be considered closer to theones taking place in fast proliferating and poorly differentiatedcancer cells.

The intrinsic features of egg extract provide some clear advan-tages and a few disadvantages for the study of important biological
processes. A major advantage is the high protein concentration andthe selective enrichment of cell cycle factors, which ensures thatproteins that might be present in limited amount in other systemsare instead well represented in the egg extract. In addition, the highdegree of genetic conservation between Xenopus and mammalian
Disadvantages

Xenopus specific depleting antibodies need to be generated in high amountWild type or mutated soluble protein components must be purified in high

amount to reconstitute depleted extract‘Quality’ of extract can vary and initial egg quality is essential

Xenopus laevis genome has not been completely sequenced (However thefully sequenced X. tropicalis genome is available)

Many mammalian pathways are not represented in yeast i.e. apoptoticpathways

Cell cycle progression in vitro is difficult to achieve

Large proteins are relatively difficult to purify

Biochemical analysis often requires large quantities of protein, which canbe difficult to achieve

Unknown steps in the pathway cannot be reconstituted

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rganisms facilitates the biochemical study of large proteins onlyresent in complex vertebrate organisms that can be easily isolatednd characterised in large quantities (Table 1).

On the other hand the absence of transcription during earlymbryonic division and in egg extract, which is itself an interest-ng yet poorly understood biological phenomenon, impedes thetudy of biological events in which transcription is involved. Oneuch example is the modulation of gene expression regulated byhe DNA damage response, which cannot be recapitulated in eggxtract. However, this provides a unique opportunity to unambigu-usly establish the role of proteins directly involved in the controlf cell cycle events such as DNA replication, mitosis or checkpointependent processes. For example a DNA replication or checkpointefect following the removal of a specific protein from extract cane directly attributed to the lack of the removed factor and not to

ndirect transcriptional effects due to its absence. This property ofgg extract has also been useful in determining the role in DNAeplication of factors previously known to be involved only in tran-cription such as c-MYC, which instead has been shown to directlyromote DNA replication in egg extract [28].

.3.1. Protein depletion: “The biochemical knockout”A significant tool available for exploitation of this system is

he possibility of depleting native proteins from the egg extractsing specific antibodies. Importantly, depleted extract can thene reconstituted with recombinant wild type or mutated proteinsTable 1). The possibility of directly supplementing egg extractith active recombinant proteins provides a unique advantage over

ell-based systems in which the introduction of active enzymesequires laborious and complex plasmid driven protein expression.his technique allows the analysis of single proteins or proteinomplexes involved in fundamental biological processes. As suchhe protein depletion and reconstitution procedure is often morenformative than other gene targeting based approaches such asnockout or knockdown. Gene knockout often results in cell lethal-ty especially when the gene encodes for a protein involved inssential processes such as DNA replication or mitosis. This impedeshe subsequent analysis of the targeted gene. In Xenopus egg extract,nstead, removal of a factor required for cell cycle events leads tohe arrest of cell cycle progression at the step in which the fac-or is involved without impairing the survival of the extract. Thisrocedure can reveal unique information about the biochemicaltep in which a given protein or protein complex is involved. Thispproach can be considered the equivalent of a “biochemical knock-ut”. In addition, protein depletion can lead to the complete andapid removal of a specific protein in the absence of any residualctivity and without the off-target effects often associated with these of RNAi based protocols.

.3.2. Xenopus egg extract and DNA damage responsesXenopus egg extracts were useful to perform the initial exper-

ments that highlighted the presence of checkpoint pathwaysontrolling cell cycle progression [29,30]. More recently this appli-ation has been expanded making the Xenopus egg extract an idealn vitro model for understanding cell cycle checkpoints in responseo DNA damage and replication stress. In the Xenopus embryo, thearly embryonic cell cycles lack cell cycle checkpoints. However,he recognition of a threshold with regard to the quantity of DNAesions and DNA molecules required to induce effective DNA dam-ge responses in egg extracts led to important achievements in these of this system to understand DNA damage signalling pathways
30–34].
Indeed using egg extract systems it is possible to moni-or responses to the introduction of a variety of aberrant DNAtructures that mimic damaged DNA and examine subsequent pro-ression into S and M phase. Different synthetic DNA templates

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have been successfully applied to egg extracts obtaining specificactivation of the ataxia telangiectasia mutated (ATM) and the ATMand Rad3 related (ATR) proteins. For example DNA containing dou-ble strand breaks (DSBs) has been shown to activate ATM whereasDNA structures made of poly-deoxy-T annealed to poly-deoxy-Aoligonucleotides, which can anneal in a staggered fashion produc-ing DNA gaps, have been shown to efficiently activate ATR [35].Similar results have been obtained with gapped DNA moleculesmade of small oligos annealed to circular single-stranded DNA(ssDNA) [36]. In this case the addition of bulky adducts interfer-ing with protein binding such as biotin moieties at the 3′ andthe 5′ of the annealed oligos led to discovery that the 5′ end ofthe oligo annealed to ssDNA is important for ATR activation. Anadditional advantage of this system is the possibility of supple-menting egg extract with active DNA modifying enzymes. Amongthese, recombinant restriction endonucleases have been used suc-cessfully to induce clean DSBs in the chromatin in the absenceof other types of DNA damage associated with the use of chem-icals or ionising radiation, providing a unique tool to assess theeffect of these lesions in the context of chromatin templates.Furthermore, the refined ability to inhibit DNA replication withchemicals that interfere with the activity of the DNA polymeraseprovides a unique tool for studying intra-S-phase responses to DNAdamage.

3. Current perspectives of checkpoint responses gainedfrom in vitro studies

3.1. Sensing DNA damage

DNA damage may be caused by a plethora of both endoge-nous and exogenous means. When a cell senses the occurrence ofDNA damage, sensor protein signalling facilitates cell cycle arrest.An understanding of the strict regulation of cell cycle phases haspaved way for our appreciation of cell cycle checkpoints. Early workin Xenopus extract systems demonstrated the importance of con-trolled cell cycle progression [30,37,38]. In the presence of DNAdamage, the signalling that relays the presence of DNA damage tothe cell cycle machinery originates from sensor proteins that func-tion in close proximity to DNA lesions themselves. One group ofproteins central to the sensor system is the phosphatidylinositol-3 kinase-like kinases (PIKKs). This group of proteins comprisesATM, ATR and the catalytic subunit of DNA protein kinase (DNA-PKcs). These proteins are considered to ‘sense’ DNA damage ina lesion-specific manner. ATM is considered the primary sensorof DSBs while ATR is predominantly activated in the presenceof single-stranded DNA (ssDNA) regions [39]. Studies of ATR andATM activation have been recently reviewed [2,40]. Here, we willdescribe findings related to these pathways obtained using in vitrosystems, focusing in particular on the ones derived from the use ofXenopus egg extract.

3.2. The ATR dependent response

ATR is an essential gene in both human and mouse models[41–43]. Seckel syndrome, a human disease characterised by devel-opmental and growth retardation has been associated with ATRmutation. Cells derived from Seckel patients have lower levels ofATR mRNA and protein, and demonstrate some defects of ATR sig-nalling [44]. It is likely that lethality in the complete absence ofATR function stems from the fact that ATR has a complex and
diverse array of physiological functions. Importantly ATR is crit-ical in the regulation of responses to replicative stress or UVirradiation [39,45] and in vitro systems have been instrumentalin aiding our understanding of ATR function in these processes.From these studies ATR emerged as a key sensor of ssDNA gen-

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rated at stalled replication forks. The recognition of ssDNA by ATRequires a dedicated DNA-binding partner protein known as ATR-nteracting protein, ATRIP. In mammals ATRIP colocalises with ATRnto nuclear foci upon DNA damage or inhibition of replication,nd is phosphorylated by ATR [43]. The ssDNA binding protein,eplication protein A (RPA) is required for ATRIP to bind sites ofNA damage and as such the recruitment and activation of ATR

equires regions of ssDNA [46,47]. In vitro systems were importantor determining the DNA structures to which ATR–ATRIP com-lexes show affinity and studies performed in Xenopus extractsave yielded much information regarding the mechanistic acti-ation of ATR. The importance of RPA covered ssDNA for ATRunction in the context of a stalled replication fork was shown byncoupling the progression of the replicative MCM (Mini Chromo-ome Maintenance) helicase from the DNA polymerase on damagedNA templates [48]. This uncoupling results in the accumulationf ssDNA at stalled replication forks that was shown to pro-ote ATR activation. ssDNA gaps obtained by digestion of nickedNA templates with Exonuclease III were also shown to activateTR [36,47,49].

Recent results obtained with DNA plasmids containing a singlerosslink replicated in nucleoplasmic extract have pinpointed thatTR activation can be achieved in the absence of DNA unwindingnd helicase uncoupling. In this case ssDNA regions activating ATRight be generated following degradation of newly synthesizedNA at replication forks stalled by the crosslink [50].

.2.1. Protein complexes required for ATR activationWhile RPA coated ssDNA is important for the recruitment of

he ATR–ATRIP complex to DNA lesions including stalled and col-apsed replication forks, the activation of ATR requires the loadingf the 9-1-1 complexes composed of RAD9, RAD1 and HUS1 and

ig. 1. DNA lesion processing, ATM/ATR activation and signal transduction. The upper pandonucleolytic activity of the MRN complex leads to the generation of single-stranded Dctivation involves the intermolecular phosphorylation of serine residue 1981 leading to thhe downstream activation of the signalling kinase Chk2. ssDNA may recruit ssDNA bindiSB lesions by MRN and MDC1 interactions in a manner that involves chromatin modificat DSB or at stalled forks by the action of ExoI or the MRN complex, Ctp1/Sae2/CtIP, andsDNA recruits the ATR–ATRIP complex to DNA lesions. Activation of ATR requires concomo include the recruitment of TopBP1 required for ATR activation. A principal recipient ofole in mediating the activation of Chk1 in this process. The activation of the effector kiinase activity. However they are pivotal in facilitating damage induced DNA repair, cell cyhromatin remodelling.

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the RAD17-RFC complex. The presence of RPA on ssDNA is thoughtto promote RAD17-RFC binding to gapped and primed ssDNA.RAD17-RFC subsequently facilitates the binding of the 9-1-1 com-plex [6,51]. In vitro systems using purified proteins have helped toclarify the mechanism of 9-1-1 loading onto gapped DNA structures[51,52]. These studies revealed that transition regions between sin-gle and double-stranded DNA, especially those containing the 5′

end of a DNA oligonucleotide annealed to ssDNA are required forproper loading of the 9-1-1 complex. The 9-1-1 complex plays amulti-faceted role in the regulation of ATR activation. The bindingof the 9-1-1 complex is crucial for ATR activation in the contextof its interaction with TopBP1 (Dpb11 in S. cerevisiae, Cut5 in S.pombe and Mus101 in Drosophila cells) (Fig. 1) [53,54]. In vitro stud-ies have demonstrated that TopBP1 contains functional domainsrequired for both binding and activation of ATR [55]. One of thesedomains is sufficient to activate ATR in vitro and in vivo [55,56].This domain is separate from the 9-1-1 complex interacting regionand is thus available to bind ATR–ATRIP upon recruitment to the9-1-1 complex. Recent work has demonstrated that TopBP1 bindsdirectly to ATRIP and that this interaction is required for ATR acti-vation [57]. Therefore, ATRIP promotes localisation of ATR to thesites of DNA damage and replication stress and is also required tomediate TopBP1 dependent activation of ATR. From these studiesTopBP1 emerges as central regulator of ATR activities. Importantly,TopBP1 is also essential for initiation of DNA replication [58]. Theinterpretation of genetic experiments in which TopBP1 is mutatedwould have been difficult without the help of in vitro dissection
of these pathways with Xenopus extract and reconstituted recom-binant complexes. In particular, the use of Xenopus egg extractallowed the identification of the C-terminal region of TopBP1 asan essential regulator of checkpoint activation independently fromreplication functions [55,56,59].
nel highlights the role of MRN dependent processing at double-strand breaks. TheNA 3′ overhang and oligonucleotides (ssDNA oligos) stimulating ATM activity. ATMe disruption of the inactive dimer. Active, phosphorylated ATM monomers facilitateng proteins such as SSB1 able to stimulate ATM activation. ATM is also recruited totions. The lower panel depicts the activation of ATR in response to ssDNA generatedDna2 nucleases following Sgs1 mediated unwinding of DNA duplexes. RPA coateditant loading of the 9-1-1 and the Rad17-RFC complex. The role of the 9-1-1 appearsactive ATRs kinase activity is the effector kinase Chk1. Claspin plays a fundamentalnases Chk1 and Chk2 represent a small sub-set of the recipients of ATM and ATRcle arrest, apoptosis and other processes such as damage induced transcription and

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As previously mentioned further details regarding the DNAtructures that activate ATR have been elucidated in Xenopusxtract. In these studies ssDNA templates with primed double-tranded DNA regions have been used to successfully recapitulateTR activation in an RPA, 9-1-1 and ATR–ATRIP dependent mannerncovering the importance of the 5′ terminus of the primed DNAdjacent to a stretch of ssDNA for ATR activation [36]. However, it isnclear how these structures arise in a more physiological context.ne possibility is that DNA structures containing a free 5′ end are

ormed by the accumulation of Okazaki fragments synthesized byolymerase � at stalled replication forks. Intriguingly, Polymerase

has been shown to be required for ATR activation [60]. Thesetructures would be also present at replication forks stalled by DNArosslinks, which are capable of activating ATR-dependent phos-horylation of Chk1 [50]. However, the link between TopBP1 andhese DNA structures in the ATR activation process remains to belarified.

.2.2. ATR signal amplificationThere are various recipients of ATR kinase activity following

ctivation, most notably Chk1, which is phosphorylated on at leastwo residues and contributes significantly to ATR-dependent DNAamage checkpoint signalling leading to cell cycle arrest, and theodulation of both origin firing and replication fork progression

61–63]. Chk1 appears to be the principle recipient of ATR kinasectivity, and Claspin has been shown to mediate the signal fromTR. The importance of Claspin was initially uncovered via in vitroxperiments using the Xenopus extract system and protein purifi-ation techniques [64–66]. The role of Claspin in other aspects ofaintaining genomic integrity has since been examined in such in

itro systems, which have revealed important roles for Claspin dur-ng S-phase and ATR dependent responses to DSBs in addition tourther aspects of efficient Chk1 phosphorylation by both ATM andTR [67–70].

The kinase activity of ATR may itself enhance the process ofTR activation in a positive feedback loop. This may be throughhe phosphorylation of TopBP1, as demonstrated in Xenopus [59].urthermore, ATM may also play a role in sustaining ATR activ-ty since ATM-dependent phosphorylation of TopBP1 contributes tohe amplification of ATR signalling [71]. DSBs have also been showno activate ATR in a manner that requires some degree of processingt the lesion [72]. Interestingly, removal of Claspin from egg extractsoes not completely abrogate the activation of Chk1 in responseo these DSB lesions. In this case depletion of BRCA1 togetherith Claspin seems to abolish this activation. Instead, the pres-

nce of BRCA1 is not necessary for activation of Chk1 in responseo stalled replication forks [70]. These different complexes helpn discriminating structurally distinct DNA lesions though check-oint mediator proteins exhibit significant functional overlap. Ofarticular interest is the observation that ATM and ATR are bothesponsible for the phosphorylation of Chk1 in response to ionis-ng radiation [73]. Indeed, DSBs formed during S and G2 phases ofhe cell cycle undergo processing in a manner that requires ATMignalling and can lead to the generation of RPA coated ssDNA ableo promote ATR activation. The Mre11/Rad50/Nbs1 (MRN) complexnd its associated nuclease activity is required for such process-ng [73]. From these observations it is clear that while ATM is stillonsidered the fundamental damage sensor of DSBs, the roles ofNA damage signalling proteins, including these sensor elements,verlap considerably.

.3. The ATM dependent response

ATM is a ubiquitously expressed protein kinase localised pre-ominantly in the nucleus [74]. One of the first demonstrations ofTM’s ability to phosphorylate proteins in vivo was the observation

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that ATM phosphorylated p53 at serine 15 in response to DNA dam-age [75]. In the absence of cellular stress, ATM molecules exist asdimers that are catalytically inactive. It is considered that changesto chromatin structure activate ATM. Activation involves the inter-molecular phosphorylation of serine 1981 causing the disruption ofdimer molecules, a process that takes place within minutes of DNAdamage [76]. Dimer dissociation releases ATM molecules whosecatalytic domains are no longer concealed and are thus liberatedto phosphorylate ATM’s many downstream substrates includingproteins involved in coordinating cell cycle arrest, DNA repair andapoptosis such as Nbs1, SMC1, Chk2 and p53 [77].

Unlike many proposed sensor proteins ATM did not appear tohave a dedicated interacting partner that directly binds to DNA,although the study of ATLD and Nijmegen breakage syndrome,which are caused by hypomorphic alleles of the Mre11 and Nbs1gene, respectively, suggested that ATM and the MRN complex,known to bind DSBs, were in the same pathway [78–80]. TheMRN complex was soon shown to be important for ATM activa-tion [69,81] though the complex roles of the MRN complex withregards to ATM activation are still being unravelled. Nbs1 is crucialfor the recruitment of ATM to the sites of DNA DSBs [82], whereasthe DSB unwinding and tethering activities of the MRN complexare essential for efficient ATM activation [1,83]. In vitro systemsproved invaluable in confirming a direct interaction between ATMand the MRN complex and the requirements for such an interactionin the activation of ATM. The MRN complex was shown to func-tion as a crucial sensor of DSBs, recruiting dimeric ATM to DSBsand promoting its activation [1]. These studies conducted usingrecombinant proteins have clarified the minimal protein and DNAstructure requirements for ATM activation showing that the MRNcomplex recruits ATM dimers onto dsDNA molecules promotingATM monomer dissociation and autophosphorylation of ATM ser-ine 1981. These studies have also excluded a direct role for serine1981 phosphorylation in ATM activation and have uncovered a rolefor the MRN complex in the initial and limited unwinding of DSBends, linking this process to ATM activation. The results obtainedwith this approach were consistent with the findings obtained inXenopus showing that extracts depleted of the MRN complex wereunable to promote ATM activation [83]. Further studies in Xenopusshowed that the process of ATM activation could be further dis-sected into a two step mechanism concerning the monomerizationof ATM dimers followed by the MRN-dependent activation of theautophosphorylation reaction of ATM on serine 1981 [84].

The MRN complex has also been shown to promote cooper-ative activation of ATM dimer molecules following recruitmentof multiple molecules onto linear DNA [85]. In contrast to datafrom in vitro studies that have predominantly detected ATM dimersbound to linear DNA, in vivo studies suggest that the monomericphosphorylated active form of ATM is bound to chromatin con-taining DSBs generated by a restriction endonuclease [86]. In suchinstances, changes in chromatin structure prior to ATM bindingappear to promote serine 1981 phosphorylation and facilitate ATMmonomerization and binding to DSBs. The nature of such changes tochromatin structure remains to be determined and perhaps in vitrostudies will aid a more unified understanding of these signallingevents. A further discrepancy is the observation that serine 1981phosphorylation seems to be required in vivo for ATM monomeriza-tion and activation in human cells whereas it is dispensable in vitroand in mouse cells [1,86,87]. This difference might be explainedby the presence of an endogenous inhibitor of ATM in human cellsthat becomes dissociated or inactive following serine 1981 phos-
phorylation. It is also possible that alternative low affinity sitescan be phosphorylated in the absence of serine 1981 phosphory-lation contributing to monomerization and activation of ATM andindeed the presence of additional phosphorylation sites has beendemonstrated for ATM [88]. However, ATM mutated in the con-

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erved autophosphorylation sites in mice can still be fully activated89]. Ultimately, while it remains unclear whether the process ofTM activation in vivo is correctly recapitulated in vitro, it is cer-ain that in vitro experiments have prompted a wealth of ingeniousxperiments designed to enhance our understanding of the earlyteps toward ATM activation.

.3.1. The link between Mre11 nuclease activity and ATMctivation

An intriguing facet to the mechanisms that contribute to ATMctivation is the importance of the nucleolytic activity of the MRNomplex, shown to possess both endonucleolytic and exonucle-lytic properties [90]. In fact nuclease deficient complexes of MRNre not able to promote the efficient activation of ATM [81]. MRNndonucleolytic activity has been shown to be involved in the pro-essing of DSBs in a manner termed resection, a process that isnhanced by CtIP (or Sae2 and Ctp1 in S. cerevisiae and S. pombeells), which also possesses nuclease activity [91–94]. Further tohis, a recent screening of chemical compounds highlighted a chem-cal inhibitor of Mre11’s nuclease activity named Mirin. Assays with

irin directly revealed a requirement for Mre11’s nuclease activityn the activation of ATM [95]. Despite these observations it has beenifficult to reconcile how the nuclease activity of the MRN complexight contribute to ATM activation. The MRN complex, togetherith ExoI nuclease, is involved in DSBs processing leading to the for-ation of 3′ overhangs important for homologous recombination

HR) based repair and for ATR activation at DSBs [96]. However, initro the MRN complex has a 3′–5′ polarity that is seemingly incom-atible with the proposed action on the 5′ strand to promote theormation of 3′ overhangs in vivo [96]. This problem could be solvedy the endonuclease activity of the MRN complex, which could beesponsible for the processing of ssDNA that progressively becomesvailable by the action of an associated helicase moving in the direc-ion of the resection. It is also possible that the MRN complex actsust at the end of the DSBs facilitating the access of other nucleasess recent studies suggest [97,98].

The in vivo role of MRN as an endonuclease was clearly shown ineiosis during which it catalyzes Spo11 elimination at sites of mei-

tic breaks [99]. Interestingly, this processing was shown to proceedhrough the formation of single-stranded DNA oligonucleotidesssDNA oligos), which remain covalently attached to Spo11 in mei-tic cells. The formation of such oligos can also be observed inomatic yeast cells in which they can be found covalently attachedo Topoisomerase II though the mechanism by which this occurs isess clear in this case [99]. Mre11 dependent endonucleolytic pro-essing of DNA ends seems to be the prevalent mode of action inivo and this is conserved in lower organisms such as P. furiosus100,101]. The possibility of endonucleolytic DNA processing fol-owing the unwinding of DSBs presents the clear advantage thatesection can proceed past damaged DNA bases in proximity of DSBshat might otherwise block the progression of processive exonu-leases. Recent results suggest that the Sgs1 helicase is involvedn the unwinding of DSBs that are undergoing resection, makingsDNA available for the action of nucleases such as Dna2 [97,98].his process seems to act redundantly with ExoI nuclease. Dna2,ike the MRN complex, seems to act predominantly as an endonu-lease yielding small ssDNA oligos as resection products [102].lthough the principles of resection are now more widely under-tood the manner by which such an activity of MRN (and otherelated endo/exonucleases responsible for DSB processing) is linkedo ATM activity is not yet clear. One possibility might be that MRN

ediated generation of ssDNA at the edge of the DSB is important totart the process of ATM activation. This model would be compati-le with the lack of extensive resection in G1-arrested cells in whichTM can be activated [73]. Recently, ATM activation has been showno require the newly identified ssDNA binding protein hSSB1 [103].

pair 8 (2009) 1025–1037

It is possible that limited ssDNA generated through MRN activity isthen able to recruit hSSB1 to promote full ATM activation explainingthe requirement for this other player in the ATM activation path-way (Fig. 1). In contrast to this data it has been shown that mousecells expressing the Mre11 mutant protein defective for the nucle-ase activity are still capable of ATM activation whereas DNA repair isseverely affected [104]. This suggests that Mre11 mediated process-ing of DNA ends is not an absolute requirement for ATM activation.However, it cannot be excluded that DNA processing mediated bydifferent nucleases such as CtIP or Dna2 replace Mre11’s functionin DSB resection and ATM activation. In addition, it is also possiblethat Mre11 nuclease activity is dispensable for initial activation ofATM but is instead involved in the amplification or maintenance ofATM activity over time.

3.3.2. Products of DNA processingWhile it is clear that the formation of overhang regions at DSBs

is crucial for efficient repair via HR the role of the by-products ofsuch processing in terms of signalling processes have only recentlybeen appreciated using Xenopus laevis egg extract. It was found thatssDNA oligo generated from endonucleolytic processing of DSBsby the MRN complex are able to influence ATM activity. The MRNcomplex appears to be required for ssDNA oligos generation as aconsequence of the resection of DSBs. ssDNA oligos then remainassociated with the MRN complex participating in the activationof ATM in response to DSBs (Fig. 1) [105]. ssDNA oligos producedat DSBs probably interact with one or more subunits of the MRNcomplex that have DNA-binding domains and promote a stableconformation capable of inducing continuous stimulation of ATMmolecules. This process potentially requires the generation of a lim-ited amount of ssDNA oligo molecules bound to MRN complexesand does not require extensive resection to activate a large numberof ATM molecules. The binding of dinucleoside polyphosphates tothe MRN complex through the Rad50 subunit has recently beendemonstrated and suggests that the activities of the MRN com-plex are regulated by different polynucleotide metabolites [106].MRN-ssDNA oligo complexes may increase the activity of ATMmolecules already bound to DSBs or facilitate the activation of inac-tive ATM molecules that have not yet engaged with DSBs. However,the absence of ATM-dependent phosphorylation of histone H2AXtriggered by ssDNA oligos alone suggests that ssDNA oligos are notsufficient to enable ATM-dependent phosphorylation of chromatin-bound targets [105]. This event probably requires the presence ofdsDNA ends onto which ATM can load [107]. Initial MRN mediatedprocessing of DSBs could promote partial activation of ATM facili-tating its monomerization and DNA binding. Alternatively, ssDNAoligos may bind other ssDNA binding proteins such as hSSB1, whichhave high affinity for short ssDNA oligos and mediate full ATM acti-vation [108]. It would be interesting to examine whether ssDNAoligos are produced under conditions that lead to autophosphory-lation of ATM in the absence of DSBs such as following structuralchromatin changes [76]. In these case ssDNA oligos could be formedvia alternative routes, for example following the processing of DNAreplication intermediates at stalled replication forks in the absenceof DSBs.

3.3.3. ssDNA oligos and DNA damage responseIt is likely that the elimination of ssDNA oligos would be required

for efficient inactivation of ATM once DSBs have been repaired,whereas the persistence of ssDNA oligos would maintain ATMactive. Interestingly, the deficiency of Trex1 exonuclease activity
in human cells leads to the accumulation of free ssDNA oligosand this correlates with chronic stimulation of the ATM-dependentDNA damage response [109]. The creation of ssDNA oligos duringthe resection of DNA undergoing repair, either from 5′ to 3′ pro-cessing of DSBs or possibly from enlarging gaps in other forms of

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sDNA oligos form only during DNA damage processing and repre-ent ideal alarm molecules signalling the presence of severe DNAamage. ssDNA oligos can also be observed following induction ofSBs in human cells, indicating that this is a conserved physiologi-al phenomenon [73]. On the basis of these findings it is plausible topeculate that ssDNA generated by limited unwinding of DSBs stim-lates the initial activation of ATM whereas ssDNA oligos arisingrom DNA processing facilitates the amplification and maintenancef ATM dependent signalling. This idea would be compatible withhe two-step model for ATM activation [84]. Notably, small ssDNAligos have been shown to activate the DNA damage response whenntroduced into human skin cells mimicking the protecting reac-ions that take place following sun light exposure [110,111]. It woulde interesting to know whether this process is also conserved in

ower organisms that lack such complex DNA damage signallingathways but that are capable of mounting a DNA damage responseuch as the SOS response [112]. Following chromosome breakagen E. coli, DNA is processed by the RecBCD complex that possesseselicase and endo/exonuclease activity [113]. Studies in vitro havehown that the SOS response can be recapitulated in the presence ofNA and active RecBCD leading to the generation of ssDNA capablef activating RecA mediated disruption of the SOS repressor LexA114]. Like hSSB1, RecA contains a ssDNA-binding domain capable ofnteracting with ssDNA. Small ssDNA oligos interacting with RecAtimulate RecA mediated inactivation of LexA [115,116]. ThereforeecA might sense ssDNA oligos produced by DNA processing as wells large regions of ssDNA arising at the site of resection triggeringhe activation of the SOS response. Although speculative, this mightepresent an interesting parallel to be explored that would estab-ish diffusible ssDNA oligos as a primordial signal of DNA damage.ltimately, it is clear that experiments using Xenopus egg extract

ystems in particular have been fundamental in aiding our under-tanding of the requirements for ATM activation, the intricacies ofhich are still being unravelled.

. Mediating and transducing checkpoint responses

Two of the most prominent effector proteins of DNA dam-ge are the protein kinases Chk1 and Chk2. DNA damage leadso the rapid phosphorylation of Chk2 by ATM [117]. This is thenollowed by further autophosphorylation facilitated by the fork-ead associated (FHA) domain of Chk2 [118]. FHA domains arehospho-specific protein–protein interaction domains [119,120].here is considerable importance for the FHA domain in termsf the phosphorylation of Chk2 and as such it is speculated thathere is a mediator protein involved that must be recognised by theHA domain. The ‘mediator of DNA damage checkpoint’ protein,DC1, is thought to play this role though evidence also exists for

he importance of 53BP1 as mediator and in fact the two bear sev-ral similarities. Both MDC1 and 53BP1 were first identified using initro binding assays as p53 interacting proteins [121] and are bothhosphorylated by ATM. Both proteins demonstrate physical andunctional interaction with phosphorylated histone H2AX. Supportf 53BP1’s mediatory role comes from the fact that in responseo ionising radiation, 53BP1 binds ATM and this binding is nec-ssary for subsequent ATM-dependent phosphorylation of Chk2122]. MDC1’s role may be somewhat more complex since MDC1lso interacts with the MRN complex and strengthens ATM bind-
ng to DSB lesions [123]. In addition to the potential role of MDC1nd 53BP1 in feedback loops to increase ATM activity in responseo DNA damage these proteins appear to mediate signal from sen-or to effector i.e. ATM to Chk2. Recently many more players withomplex roles have emerged in the pathway that leads to signal
pair 8 (2009) 1025–1037 1031

amplification at DSBs. Among these there are RNF8 and Ubc13responsible for histone ubiquitination and chromatin modificationsfollowing recruitment on DSBs mediated by phosphorylated MDC1.These modifications lead to the recruitment of ubiquitin bindingmolecules such as Rap80, Abraxas and BRCA1. All these moleculesare probably required for different tasks such as DSB processingand repair or efficient activation of cellular checkpoints (reviewedin Ref. [124]). Reconstitution of this pathway using in vitro systemswould undoubtedly aid a better understanding of their function.

As described, while Chk2 is downstream of ATM, Chk1 is themajor effector for signal transmission from ATR to cell cycle regula-tory substrates. Chk1 plays a pivotal role in coordinating a G2/Mcheckpoint and may be important in replication fork recoveryand monitoring origin firing [63,125,126]. Ultimately, the effectorroles of Chk1 and Chk2 may overlap but they display individualcharacteristics of function that are distinct from one another. Theunderstanding of downstream events following Chk1 and Chk2activation is limited due to the fact that only a few targets of thesekinases have been identified so far. Due to the importance of Chk1and Chk2 in mediating checkpoint responses it will be essential toidentify their substrates to better understand their role.

5. In vitro studies on the roles of ATM and ATR inmaintaining genome stability

5.1. ATM and ATR mediated regulation of S-phase progression

A major response following the DNA damage-induced activa-tion of ATM and ATR and the subsequent activation of the effectorkinases Chk1 and Chk2 is cell cycle arrest. Phosphorylation andstabilization of p53 and the increased transcription of various tar-gets of p53’s transcriptional activity plays a major role in this event[127]. Increased transcription of the p21 gene product is an impor-tant facet of maintenance of the G1/S checkpoint as p21 proteinis a broad range Cdk inhibitor and as a consequence of inhibitionof Cdk activity prevents pRb phosphorylation and thus transitioninto S-phase. However, this stream of signalling is not the onlymeans by which G1/S checkpoint arrest is achieved in vertebrates.During S-phase the cellular genome is highly vulnerable to dam-age, particularly from replication fork inhibition or collapse, andit is important that the cell be prepared to arrest and be primedfor repair. Transcription mediated responses appear to be too slowto counteract sudden DNA damage occurring prior to or during S-phase. Therefore a more rapid, transcription-independent pathwayhalting S-phase is more suited for this rapid response. Responses toDNA damage can in fact be observed within minutes of its occur-rence but it was once unclear whether non-transcriptional, morerapid ATM responses were activated to achieve a G1/S checkpointarrest. Xenopus egg extract, in which transcription is absent, pointedat the existence of such ATM dependent responses that facilitatethe down-regulation of Cdk2 activity and consequently G1/S-phasearrest in the absence of transcription [34]. This simplified system,capable of recapitulating a G1/S checkpoint response allowed theprecise molecular dissection of an ATM-dependent pathway thatinhibits the loading of Cdc45 at replication origins via inhibitionof cyclin E/Cdk2 activity [34]. In particular it was demonstratedthat ATM activation leads to the inhibition of the CDC25A phos-phatase that is usually responsible for removal of the inhibitorytyrosine 15 phosphorylation on Cdk2. ATM and Chk2 dependentinhibition of CDC25A and Cdk2 activity was subsequently shown
to affect initiation of DNA replication also in the context of mam-malian cell responses [128]. The elucidation of this checkpoint wasachieved through direct activation of ATM by the addition of lin-ear DNA fragments mimicking DSBs to egg extract followed by anelaborate purification scheme to isolate the biochemical fractions

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ble to impose the block on DNA replication initiation. This strategyllowed the separation of the activation from the effector steps ofhis ATM dependent checkpoint [34].

Furthermore, experiments in the Xenopus system have revealedTR and RPA dependent mechanisms to prevent origin firing andhus DNA replication in the presence of DNA damage [129]. Thexistence of both ATM and ATR dependent blocks to origin firingere further confirmed by studies that employed the use of restric-

ion enzymes to generate lesions in the replicating templates [130].tudies in Xenopus also revealed that the ATM and ATR checkpointsegulate origin firing even in the absence of apparent DNA dam-ge [63]. As far as the targets of this checkpoint are concerned initro work in combination with observations in other systems haveevealed a plethora of recipients of checkpoint signalling such asbs1, the structural maintenance of chromosomes protein SMC1,RCA1, and FANCD2 in addition to the Chk2-CDC25A mediated

nhibition of Cdk2 activity [131–134]. The implication of these phos-horylation events on the activity of the single protein is far fromeing understood and will surely gain advantage from the develop-ent of in vitro assays recapitulating these pathways.

.2. The role of ATM and ATR in maintaining replication forktability

As previously discussed, experiments with conditional knock-ut mice have shown that ATR is an essential gene. Cells lackingTR undergo chromosome fragmentation during S-phase and sub-equent cell death in the absence of apparent exogenous sources ofNA damage [42]. In fact cells are highly sensitive even to partialTR deficiency, as shown by the Seckel syndrome phenotype [135].TR is essential to maintain chromosome integrity by promot-

ng stabilization of replication forks. Cells with a mutant allele ofec1, the ATR ortholog in budding yeast, accumulate chromosomal

reaks [136]. Mec1 probably prevents replication fork collapse byodulating Rad53 kinase activity required to stabilize stalled repli-

ation forks [137–139]. Recently, the extensive crosstalk between
TM and ATR in maintaining replication fork stability has beenppreciated [72]. In addition to the proposed role of fork stabiliza-ion, the use of Xenopus extracts revealed that ATM and ATR areoth required to prevent DSB accumulation during DNA replica-ion by promoting the restart of collapsed replication forks [140].
ig. 2. The role of ATM, ATR and the MRN complex in replication fork stability. ATM and ATy stabilizing and promoting restart of collapsed replication forks. The MRN complex facight tether DNA fragments at collapsed forks stabilizing them and promoting function

olymerase at stalled forks and facilitate reloading of Pol � onto restarting replication forksrigin firing and entry into mitosis. This process is overcome in the by Plx1 recruitment ontn the case of polymerase inhibition the Tim–Tipin complex, as opposed to ATR and ATR,

pair 8 (2009) 1025–1037

In order to examine the specific roles of ATM and ATR in repair-ing and restarting disrupted replication forks, assays using Xenopusegg extract to monitor recovery of stalled and collapsed replica-tion forks were developed. One such assay involves two steps: areplication inhibition step followed by a recovery step. Essentiallychromatin is introduced into a replication competent extract thathas been depleted of ATM or ATR. Replication fork collapse is theninduced by different DNA damaging agents before chromatin istransferred to restarting extracts. If restarting extracts lack ATMor ATR, the resulting replication fork recovery is deficient [140].As such these data have led to a model in which ATM and ATRare integral parts of the moving replication fork that facilitate nor-mal fork progression by preventing replication fork collapse and byprompting repair and restart of collapsed forks (Fig. 2).

In terms of the molecular mechanism behind replication forkcollapse and recovery in lower eukaryotes it has been shown thatin the event of replication fork collapse, replication proteins suchas DNA polymerases and components of the MCM complex are dis-assembled from the collapsed fork [138,141]. However, in Xenopusegg extract, even in the absence of ATM and ATR, MCM proteins arenot removed from chromatin in the event of DNA damage [140].Higher eukaryotes differ from yeast for example, in that multipleMCM complexes are loaded onto chromatin for every active originleading to considerable redundancy [142]. Such redundancy maybe of significance for restart of collapsed forks without reassem-bling new replication complexes. In contrast to the behavior of MCMproteins, but rather analogously to the yeast system, in Xenopusin the absence of ATM or ATR the polymerase Pol � is lost fromreplication forks that have been damaged [140]. It is known thatPol � is required for efficient DNA replication, DNA repair, and cellcycle checkpoint control in eukaryotic cells and indeed Xenopus eggextracts that are depleted of Pol � are unable to assemble a properDNA synthesis machinery at the replication fork [143]. In yeast,Mec1 has been shown to be important for maintaining the pres-ence of Pol � at stalled replication forks [144]. Results from Xenopusextract systems have demonstrated that both ATM and ATR promote
the reloading of Pol � onto recovering replication forks thus reveal-ing a fundamental link between ATM, ATR and Pol � in maintainingreplication fork stability. Consistent with these results, checkpointdeficient cells have been shown to be defective in re-establishingDNA synthesis on damaged leading strand templates [145]. It is also
R are required to prevent double-strand break accumulation during DNA replicationilitates the activation of ATM and also localises to stalled forks. The MRN complexal fork reassembly. The activity of ATM and ATR is thought to help to maintain the. Furthermore, ATR mediated activation of Chk1 following replication stress inhibitso stalled replication forks that facilitate de-repression and firing of dormant origins.plays a fundamental role in maintaining fork structure.

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nteresting to point out that Pol � is directly phosphorylated by bothTM and ATR and it will be important to assess the functional role ofhis phosphorylation [146]. A further link between Mrc1, a check-oint protein required for replication fork stability and Pol � hasecently been established [147].

.3. MRN dependent replication fork stabilization and restart

All components of the MRN complex are essential for lifehereas cells tolerate ATM deficiency [148–151]. This suggests

hat the MRN complex harbours additional functions in additiono its role in ATM activation. One such example is the observed

re11 localisation to stalled replication forks suggesting a roleor Mre11 during DNA replication [152]. Experiments in Xenopusemonstrated the requirement for the MRN complex in prevent-

ng the accumulation of DSBs during DNA replication [153]. Thesebservations indicated that the MRN complex is not only requiredo respond to DSBs but also to resolve aberrant structures thatrise during normal DNA replication that may themselves lead tohe formation of DSBs. Interestingly, Mre11 is phosphorylated incaffeine-sensitive manner during S-phase in Xenopus egg extract

ndicating that Mre11 is normally under the control of ATM and ATRven in the absence of detectable DNA damage [153]. Recent obser-ations clearly showed that the MRN complex is required to restartollapsed replication forks and this activity is influenced by ATMnd ATR [140]. ATM, ATR and the MRN complex might promote theecapture of broken DNA molecules at collapsed replication forksacilitating fork reassembly. This hypothesis would be compatibleith the observations that the MRN complex is involved in the teth-

ring of DNA fragments [83,154,155]. Alternatively, it is possiblehat ATM, ATR and the MRN complex prevent the collapse of stalledorks stabilizing the DNA replication intermediates. As such it can bepeculated that the essential role of the MRN complex in vertebratesight be to prevent the collapse of stalled replication forks and to

romote the restart of replication forks that have already collapsed.role for the Mre11 in the restart of replication forks is consistentith elegant structural studies that revealed how Mre11 dimers are

apable of recognizing and bridging ssDNA–dsDNA structures typ-cally present at collapsed or restarting replication forks [101]. Inddition spontaneous DNA aberrations arising during S-phase inhe absence of Mre11 nuclease activity suggest an active role forhe MRN complex in preventing genome instability during chro-

osome replication. Taken together these results indicate thathe common features observed in the phenotypes of A-T, ATLDnd Seckel syndrome (in which ATM, Mre11 or ATR are deficient,espectively) might be explained by the accumulation of collapsedeplication forks during embryonic DNA replication. Conversely, it islso important to highlight that during embryonic DNA replicationn Xenopus, when replication forks stall in the absence of physicalamage in the template via the inhibition of polymerase activityy aphidicolin ATM and ATR are not required for replication forktabilization and restart [140]. This is different from somatic cellsnd might point at a fundamental difference in the way embry-nic replication machinery overcomes DNA replication stress. Itould appear that in such instances of polymerase inhibition alter-ative mechanisms are employed for replication fork restart. Inhese conditions the Tim–Tipin complex, which is associated withhe replicative helicase [156], seems to play a fundamental role in

aintaining the integrity of the replication fork structure [67].

. Adaptation to the checkpoint signal and genome stability

Eukaryotic cells make use of multiple origins of replicationuring S-phase to ensure the complete replication of all DNAuring a single S-phase prior to the onset of mitosis. DNA repli-

pair 8 (2009) 1025–1037 1033

cation is achieved through the coordinated activation of multiplereplication origins distributed throughout the DNA. Replication ini-tiation requires both Cdk2- and Cdc7-mediated activation of thepre-replicative complexes to unwind the DNA and to start DNApolymerization [157]. The process of DNA replication is tightly reg-ulated to prevent genetic aberrations and any perturbations toreplication fork progression induce the potent activation of ATMand ATR kinases. In metazoan organisms the number of replicationorigins available exceeds the number of origins effectively used forthe replication of a chromosomal region. This correlates with anexcess of MCM complex that makes up part of the pre-replicativecomplex bound to DNA [142]. The excess loading of MCM complexesrepresents an important mode for maintaining genomic stabilityin the event of DNA damage or replicative stress. The recruit-ment of supplementary replication origins, which would otherwiseremain dormant, in chromosomal regions affected by the presenceof stalled or collapsed replication forks seems to ensure effectivereplication of damaged DNA contributing to the maintenance ofgenome stability [158–160]. Importantly, for supplementary originactivation to take place a transient suppression of the intra-S-phasecheckpoint that maintains supplementary origins inactive is neces-sary [158].

It was previously unclear what caused the transient suppres-sion of the checkpoint allowing the recruitment of dormant originssince DNA damage further stimulates the activation of ATM andATR, which normally inhibit the firing of redundant origins andmaintain them in a dormant state [63]. The mechanism that turnsoff the checkpoint signal during S-phase to allow completion ofDNA replication through the recruitment of dormant supplemen-tary origins and MCM complexes was studied using Xenopus eggextract and was shown to require the Polo-like kinase, Plx1 [161].It was known that unicellular organisms can overcome checkpointresponse signals by a process known as adaptation, in which yeastCdc5 was shown to be essential [162]. Adaptation was believedto play a minor role in higher organisms because it appears toallow the potentially dangerous bypass of DNA damage signalsenabling the propagation of DNA damage to dividing cells. Instead,adaptation to the ATR-dependent checkpoint response that inhibitsmitotic entry in the presence of unreplicated DNA was demon-strated in vertebrate organisms such as Xenopus laevis and wasshown to be regulated by Plx1 [163]. In particular it was demon-strated that ATR-dependent phosphorylation of Claspin creates adocking site for Plx1, which phosphorylates Claspin and induces itsdisplacement from chromatin promoting the attenuation of Chk1activity [163]. This process allows progression into mitosis even inthe presence of unreplicated DNA. Consistent with the role of Plx1in mitosis, Plx1 was shown to attenuate checkpoint signals duringDNA replication in the presence of DNA damage and stalled repli-cation forks. This attenuation results in the de-repression of originfiring otherwise suppressed by ATM and ATR allowing recruitmentof supplementary dormant origins [161]. Essential for such Plx1mediated function is the ATR and ATM-dependent phosphoryla-tion of MCM2 [130,164], which promotes Plx1 binding to the MCMcomplex through the Polo box domain [161]. This suggests a directcoordination of MCM activity in the presence of DNA lesions activat-ing an intra-S-phase checkpoint. This mechanism works at the levelof stalled replication forks leading to the release of Chk1-mediatedsuppression of nearby dormant origins (Fig. 2) [161]. It remainsunclear how Plx1 suppresses Chk1 activity in this process. Phos-phorylation of adaptors required for Chk1 activation such as Tipin,which has been shown to be required for Chk1 activity [67], might
be involved in this process. Overall these data suggest that MCM-recruited Plx1 promotes replication progression in the presence ofreplication stress [161], and that Plx1-mediated Claspin inactiva-tion is necessary to regulate the transition from S-phase to mitosis[165]. Intriguingly, DNA replication in the absence of Plx1 leads to

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he accumulation of DNA DSBs further supporting the role of Plx1n promoting genome stability during S-phase.

. The future of in vitro systems for investigatingheckpoint responses

In vitro experimental models, particularly Xenopus egg extract,rovide a versatile tool within which to examine many aspects ofheckpoint activation, regulation and control. Observations madesing such in vitro models have so far proven to represent the anal-gous systems and mechanisms present in mammalian systems.any of the aspects of checkpoint signalling might not yet have

een uncovered if not for the flexibility of such in vitro systems thatllow the study of the specific details of rapid checkpoint responses.ecent studies revealed that many ATM and ATR substrates are

nvolved in DNA replication and DNA repair/recombination [146].s genetic studies with knockout/knock-in approaches are time-onsuming and difficult to perform, in vitro studies using purifiedroteins with mutated phosphorylation sites will be helpful tonderstand the effects of these phosphorylations on the activity ofingle proteins. Where these phosphorylation sites are conserved,he Xenopus system will be useful to study the effects of ATMnd ATR phosphorylation in a more complex biological contexty replacing endogenous protein complexes with proteins bear-ng mutated sites. For this, novel assays will need to be developedo understand the intricacy of ATM and ATR mediated regulationf basic processes such as DNA replication and DNA repair. Inter-stingly, phosphorylation studies have revealed the presence ofTM and ATR targets involved in the regulation of critical cellularrocesses such as mitotic progression, spindle assembly, micro-ubule dynamics, oxidative stress responses, energy metabolism,nd many others in addition to DNA replication and DNA repair.he Xenopus system might be useful to dissect the effects of thesehosphorylations as it has already proven to be an extremely pow-rful tool for aiding our understanding of some of these complexiological events [21,166]. In conclusion it is clear that our exploita-ion of in vitro systems is just at the beginning and will surely bringew and exciting discoveries in the field of DNA damage responses.

cknowledgements

We thank members of the Clare Hall Laboratories and of theenome Stability Unit for helpful discussions. The Genome Stabilitynit is funded by Cancer Research UK, the Lister Institute for Preven-

ive Medicine, the EMBO Yip program and the European Researchouncil (ERC) start up grant for young investigators awarded to VC.

eferences

[1] J.H. Lee, T.T. Paull, ATM activation by DNA double-strand breaks through theMre11-Rad50-Nbs1 complex, Science 308 (2005) 551–554.

[2] J.H. Lee, T.T. Paull, Activation and regulation of ATM kinase activity in responseto DNA double-strand breaks, Oncogene 26 (2007) 7741–7748.

[3] J. Majka, S.K. Binz, M.S. Wold, P.M. Burgers, Burgers Replication protein Adirects loading of the DNA damage checkpoint clamp to 5′-DNA junctions,J. Biol. Chem. 281 (2006) 27855–27861.

[4] J. Majka, A. Niedziela-Majka, P.M. Burgers, The checkpoint clamp activatesMec1 kinase during initiation of the DNA damage checkpoint, Mol. Cell 24(2006) 891–901.

[5] V.P. Bermudez, L.A. Lindsey-Boltz, A.J. Cesare, Y. Maniwa, J.D. Griffith, J. Hur-witz, A. Sancar, Loading of the human 9-1-1 checkpoint complex onto DNAby the checkpoint clamp loader hRad17-replication factor C complex in vitro,Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 1633–1638.

[6] L.A. Lindsey-Boltz, V.P. Bermudez, J. Hurwitz, A. Sancar, Purification and char-acterization of human DNA damage checkpoint Rad complexes, Proc. Natl.Acad. Sci. U.S.A. 98 (2001) 11236–11241.

[7] J.H. Choi, L.A. Lindsey-Boltz, A. Sancar, Reconstitution of a human ATR-mediated checkpoint response to damaged DNA, Proc. Natl. Acad. Sci. U.S.A.104 (2007) 13301–13306.

[8] S.J. Araujo, E.A. Nigg, R.D. Wood, Strong functional interactions of TFIIH withXPC and XPG in human DNA nucleotide excision repair, without a preassem-bled repairosome, Mol. Cell Biol. 21 (2001) 2281–2291.

pair 8 (2009) 1025–1037

[9] M. Biggerstaff, R.D. Wood, Assay for nucleotide excision repair protein activityusing fractionated cell extracts and UV-damaged plasmid DNA, Methods Mol.Biol. 113 (1999) 357–372.

[10] M.K. Shivji, J.G. Moggs, I. Kuraoka, R.D. Wood, Dual-incision assays fornucleotide excision repair using DNA with a lesion at a specific site, MethodsMol. Biol. 113 (1999) 373–392.

[11] Y. Kubota, R.A. Nash, A. Klungland, P. Schar, D.E. Barnes, T. Lindahl, Reconsti-tution of DNA base excision-repair with purified human proteins: interactionbetween DNA polymerase beta and the XRCC1 protein, EMBO J. 15 (1996)6662–6670.

[12] J. Hansson, L. Grossman, T. Lindahl, R.D. Wood, Complementation of the xero-derma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABCproteins in a cell-free system, Nucleic Acids Res. 18 (1990) 35–40.

[13] J. Gerhart, M. Wu, M. Cyert, M. Kirschner, M-phase promoting factors fromeggs of Xenopus laevis, Cytobios 43 (1985) 335–347.

[14] J. Gerhart, M. Wu, M. Kirschner, Cell cycle dynamics of an M-phase-specificcytoplasmic factor in Xenopus laevis oocytes and eggs, J. Cell Biol. 98 (1984)1247–1255.

[15] T. Evans, E.T. Rosenthal, J. Youngblom, D. Distel, T. Hunt, Cyclin: a protein spec-ified by maternal mRNA in sea urchin eggs that is destroyed at each cleavagedivision, Cell 33 (1983) 389–396.

[16] R.H. Chen, A. Murray, Characterization of spindle assembly checkpoint inXenopus egg extracts, Methods Enzymol. 283 (1997) 572–584.

[17] J.J. Blow, S.M. Dilworth, C. Dingwall, A.D. Mills, R.A. Laskey, Chromosome repli-cation in cell-free systems from Xenopus eggs, Philos. Trans. R. Soc. Lond. B:Biol. Sci. 317 (1987) 483–494.

[18] J.J. Blow, R.A. Laskey, Initiation of DNA replication in nuclei and purified DNAby a cell-free extract of Xenopus eggs, Cell 47 (1986) 577–587.

[19] C.J. Hutchison, R. Cox, R.S. Drepaul, M. Gomperts, C.C. Ford, Periodic DNAsynthesis in cell-free extracts of Xenopus eggs, EMBO J. 6 (1987) 2003–2010.

[20] M.J. Lohka, Y. Masui, Formation in vitro of sperm pronuclei and mitotic chro-mosomes induced by amphibian ooplasmic components, Science 220 (1983)719–721.

[21] A. Desai, A. Murray, T.J. Mitchison, C.E. Walczak, The use of Xenopus eggextracts to study mitotic spindle assembly and function in vitro, Methods CellBiol. 61 (1999) 385–412.

[22] T. Stearns, M. Kirschner, In vitro reconstitution of centrosome assembly andfunction: the central role of gamma-tubulin, Cell 76 (1994) 623–637.

[23] R. Miake-Lye, M.W. Kirschner, Induction of early mitotic events in a cell-freesystem, Cell 41 (1985) 165–175.

[24] G. Almouzni, M. Mechali, Xenopus egg extracts: a model system for chromatinreplication, Biochim. Biophys. Acta 951 (1988) 443–450.

[25] A.W. Murray, Cell cycle extracts, Methods Cell Biol. 36 (1991) 581–605.[26] J. Gautier, J. Minshull, M. Lohka, M. Glotzer, T. Hunt, J.L. Maller, Cyclin is a com-

ponent of maturation-promoting factor from Xenopus, Cell 60 (1990) 487–494.[27] J. Walter, L. Sun, J. Newport, Regulated chromosomal DNA replication in the

absence of a nucleus, Mol. Cell 1 (1998) 519–529.[28] D. Dominguez-Sola, C.Y. Ying, C. Grandori, L. Ruggiero, B. Chen, M. Li, D.A.

Galloway, W. Gu, J. Gautier, R. Dalla-Favera, Non-transcriptional control ofDNA replication by c-Myc, Nature 448 (2007) 445–451.

[29] A. Kumagai, W.G. Dunphy, Control of the Cdc2/cyclin B complex in Xenopusegg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors,Mol. Biol. Cell 6 (1995) 199–213.

[30] M. Dasso, J.W. Newport, Completion of DNA replication is monitored by a feed-back system that controls the initiation of mitosis in vitro: studies in Xenopus,Cell 61 (1990) 811–823.

[31] J. Greenwood, V. Costanzo, K. Robertson, C. Hensey, J. Gautier, Responses toDNA damage in Xenopus: cell death or cell cycle arrest, Novartis Found Symp.237 (2001) 221–230 (discussion 230–224).

[32] Z. Guo, W.G. Dunphy, Response of Xenopus Cds1 in cell-free extracts toDNA templates with double-stranded ends, Mol. Biol. Cell 11 (2000) 1535–1546.

[33] C.W. Conn, A.L. Lewellyn, J.L. Maller, The DNA damage checkpoint in embryoniccell cycles is dependent on the DNA-to-cytoplasmic ratio, Dev. Cell 7 (2004)275–281.

[34] V. Costanzo, K. Robertson, C.Y. Ying, E. Kim, E. Avvedimento, M. Gottesman,D. Grieco, J. Gautier, Reconstitution of an ATM-dependent checkpoint thatinhibits chromosomal DNA replication following DNA damage, Mol. Cell 6(2000) 649–659.

[35] Z. Guo, A. Kumagai, S.X. Wang, W.G. Dunphy, Requirement for Atr in phos-phorylation of Chk1 and cell cycle regulation in response to DNA replicationblocks and UV-damaged DNA in Xenopus egg extracts, Genes Dev. 14 (2000)2745–2756.

[36] C.A. MacDougall, T.S. Byun, C. Van, M.C. Yee, K.A. Cimprich, The structuraldeterminants of checkpoint activation, Genes Dev. 21 (2007) 898–903.

[37] M. Dasso, C. Smythe, K. Milarski, S. Kornbluth, J.W. Newport, DNA replicationand progression through the cell cycle, Ciba Found Symp. 170 (1992) 161–180(discussion 180–166).

[38] J. Newport, M. Dasso, On the coupling between DNA replication and mitosis,J. Cell Sci. 12 (Suppl.) (1989) 149–160.

[39] R.T. Abraham, Cell cycle checkpoint signaling through the ATM and ATRkinases, Genes Dev. 15 (2001) 2177–2196.

[40] K.A. Cimprich, D. Cortez, ATR: an essential regulator of genome integrity, Nat.Rev. Mol. Cell Biol. 9 (2008) 616–627.

[41] A. de Klein, M. Muijtjens, R. van Os, Y. Verhoeven, B. Smit, A.M. Carr, A.R.Lehmann, J.H.J. Hoeijmakers, Targeted disruption of the cell-cycle checkpoint

NA Re
gene ATR leads to early embryonic lethality in mice, Curr. Biol. 10 (2000)479–482.

[42] E.J. Brown, D. Baltimore, ATR disruption leads to chromosomal fragmentationand early embryonic lethality, Genes Dev. 14 (2000) 397–402.

[43] D. Cortez, S. Guntuku, J. Qin, S.J. Elledge, ATR and ATRIP: partners in checkpointsignaling, Science 294 (2001) 1713–1716.

[44] G.K. Alderton, H. Joenje, R. Varon, A.D. Borglum, P.A. Jeggo, M. O’Driscoll,Seckel syndrome exhibits cellular features demonstrating defects in the ATR-signalling pathway, Hum. Mol. Genet. 13 (2004) 3127–3138.

[45] Y. Shiloh, ATM and ATR: networking cellular responses to DNA damage, Curr.Opin. Genet. Dev. 11 (2001) 71–77.

[46] L. Zou, S.J. Elledge, Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes, Science 300 (2003) 1542–1548.

[47] V. Costanzo, J. Gautier, Single-strand DNA gaps trigger an ATR- and Cdc7-dependent checkpoint, Cell Cycle 2 (2003) 17.

[48] T.S. Byun, M. Pacek, M.C. Yee, J.C. Walter, K.A. Cimprich, Functional uncouplingof MCM helicase and DNA polymerase activities activates the ATR-dependentcheckpoint, Genes Dev. 19 (2005) 1040–1052.

[49] P.J. Lupardus, C. Van, K.A. Cimprich, Analyzing the ATR-mediated checkpointusing Xenopus egg extracts, Methods 41 (2007) 222–231.

[50] M. Raschle, P. Knipsheer, M. Enoiu, T. Angelov, J. Sun, J.D. Griffith, T.E. Ellen-berger, O.D. Scharer, J.C. Walter, Mechanism of replication-coupled DNAinterstrand crosslink repair, Cell 134 (2008) 969–980.

[51] L. Zou, D. Liu, S.J. Elledge, Replication protein A-mediated recruitment andactivation of Rad17 complexes, Proc. Natl. Acad. Sci. U.S.A. 100 (2003)13827–13832.

[52] V. Ellison, B. Stillman, Biochemical characterization of DNA damage check-point complexes: clamp loader and clamp complexes with specificity for 5′

recessed DNA, PLoS Biol. 1 (2003) E33.[53] S. Delacroix, J.M. Wagner, M. Kobayashi, K. Yamamoto, L.M. Karnitz, The Rad9-

Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1, GenesDev. 21 (2007) 1472–1477.

[54] J. Lee, A. Kumagai, W.G. Dunphy, The Rad9-Hus1-Rad1 checkpoint clamp reg-ulates interaction of TopBP1 with ATR, J. Biol. Chem. 282 (2007) 28036–28044.

[55] A. Kumagai, J. Lee, H.Y. Yoo, W.G. Dunphy, TopBP1 activates the ATR–ATRIPcomplex, Cell 124 (2006) 943–955.

[56] L.I. Toledo, M. Murga, P. Gutierrez-Martinez, R. Soria, O. Fernandez-Capetillo,ATR signaling can drive cells into senescence in the absence of DNA breaks,Genes Dev. 22 (2008) 297–302.

[57] D.A. Mordes, G.G. Glick, R. Zhao, D. Cortez, TopBP1 activates ATR through ATRIPand a PIKK regulatory domain, Genes Dev. 22 (2008) 1478–1489.

[58] R.A. Van Hatten, A.V. Tutter, A.H. Holway, A.M. Khederian, J.C. Walter, W.M.Michael, The Xenopus Xmus101 protein is required for the recruitment ofCdc45 to origins of DNA replication, J. Cell Biol. 159 (2002) 541–547.

[59] Y. Hashimoto, T. Tsujimura, A. Sugino, H. Takisawa, The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activationof Chk1, Genes Cells 11 (2006) 993–1007.

[60] W.M. Michael, R. Ott, E. Fanning, J. Newport, Activation of the DNA replicationcheckpoint through RNA synthesis by primase, Science 289 (2000) 2133–2137.

[61] Y. Sanchez, C. Wong, R.S. Thoma, R. Richman, Z. Wu, H. Piwnica-Worms, S.J.Elledge, Conservation of the Chk1 checkpoint pathway in mammals: linkage ofDNA damage to Cdk regulation through Cdc25, Science 277 (1997) 1497–1501.

[62] B. Furnari, N. Rhind, P. Russell, Cdc25 mitotic inducer targeted by chk1 DNAdamage checkpoint kinase, Science 277 (1997) 1495–1497.

[63] D. Shechter, V. Costanzo, J. Gautier, ATR and ATM regulate the timing of DNAreplication origin firing, Nat. Cell Biol. 6 (2004) 648–655.

[64] A. Kumagai, W.G. Dunphy Claspin, A novel protein required for the activationof Chk1 during a DNA replication checkpoint response in Xenopus egg extracts,Mol. Cell 6 (2000) 839–849.

[65] A. Kumagai, W.G. Dunphy, Repeated phosphopeptide motifs in Claspin medi-ate the regulated binding of Chk1, Nat. Cell Biol. 5 (2003) 161–165.

[66] J. Lee, A. Kumagai, W.G. Dunphy, Claspin, a Chk1-regulatory protein, monitorsDNA replication on chromatin independently of RPA, ATR, and Rad17, Mol. Cell11 (2003) 329–340.

[67] A. Errico, V. Costanzo, T. Hunt, Tipin is required for stalled replication forks toresume DNA replication after removal of aphidicolin in Xenopus egg extracts,Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 14929–14934.

[68] S.M. Kim, A. Kumagai, J. Lee, W.G. Dunphy, Phosphorylation of Chk1 by ATM-and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP toATR but not the stable DNA-binding or coiled-coil domains of ATRIP, J. Biol.Chem. 280 (2005) 38355–38364.

[69] C.T. Carson, R.A. Schwartz, T.H. Stracker, C.E. Lilley, D.V. Lee, M.D. Weitzman,The Mre11 complex is required for ATM activation and the G2/M checkpoint,EMBO J. 22 (2003) 6610–6620.

[70] H.Y. Yoo, S.Y. Jeong, W.G. Dunphy, Site-specific phosphorylation of a check-point mediator protein controls its responses to different DNA structures,Genes Dev. 20 (2006) 772–783.

[71] H.Y. Yoo, A. Kumagai, A. Shevchenko, A. Shevchenko, W.G. Dunphy, Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs throughphosphorylation of TopBP1 by ATM, J. Biol. Chem. 282 (2007) 17501–17506.

[72] A. Jazayeri, J. Falck, C. Lukas, J. Bartek, G.C. Smith, J. Lukas, S.P. Jackson, ATM-and cell cycle-dependent regulation of ATR in response to DNA double-strandbreaks, Nat. Cell Biol. 8 (2006) 37–45.

[73] A. Jazayeri, J. Falck, C. Lukas, J. Bartek, G.C. Smith, J. Lukas, S.P. Jackson, ATM-and cell cycle-dependent regulation of ATR in response to DNA double-strandbreaks, Nat. Cell Biol. (2005).

pair 8 (2009) 1025–1037 1035

[74] G. Rotman, Y. Shiloh, ATM: a mediator of multiple responses to genotoxicstress, Oncogene 18 (1999) 6135–6144.

[75] S. Banin, L. Moyal, S. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky,C. Prives, Y. Reiss, Y. Shiloh, Y. Ziv, Enhanced phosphorylation of p53 by ATMin response to DNA damage, Science 281 (1998) 1674–1677.

[76] C.J. Bakkenist, M.B. Kastan, DNA damage activates ATM through intermolecu-lar autophosphorylation and dimer dissociation, Nature 421 (2003) 499–506.

[77] J. Bartek, J. Lukas, DNA repair: damage alert, Nature 421 (2003) 486–488.[78] J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, M. Le Beau, J.R. Yates 3rd, L. Hays,

W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegenbreakage syndrome: linkage of double-strand break repair to the cellular DNAdamage response, Cell 93 (1998) 477–486.

[79] G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers,A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand breakrepair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder, Cell 99 (1999) 577–587.

[80] R. Varon, C. Vissinga, M. Platzer, K.M. Cerosaletti, K.H. Chrzanowska, K. Saar, G.Beckmann, E. Seemanova, P.R. Cooper, N.J. Nowak, M. Stumm, C.M. Weemaes,R.A. Gatti, R.K. Wilson, M. Digweed, A. Rosenthal, K. Sperling, P. Concannon,A. Reis Nibrin, A novel DNA double-strand break repair protein, is mutated inNijmegen breakage syndrome, Cell 93 (1998) 467–476.

[81] T. Uziel, Y. Lerenthal, L. Moyal, Y. Andegeko, L. Mittelman, Y. Shiloh, Require-ment of the MRN complex for ATM activation by DNA damage, EMBO J. 22(2003) 5612–5621.

[82] J. Falck, J. Coates, S.P. Jackson, Conserved modes of recruitment of ATM, ATRand DNA-PKcs to sites of DNA damage, Nature 434 (2005) 605–611.

[83] V. Costanzo, T. Paull, M. Gottesman, J. Gautier, Mre11 assembles linear DNAfragments into DNA damage signaling complexes, PLoS Biol. 2 (2004) E110.

[84] A. Dupre, L. Boyer-Chatenet, J. Gautier, Two-step activation of ATM by DNA andthe Mre11-Rad50-Nbs1 complex, Nat. Struct. Mol. Biol. 13 (2006) 451–457.

[85] Z. You, J.M. Bailis, S.A. Johnson, S.M. Dilworth, T. Hunter, Rapid activation ofATM on DNA flanking double-strand breaks, Nat. Cell Biol. 9 (2007) 1311–1318.

[86] E. Berkovich, R.J. Monnat Jr., M.B. Kastan, Roles of ATM and NBS1 in chromatinstructure modulation and DNA double-strand break repair, Nat. Cell Biol. 9(2007) 683–690.

[87] M. Pellegrini, A. Celeste, S. Difilippantonio, R. Guo, W. Wang, L. Feigenbaum, A.Nussenzweig, Autophosphorylation at serine 1987 is dispensable for murineATM activation in vivo, Nature 443 (2006) 222–225.

[88] S.V. Kozlov, M.E. Graham, C. Peng, P. Chen, P.J. Robinson, M.F. Lavin, Involve-ment of novel autophosphorylation sites in ATM activation, EMBO J. 25 (2006)3504–3514.

[89] J.A. Daniel, M. Pellegrini, J.H. Lee, T.T. Paull, L. Feigenbaum, A. Nussenzweig,Multiple autophosphorylation sites are dispensable for murine ATM activa-tion in vivo, J. Cell Biol. 183 (2008) 777–783.

[90] T.T. Paull, M. Gellert, The 3′ to 5′ exonuclease activity of Mre 11 facilitatesrepair of DNA double-strand breaks, Mol. Cell 1 (1998) 969–979.

[91] O. Limbo, C. Chahwan, Y. Yamada, R.A. de Bruin, C. Wittenberg, P. Russell, Ctp1is a cell-cycle-regulated protein that functions with Mre11 complex to controldouble-strand break repair by homologous recombination, Mol. Cell 28 (2007)134–146.

[92] A.A. Sartori, C. Lukas, J. Coates, M. Mistrik, S. Fu, J. Bartek, R. Baer, J. Lukas,S.P. Jackson, Human CtIP promotes DNA end resection, Nature 450 (2007)509–514.

[93] S. Takeda, K. Nakamura, Y. Taniguchi, T.T. Paull, Ctp1/CtIP and the MRN com-plex collaborate in the initial steps of homologous recombination, Mol. Cell28 (2007) 351–352.

[94] M. Clerici, D. Mantiero, G. Lucchini, M.P. Longhese, The Saccharomyces cere-visiae Sae2 protein promotes resection and bridging of double strand breakends, J. Biol. Chem. 280 (2005) 38631–38638.

[95] A. Dupre, L. Boyer-Chatenet, R.M. Sattler, A.P. Modi, J.H. Lee, M.L. Nicolette, L.Kopelovich, M. Jasin, R. Baer, T.T. Paull, J. Gautier, A forward chemical geneticscreen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex, Nat. Chem.Biol. 4 (2008) 119–125.

[96] J.E. Haber, The many interfaces of Mre11, Cell 95 (1998) 583–586.[97] E.P. Mimitou, L.S. Symington, Sae2, Exo1 and Sgs1 collaborate in DNA double-

strand break processing, Nature (2008).[98] Z. Zhu, W.H. Chung, E.Y. Shim, S.E. Lee, G. Ira, Sgs1 helicase and two nucle-

ases Dna2 and Exo1 resect DNA double-strand break ends, Cell 134 (2008)981–994.

[99] M.J. Neale, J. Pan, S. Keeney, Endonucleolytic processing of covalent protein-linked DNA double-strand breaks, Nature 436 (2005) 1053–1057.

[100] B.B. Hopkins, T.T. Paull, The P. furiosus mre11/rad50 complex promotes5′ strand resection at a DNA double-strand break, Cell 135 (2008) 250–260.

[101] R.S. Williams, G. Moncalian, J.S. Williams, Y. Yamada, O. Limbo, D.S. Shin, L.M.Groocock, D. Cahill, C. Hitomi, G. Guenther, D. Moiani, J.P. Carney, P. Russell, J.A.Tainer, Mre11 dimers coordinate DNA end bridging and nuclease processingin double-strand-break repair, Cell 135 (2008) 97–109.

[102] M.E. Budd, W. Choe, J.L. Campbell, The nuclease activity of the yeast Dna2protein, which is related to the RecB-like nucleases, is essential in vivo, J. Biol.
Chem. 275 (2000) 16518–16529.
[103] D.J. Richard, E. Bolderson, L. Cubeddu, R.I. Wadsworth, K. Savage, G.G. Sharma,M.L. Nicolette, S. Tsvetanov, M.J. McIlwraith, R.K. Pandita, S. Takeda, R.T. Hay,J. Gautier, S.C. West, T.T. Paull, T.K. Pandita, M.F. White, K.K. Khanna, Single-stranded DNA-binding protein hSSB1 is critical for genomic stability, Nature453 (2008) 677–681.

1 NA Re

[

[

[

[

[

[


[104] J. Buis, Y. Wu, Y. Deng, J. Leddon, G. Westfield, M. Eckersdorff, J.M. Sekiguchi,S. Chang, D.O. Ferguson, Mre11 nuclease activity has essential roles in DNArepair and genomic stability distinct from ATM activation, Cell 135 (2008)85–96.

[105] A. Jazayeri, A. Balestrini, E. Garner, J.E. Haber, V. Costanzo, Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides thatstimulate ATM activity, EMBO J. 27 (2008) 1953–1962.

[106] V. Bhaskara, A. Dupre, B. Lengsfeld, B.B. Hopkins, A. Chan, J.H. Lee, X. Zhang,J. Gautier, V. Zakian, T.T. Paull, Rad50 adenylate kinase activity regulates DNAtethering by Mre11/Rad50 complexes, Mol. Cell 25 (2007) 647–661.

[107] Z. You, J.M. Bailis, S.A. Johnson, S.M. Dilworth, T. Hunter, Rapid activation ofATM on DNA flanking double-strand breaks, Nat. Cell Biol. (2007).

[108] D.J. Richard, E. Bolderson, L. Cubeddu, R.I. Wadsworth, K. Savage, G.G. Sharma,M.L. Nicolette, S. Tsvetanov, M.J. McIlwraith, R.K. Pandita, S. Takeda, R.T. Hay,J. Gautier, S.C. West, T.T. Paull, T.K. Pandita, M.F. White, K.K. Khanna, Single-stranded DNA-binding protein hSSB1 is critical for genomic stability, Nature(2008).

[109] Y.J. Crow, B.E. Hayward, R. Parmar, P. Robins, A. Leitch, M. Ali, D.N. Black, H.van Bokhoven, H.G. Brunner, B.C. Hamel, P.C. Corry, F.M. Cowan, S.G. Frints,J. Klepper, J.H. Livingston, S.A. Lynch, R.F. Massey, J.F. Meritet, J.L. Michaud,G. Ponsot, T. Voit, P. Lebon, D.T. Bonthron, A.P. Jackson, D.E. Barnes, T. Lin-dahl, Mutations in the gene encoding the 3′–5′ DNA exonuclease TREX1 causeAicardi-Goutieres syndrome at the AGS1 locus, Nat. Genet. 38 (2006) 917–920.

[110] M.S. Eller, T. Maeda, C. Magnoni, D. Atwal, B.A. Gilchrest, Enhancement of DNArepair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response, Proc. Natl. Acad. Sci. U.S.A. 94 (1997)12627–12632.

[111] M.S. Eller, K. Ostrom, B.A. Gilchrest, DNA damage enhances melanogenesis,Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 1087–1092.

[112] M.D. Sutton, B.T. Smith, V.G. Godoy, G.C. Walker, The SOS response: recentinsights into umuDC-dependent mutagenesis and DNA damage tolerance,Annu. Rev. Genet. 34 (2000) 479–497.

[113] M.R. Singleton, M.S. Dillingham, M. Gaudier, S.C. Kowalczykowski, D.B. Wigley,Crystal structure of RecBCD enzyme reveals a machine for processing DNAbreaks, Nature 432 (2004) 187–193.

[114] D.G. Anderson, S.C. Kowalczykowski, Reconstitution of an SOS response path-way: derepression of transcription in response to DNA breaks, Cell 95 (1998)975–979.

[115] N.L. Craig, J.W. Roberts, E. coli recA protein-directed cleavage of phage lambdarepressor requires polynucleotide, Nature 283 (1980) 26–30.

[116] N.L. Craig, J.W. Roberts, Function of nucleoside triphosphate and polynu-cleotide in Escherichia coli recA protein-directed cleavage of phage lambdarepressor, J. Biol. Chem. 256 (1981) 8039–8044.

[117] S. Matsuoka, M. Huang, S.J. Elledge, Linkage of ATM to cell cycle regulation bythe Chk2 protein kinase, Science 282 (1998) 1893–1897.

[118] C.H. Lee, J.H. Chung, The hCds1 (Chk2)-FHA domain is essential for a chain ofphosphorylation events on hCds1 that is induced by ionizing radiation, J. Biol.Chem. 276 (2001) 30537–30541.

[119] Z. Sun, J. Hsiao, D.S. Fay, D.F. Stern, Rad53 FHA domain associated with phos-phorylated Rad9 in the DNA damage checkpoint, Science 281 (1998) 272–274.

[120] D. Durocher, J. Henckel, A.R. Fersht, S.P. Jackson, The FHA domain is a modularphosphopeptide recognition motif, Mol. Cell 4 (1999) 387–394.

[121] K. Iwabuchi, P.L. Bartel, B. Li, R. Marraccino, S. Fields, Two cellular proteins thatbind to wild-type but not mutant p53, Proc. Natl. Acad. Sci. U.S.A. 91 (1994)6098–6102.

122] R.A. DiTullio Jr., T.A. Mochan, M. Venere, J. Bartkova, M. Sehested, J. Bartek, T.D.Halazonetis, 53BP1 functions in an ATM-dependent checkpoint pathway thatis constitutively activated in human cancer, Nat. Cell Biol. 4 (2002) 998–1002.

123] C. Lukas, F. Melander, M. Stucki, J. Falck, S. Bekker-Jensen, M. Goldberg, Y.Lerenthal, S.P. Jackson, J. Bartek, J. Lukas, Mdc1 couples DNA double-strandbreak recognition by Nbs1 with its H2AX-dependent chromatin retention,EMBO J. 23 (2004) 2674–2683.

[124] K. Iijima, M. Ohara, R. Seki, H. Tauchi, Dancing on damaged chromatin: func-tions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses toDNA damage, J. Radiat. Res. (Tokyo) (2008).

125] C. Feijoo, C. Hall-Jackson, R. Wu, D. Jenkins, J. Leitch, D.M. Gilbert, C. Smythe,Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1in the intra-S phase checkpoint monitoring replication origin firing, J. Cell Biol.154 (2001) 913–923.

126] A. Kumagai, Z. Guo, K.H. Emami, S.X. Wang, W.G. Dunphy, The Xenopus Chk1protein kinase mediates a caffeine-sensitive pathway of checkpoint control incell-free extracts, J. Cell Biol. 142 (1998) 1559–1569.

[127] D.P. Lane, Cancer. p53, guardian of the genome, Nature 358 (1992) 15–16.128] J. Falck, N. Mailand, R.G. Syljuasen, J. Bartek, J. Lukas, The ATM-Chk2-Cdc25A

checkpoint pathway guards against radioresistant DNA synthesis, Nature 410(2001) 842–847.

129] V. Costanzo, D. Shechter, P.J. Lupardus, K.A. Cimprich, M. Gottesman, J. Gautier,An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiationof DNA replication, Mol. Cell 11 (2003) 203–213.

[130] H.Y. Yoo, A. Shevchenko, W.G. Dunphy, Mcm2 is a direct substrate of ATM andATR during DNA damage and DNA replication checkpoint responses, J. Biol.Chem. 279 (2004) 53353–53364.

[131] P.T. Yazdi, Y. Wang, S. Zhao, N. Patel, E.Y. Lee, J. Qin, SMC1 is a downstreameffector in the ATM/NBS1 branch of the human S-phase checkpoint, GenesDev. 16 (2002) 571–582.

pair 8 (2009) 1025–1037

[132] R. Jessberger, P. Berg, Repair of deletions and double-strand gaps by homolo-gous recombination in a mammalian in vitro system, Mol. Cell Biol. 11 (1991)445–457.

[133] D. Cortez, Y. Wang, J. Qin, S.J. Elledge, Requirement of ATM-dependent phos-phorylation of brca1 in the DNA damage response to double-strand breaks,Science 286 (1999) 1162–1166.

[134] T. Taniguchi, I. Garcia-Higuera, B. Xu, P.R. Andreassen, R.C. Gregory, S.T. Kim,W.S. Lane, M.B. Kastan, A.D. D’Andrea, Convergence of the Fanconi anemia andataxia telangiectasia signaling pathways, Cell 109 (2002) 459–472.

[135] M. O’Driscoll, V.L. Ruiz-Perez, C.G. Woods, P.A. Jeggo, J.A. Goodship, A splic-ing mutation affecting expression of ataxia-telangiectasia and Rad3-relatedprotein (ATR) results in Seckel syndrome, Nat. Genet. 33 (2003) 497–501.

[136] R.S. Cha, N. Kleckner, ATR homolog Mec1 promotes fork progression, thusaverting breaks in replication slow zones, Science 297 (2002) 602–606.

[137] M. Lopes, C. Cotta-Ramusino, A. Pellicioli, G. Liberi, P. Plevani, M. Muzi-Falconi,C.S. Newlon, M. Foiani, The DNA replication checkpoint response stabilizesstalled replication forks, Nature 412 (2001) 557–561.

[138] J.M. Sogo, M. Lopes, M. Foiani, Fork reversal and ssDNA accumulation at stalledreplication forks owing to checkpoint defects, Science 297 (2002) 599–602.

[139] J.A. Tercero, M.P. Longhese, J.F. Diffley, A central role for DNA replication forksin checkpoint activation and response, Mol. Cell 11 (2003) 1323–1336.

[140] K. Trenz, E. Smith, S. Smith, V. Costanzo, ATM and ATR promote Mre11 depen-dent restart of collapsed replication forks and prevent accumulation of DNAbreaks, EMBO J. 25 (2006) 1764–1774.

[141] C. Lucca, F. Vanoli, C. Cotta-Ramusino, A. Pellicioli, G. Liberi, J. Haber, M. Foiani,Checkpoint-mediated control of replisome-fork association and signalling inresponse to replication pausing, Oncogene 23 (2004) 1206–1213.

[142] M.C. Edwards, A.V. Tutter, C. Cvetic, C.H. Gilbert, T.A. Prokhorova, J.C. Walter,MCM2-7 complexes bind chromatin in a distributed pattern surrounding theorigin recognition complex in Xenopus egg extracts, J. Biol. Chem. 277 (2002)33049–33057.

[143] S. Waga, T. Masuda, H. Takisawa, A. Sugino, DNA polymerase epsilon is requiredfor coordinated and efficient chromosomal DNA replication in Xenopus eggextracts, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 4978–4983.

[144] J.A. Cobb, L. Bjergbaek, K. Shimada, C. Frei, S.M. Gasser, DNA polymerase stabi-lization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1,EMBO J. 22 (2003) 4325–4336.

[145] M. Lopes, M. Foiani, J.M. Sogo, Multiple mechanisms control chromosomeintegrity after replication fork uncoupling and restart at irreparable UVlesions, Mol. Cell 21 (2006) 15–27.

[146] S. Matsuoka, B.A. Ballif, A. Smogorzewska, E.R. McDonald 3rd, K.E. Hurov, J.Luo, C.E. Bakalarski, Z. Zhao, N. Solimini, Y. Lerenthal, Y. Shiloh, S.P. Gygi, S.J.Elledge, ATM and ATR substrate analysis reveals extensive protein networksresponsive to DNA damage, Science 316 (2007) 1160–1166.

[147] H. Lou, M. Komata, Y. Katou, Z. Guan, C.C. Reis, M. Budd, K. Shirahige, J.L.Campbell, Mrc1 and DNA polymerase epsilon function together in linking DNAreplication and the S phase checkpoint, Mol. Cell 32 (2008) 106–117.

[148] J.H. Petrini, M.E. Walsh, C. DiMare, X.N. Chen, J.R. Korenberg, D.T. Weaver, Iso-lation and characterization of the human MRE11 homologue, Genomics 29(1995) 80–86.

[149] Y. Xiao, D.T. Weaver, Conditional gene targeted deletion by Cre recombinasedemonstrates the requirement for the double-strand break repair Mre11 pro-tein in murine embryonic stem cells, Nucleic Acids Res. 25 (1997) 2985–2991.

[150] J. Kang, R.T. Bronson, Y. Xu, Targeted disruption of NBS1 reveals itsroles in mouse development and DNA repair, EMBO J. 21 (2002) 1447–1455.

[151] G. Luo, M.S. Yao, C.F. Bender, M. Mills, A.R. Bladl, A. Bradley, J.H. Petrini, Dis-ruption of mRad50 causes embryonic stem cell lethality, abnormal embryonicdevelopment, and sensitivity to ionizing radiation, Proc. Natl. Acad. Sci. U.S.A.96 (1999) 7376–7381.

[152] O.K. Mirzoeva, J.H. Petrini, DNA replication-dependent nuclear dynamics ofthe Mre11 complex, Mol. Cancer Res. 1 (2003) 207–218.

[153] V. Costanzo, K. Robertson, M. Bibikova, E. Kim, D. Grieco, M. Gottesman, D.Carroll, J. Gautier, Mre11 protein complex prevents double-strand break accu-mulation during chromosomal DNA replication, Mol. Cell 8 (2001) 137–147.

[154] M. de Jager, J. van Noort, D.C. van Gent, C. Dekker, R. Kanaar, C. Wyman, HumanRad50/Mre11 is a flexible complex that can tether DNA ends, Mol. Cell 8 (2001)1129–1135.

[155] K.P. Hopfner, A. Karcher, L. Craig, T.T. Woo, J.P. Carney, J.A. Tainer, Structuralbiochemistry and interaction architecture of the DNA double-strand breakrepair Mre11 nuclease and Rad50-ATPase, Cell 105 (2001) 473–485.

[156] D.M. Chou, S.J. Elledge, Tipin and timeless form a mutually protective complexrequired for genotoxic stress resistance and checkpoint function, Proc. Natl.Acad. Sci. U.S.A. 103 (2006) 18143–18147.

[157] J.F. Diffley, Eukaryotic DNA replication, Curr. Opin. Cell. Biol. 6 (1994) 368–372.[158] A.M. Woodward, T. Gohler, M.G. Luciani, M. Oehlmann, X. Ge, A. Gartner, D.A.

Jackson, J.J. Blow, Excess Mcm2-7 license dormant origins of replication thatcan be used under conditions of replicative stress, J. Cell Biol. 173 (2006)673–683.

[159] X.Q. Ge, D.A. Jackson, J.J. Blow, Dormant origins licensed by excess Mcm2-7
are required for human cells to survive replicative stress, Genes Dev. 21 (2007)3331–3341.
[160] J.J. Blow, P.J. Gillespie, Replication licensing and cancer—a fatal entanglement?Nat. Rev. Cancer (2008).

[161] K. Trenz, A. Errico, V. Costanzo, Plx1 is required for chromosomal DNA repli-cation under stressful conditions, EMBO J. 27 (2008) 876–885.

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[165] H.Y. Yoo, A. Kumagai, A. Shevchenko, A. Shevchenko, W.G. Dunphy, Adapta-tion of a DNA replication checkpoint response depends upon inactivation of


162] D.P. Toczyski, D.J. Galgoczy, L.H. Hartwell, CDC5 and CKII control adaptationto the yeast DNA damage checkpoint, Cell 90 (1997) 1097–1106.

163] H.Y. Yoo, A. Shevchenko, A. Shevchenko, W.G. Dunphy, Mcm2 is a direct sub-strate of ATM and ATR during DNA damage and DNA replication checkpointresponses, J. Biol. Chem. 279 (2004) 53353–53364.

164] D. Cortez, G. Glick, S.J. Elledge, Minichromosome maintenance proteins aredirect targets of the ATM and ATR checkpoint kinases, Proc. Natl. Acad. Sci.U.S.A. 101 (2004) 10078–10083.

pair 8 (2009) 1025–1037 1037

Claspin by the Polo-like kinase, Cell 117 (2004) 575–588.[166] Z. Tang, H. Yu, Functional analysis of the spindle-checkpoint proteins using

an in vitro ubiquitination assay, Methods Mol. Biol. 281 (2004) 227–242.

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Studying the DNA damage response using in vitro model systems

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