Single-Stranded DNA Orchestrates an ATM-to-ATR Switch at DNA Breaks

Single-Stranded DNA Orchestrates an ATM-to-ATR Switch atDNA Breaks

Bunsyo Shiotani1 and Lee Zou1,2,31 Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA021292 Department of Pathology, Harvard Medical School, Boston, MA 02115

SUMMARYATM and ATR are two master checkpoint kinases activated by double-strand DNA breaks(DSBs). ATM is critical for the initial response and the subsequent ATR activation. Here, weshow that ATR activation is coupled with loss of ATM activation, an unexpected ATM-to-ATRswitch during the biphasic DSB response. ATM is activated by DSBs with blunts ends or shortsingle-strand overhangs (SSOs). Surprisingly, the activation of ATM in the presence of SSOs, likethat of ATR, relies on single- and double-stranded DNA junctions. In a length-dependent manner,SSOs attenuate ATM activation and potentiate ATR activation through a swap of DNA damagesensors. Progressive resection of DSBs directly promotes the ATM-to-ATR switch in vitro. Incells, the ATM-to-ATR switch is driven by both ATM and the nucleases participating in DSBresection. Thus, single-stranded DNA orchestrates ATM and ATR to function orderly andreciprocally in two distinct phases of DSB response.

INTRODUCTIONDouble-strand DNA breaks (DSBs) are among the most deleterious DNA lesions thatthreaten genomic integrity. DSBs are generated not only by exogenous DNA-damagingagents, but also by normal cellular processes such as V(D)J recombination, meiosis, andDNA replication. Furthermore, increased amounts of DSBs are induced by oncogenicstresses during the early stage of tumorigenesis (Bartkova et al., 2005). In response to DSBs,the ATM kinase phosphorylates and regulates a large number of substrates involved in DNArepair, DNA replication, and other cellular processes important for genomic stability(Matsuoka et al., 2007). In addition to DSBs, ATM also responds to other cellular stressessuch as hypoxia and chromatin alterations (Bakkenist and Kastan, 2003; Bencokova et al.,2008; Gibson et al., 2005). Mutations of ATM in humans result in ataxia-telangiectasia(AT), a genetic disorder associated with radiation sensitivity, neuron degeneration, immunedeficiencies, premature aging, and predisposition to cancers (Shiloh and Kastan, 2001).ATM is also one of the most frequently mutated kinases in human cancers (Greenman et al.,2007). All evidence points to that ATM is a crucial guardian of genomic integrity.

3Corresponding Author: Email: [email protected], Phone: (617) 724-9534, Fax: (617) 726-7808.SUPPLEMENTAL DATASupplemental Data include six figures and Supplemental Experimental Procedures.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptMol Cell. Author manuscript; available in PMC 2010 March 13.

Published in final edited form as:Mol Cell. 2009 March 13; 33(5): 547–558. doi:10.1016/j.molcel.2009.01.024.

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The mechanisms by which ATM is activated have been under intensive investigation(Harper and Elledge, 2007). The activation of ATM coincides with the autophosphorylationof ATM at Ser1981 and the conversion of ATM oligomers to monomers (Bakkenist andKastan, 2003). The Mre11-Rad50-Nbs1 (MRN) complex is a sensor of DSBs and a directactivator of the ATM kinase (Lee and Paull, 2005). While ATM is not solely regulated byMRN in vivo (Kanu and Behrens, 2007), its activation at DSBs is primarily mediated byMRN (Berkovich et al., 2007; Falck et al., 2005; Kitagawa et al., 2004; You et al., 2005).After the initial ATM activation by DSBs, ATM executes specific functions around thebreaks through a chromatin-mediated mechanism involving H2AX, Mdc1, and otherproteins (Lou et al., 2006; Stewart et al., 2003; Stucki et al., 2005). Direct tethering of alarge number of ATM molecules or its regulators to an array of binding sites activates ATMeven in the absence of DSBs (Soutoglou and Misteli, 2008), indicating that a criticalfunction of DSBs in ATM activation is to nucleate ATM and its regulators at sites of DNAdamage. The activation of ATM at and around actual DSBs is a step-wise process initiatedby the breaks. Despite the clear involvement of DSBs in ATM activation, the exact DNAstructural determinants for ATM activation have not been clearly defined. Furthermore, howthe structures of DNA at DSBs contribute to ATM activation is not well understood.

In addition to ATM, DSBs also activate ATR, another master checkpoint kinase that hasoverlapping substrate specificity with ATM. Like ATM, ATR is critical for the fullcheckpoint response to DSBs (Brown and Baltimore, 2003; Cortez et al., 2001), indicatingthat ATM and ATR have non-redundant functions in this process. Unlike ATM, however,ATR also responds to a broad spectrum of DNA damage besides DSBs, especially thedamage interfering with DNA replication. The recruitment of ATR to DSBs requires RPA-coated single-stranded DNA (RPA-ssDNA), a structure generated by the nuclease-mediatedresection of DSBs (Zou and Elledge, 2003). The junctions between single- and double-stranded DNA, another structure associated with resected DSBs, are also important for ATRactivation (MacDougall et al., 2007; Zou, 2007). Several nucleases and helicases, includingMRN, CtIP, Exo1, and BLM have been implicated in the resection of DSBs (Gravel et al.,2008; Lengsfeld et al., 2007; Limbo et al., 2007; Mimitou and Symington, 2008; Sartori etal., 2007; Schaetzlein et al., 2007; Zhu et al., 2008). Interestingly, ATM is required for theefficient resection of DSBs and the activation of ATR by DSBs (Jazayeri et al., 2006; Myersand Cortez, 2006; Yoo et al., 2007).

The sequential activation of ATM and ATR by DSBs suggests that the checkpoint responseto DSBs is biphasic. Although ATM is clearly critical for the initial response to DSBs, howATM and ATR orchestrate the second phase of checkpoint response is unclear. Aparticularly interesting question is how ATM and ATR are coordinated at the DSBsundergoing resection, a dynamic structure that integrates checkpoint signaling with DNArepair. While the respective activation of ATM and ATR by DSBs has been extensivelystudied, these kinases and the DNA structures regulating them have rarely beencharacterized as a whole. Furthermore, the fundamental question of how exactly ATM andATR distinguish DNA damage structures remains to be addressed.

In this study, using a newly developed ATM/ATR activation assay, we show that theactivation of ATM is regulated by multiple DNA structural elements of DSBs. Moreimportantly, we reveal that ATM and ATR are activated by similar yet distinct DNAstructures at resected DSBs. While both ATM and ATR depend on the junctions of single-and double-stranded DNA for activation, they are oppositely regulated by the lengthening ofsingle-stranded overhangs (SSOs). SSOs simultaneously attenuate ATM activation andpotentiate ATR activation, thereby promoting an ATM-to-ATR switch during the process ofDSB resection. These findings provide mechanistic insights into how the DNA-damage

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specificities of ATM and ATR are distinct from each other and, furthermore, how ATM andATR function in concert to bring about the biphasic DSB response.

RESULTSDouble-stranded DNA- and Nbs1-dependent ATM activation in vitro

Biochemical studies using purified proteins or Xenopus extracts have shown that ATM canbe activated by DNA fragments in vitro (Dupre et al., 2006; Lee and Paull, 2005; Yoo et al.,2004; You et al., 2007). To reveal the DNA structural determinants for ATM activation, wedevised an in vitro ATM activation assay using human cell extracts and defined DNAstructures. A 70-bp dsDNA fragment with blunt ends was generated using twocomplementary ssDNA oligomers. In HeLa cell nuclear extracts, dsDNA but not ssDNAinduced the phosphorylation of ATM at Ser1981 in a concentration-dependent manner (Fig.1A). The phosphorylation of Chk2 at Thr68, a known ATM substrate site in cells, was alsoinduced by dsDNA (Fig. 1A). The dsDNA-induced phosphorylation of ATM and Chk2 wasinhibited by KU-55933, a specific ATM inhibitor, suggesting that these phosphorylationevents are ATM-dependent (Fig. 1B). The dsDNA-induced phosphorylation of ATM andChk2 was not detected in AT cell extracts, but was detected in the extracts of the AT cellscomplemented with ATM (Fig. 1C), confirming that the phosphorylation of Chk2 is ATM-dependent.

To further assess if the dsDNA-induced phosphorylation of ATM and Chk2 indeed reflectsthe activation of ATM in extracts, we asked if it is dependent on Nbs1 or Ku70. In cells,Nbs1 is critical for the activation of ATM at DSBs, whereas Ku70 is required for theactivation of DNA-PKcs, another kinase responsive to DSBs. We generated extracts fromthe HeLa cells in which Nbs1 or Ku70 was depleted by siRNA. The induction of ATM andChk2 phosphorylation by dsDNA was significantly diminished in the Nbs1-depleted extractscompared to the controls (Fig. 1D). In marked contrast, in the extracts with reduced levels ofKu70, ATM and Chk2 were substantially phosphorylated even when no dsDNA was added(Fig. 1E). This phosphorylation of ATM and Chk2 may be due to the genomic instability inKu70-depleted cells, or the binding of MRN to the residual genomic DNA in extracts whenKu70 was removed. Despite this basal phosphorylation, ATM and Chk2 were furtherphosphorylated when dsDNA was added to the Ku70-depleted extracts. These resultssuggest that the DSB-induced phosphorylation of ATM and Chk2 in extracts, like that incells, is dependent on Nbs1 but not DNA-PKcs.

To directly determine if ATM is activated by dsDNA in extracts, we measured the kinaseactivity of ATM. As revealed by in vitro kinase assays with immunoprecipitated ATM,dsDNA stimulated the kinase activity of ATM by approximately 2-fold in extracts (Fig. S1).Similar elevations of ATM kinase activity were observed in cells treated with ionizingradiation (IR) (Pandita et al., 2000). Collectively, these results suggest that the activation ofATM by dsDNA in extracts closely resembles the activation of ATM by DSBs in cells.

dsDNA regulates ATM activation through length- and end-dependent mechanismsUsing the in vitro assay above, we sought to systematically characterize the DNA structuraldeterminants for ATM activation. Studies using purified proteins or Xenopus extracts haveshown that ATM is activated by dsDNA in a length-dependent manner (Lee and Paull, 2005;You et al., 2007). In these studies, only the DNA fragments longer than 200 bp efficientlyactivated ATM (Lee and Paull, 2005; You et al., 2007). In HeLa extracts, however, even12.5 nM of 70-bp dsDNA (1.5×1010 DNA ends μl−1) induced substantial ATMphosphorylation (Fig. 2A). The high sensitivity of this assay allowed us to analyze shortdsDNA fragments with defined structural features.



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We first asked whether and how ATM is activated by dsDNA in a length-dependent mannerin human cell extracts. When present at the same molar concentrations or the same DNAmass, dsDNA of 70 bp, 40 bp, or 20 bp induced ATM phosphorylation in a length-dependent manner (Fig. 2A). Since these dsDNA fragments are much shorter than the DNAof a single nucleosome, a length-dependent mechanism for ATM activation may operate onthe nucleosome-free dsDNA immediately flanking the breaks. To investigate how the lengthof dsDNA contributes to ATM activation, we generated a “bubble” DNA structure byconverting an internal 30-bp region of the 70-bp dsDNA into a single-stranded region (Fig.2B). The ability of the bubble structure to induce ATM phosphorylation was between thoseof the 70-bp and the 40-bp dsDNA (Fig. 2B), showing that the internal region of the 70-bpdsDNA contributes to the length-dependent activation of ATM. Using purified MRNcomplexes, we found that greater amounts of Nbs1 and Rad50 associated with 70-bpdsDNA than 40- and 20-bp dsDNA (Fig. 2C). These results suggest that the MRN complexassociates with nucleosome-free dsDNA in a length-dependent manner, providing a possiblemechanism for ATM activation along dsDNA.

To assess if the ends of dsDNA are critical for ATM activation, we biotinylated all fourDNA ends of the 20- and 70-bp fragments (5′ and 3′ ends of both strands). The biotinylateddsDNA efficiently induced ATM phosphorylation in the absence of streptavidin, but lost thisactivity when the ends were blocked by streptavidin (Fig. 2D). When only the 5′ or 3′ endsof 70-bp dsDNA were blocked, the ability of the fragment to activate ATM wassubstantially reduced (Fig. S2). Since the streptavidin on one DNA strand may block accessto both strands, it was not possible to resolve how 5′ or 3′ ends contribute to ATMactivation. Nonetheless, blockage of DNA ends inhibited ATM activation regardless of thelength of dsDNA, suggesting that the length-dependent mechanism for ATM activationneeds to be initiated from DNA ends, or act through the ends.

The ends of dsDNA could potentially be processed by helicases and/or nucleases in extracts.To assess how unwinding of dsDNA affects ATM activation in extracts, we generated afork-like DNA structure that possesses both paired and unpaired DNA ends (Fig. 2E). Theability of the fork structure to activate ATM was lost when the paired ends were blocked,but was unaffected when the unpaired ends were blocked (Fig. 2E). Therefore, paired DNAends are required for initiating ATM activation in extracts. These results suggest that ATMcannot be directly activated by unwound DNA ends or by the fork-like DNA structuresassociated with DNA replication or DNA repair.

Single-strand overhangs interfere with ATM activation by attenuating MRN bindingDSBs are not always blunt-ended in cells. The DSBs generated by the HO or I-SceIendonuclease initially have 4-nt 3′ single-strand overhangs (SSOs) (Colleaux et al., 1988;Kostriken et al., 1983). V(D)J recombination and meiosis produce DSBs with 3′ and 5′SSOs, respectively (Schlissel, 1998; Xu and Kleckner, 1995). The DSBs resulting fromcollapsed replication forks or broken ssDNA gaps may possess either 3′ or 5′ SSOs (Lopeset al., 2006). The “uncapped” telomeres resemble DSBs with 3′ SSOs (Celli and de Lange,2005). When exposed in cells, DSBs can be resected by exo- or endonucleases in the 5′-to-3′direction (Lee et al., 1998). ATM has been implicated in the response to the various types ofDSBs above, indicating that it can be activated by DSBs with SSOs. In extracts, while thebulk of dsDNA appeared unaltered (Fig. S3), a small fraction of it might be processed bynucleases. The in vivo functions of ATM in the response to SSO-bearing DSBs prompted usto investigate the role of SSOs in ATM activation.

To directly assess the effects of SSOs on ATM activation, we analyzed the 20- and 70-bpdsDNA bearing either 5′ or 3′ SSOs of random sequences (Figs. 3A and 3B). Both 5′ and 3′SSOs of 5 nt slightly enhanced ATM phosphorylation. Interestingly, both 5′ and 3′ SSOs of



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25 or 50 nt attenuated ATM and Chk2 phosphorylation (Figs. 3A and 3B), suggesting thatSSOs interfere with ATM activation in a length-dependent manner. SSOs of poly A alsohindered ATM activation in a length-dependent manner (Fig. 3C). SSOs not only attenuatedthe activation of ATM by 20- and 70-bp dsDNA, but also that by linear plasmids (see Fig.5). Together, these results suggest that SSOs may interfere with a DNA end-dependent eventin ATM activation, which is independent of the length of dsDNA.

The ssDNA generated by resection may interfere with ATM activation in cis or in trans.When blunt-ended 70-bp dsDNA was added to extracts with 25-nt ssDNA at 1:2 or 1:5molar ratios, a modest reduction of ATM activation was observed (Fig. 3D). When the 25-ntssDNA was linked to 70-bp dsDNA as overhangs, it interfered with ATM activation moreeffectively. Thus, while ssDNA can interfere with ATM activation both in cis and in trans,SSOs are more potent than free ssDNA for this function.

To reveal the mechanism by which SSOs interfere with ATM activation, we asked if SSOsaffect the binding of MRN to dsDNA. Indeed, 3′ SSOs of 25 nt substantially reduced theamounts of Nbs1 and Mre11 associated with 70-bp dsDNA in extracts (Fig. 3E). However,purified MRN bound to dsDNA efficiently regardless of the presence or absence of SSOs(Fig. 3E). Together, these results suggest that SSOs do not directly interfere with the bindingof MRN to dsDNA, but they reduce MRN binding in the presence of other proteins.

ATM activation requires junctions of single- and double-stranded DNAAlthough less potent than blunt-ended dsDNA, dsDNA bearing short SSOs retain someability to associate with MRN and to active ATM in extracts. Our analysis of blunt-endeddsDNA suggest that ATM activation is dependent on DNA ends (Fig. 2). Two types of DNAends are present in the DNA fragments with SSOs: the ends of the dsDNA region (thejunctions of dsDNA/ssDNA) and the ends of SSOs (Fig. 4A). To assess how these DNAends contribute to ATM activation, we tested three sets of DNA structures (20-bp dsDNAwith 5′ or 3′ 25-nt SSOs and 70-bp dsDNA with 3′ 25-nt SSOs) in which either the junctionsor the SSO ends were biotinylated (Figs. 4A–C). In the absence of streptavidin, all of theDNA structures with SSOs induced ATM phosphorylation at reduced levels compared toblunt-ended dsDNA (Figs. 3A–D). When the ends of the 5′ or 3′ SSOs were blocked bystreptavidin, the ability of the DNA fragments to activate ATM and Chk2 was not affected(Figs. 4A–C). In striking contrast, when the 5′ or 3′ junctions of dsDNA/ssDNA wereblocked by streptavidin, the DNA fragments failed to activate ATM and Chk2 (Figs. 4A–C).These results suggest that the junctions of dsDNA/ssDNA, but not the ends of SSOs, arecritical for ATM activation. Furthermore, the junctions of dsDNA/ssDNA are required forATM activation regardless of the length of dsDNA (Figs. 4A–C), suggesting that these endsare involved in an initiating event for ATM activation, possibly the DNA recognition byMRN.

The junctions of dsDNA/ssDNA are present not only at DSBs, but also at single-strandDNA breaks, gaps, and DNA replication forks. To assess if dsDNA/ssDNA junctions aresufficient to activate ATM, we generated a plasmid carrying a single cleavage site of thenicking enzyme N. BbvCI (Fig. S4A). Using the nicking enzyme or a restriction enzyme thatcuts the plasmid in both DNA strands, we generated nicked plasmids and linear plasmidsbearing blunt ends (Fig. S4B). Like the short dsDNA fragments, linear plasmids inducedATM phosphorylation (Fig. 4D). In contrast, nicked plasmids were unable to induce anyATM phosphorylation (Fig. 4D). Moreover, when the DNA nicks were extended intossDNA gaps by Exonuclease III (Fig. S4C), the gap-carrying plasmids were still unable toactivate ATM (Fig. 4D). Thus, while the junctions of dsDNA/ssDNA are required for ATMactivation at DSBs, they are not sufficient to elicit ATM response when present internally onDNA. These internal junctions may be recognized by proteins that inhibit ATM activation.



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Alternatively, additional structural features of DSBs, such as the topological state of DNA(Fig. S4B), may be involved in ATM activation.

Resection of DNA ends promotes an ATM-to-ATR switchThe involvement of dsDNA/ssDNA junctions in ATM activation is surprising because thesestructures have been implicated in the activation of ATR (MacDougall et al., 2007; Zou,2007). These results raise the question as to how the DNA-damage specificities of ATM andATR are distinct from each other at DSBs, and how ATM and ATR are coordinated at theDSBs undergoing resection. In human cells, ATM is required for DSB resection and ATRactivation (Jazayeri et al., 2006). In this study, we show that SSOs interfere with ATMactivation in a length-dependent manner (Fig. 3). In addition, we have previously shown thatssDNA coated by RPA binds to ATRIP in a length-dependent manner, allowing the ATR-ATRIP kinase complex to recognize DSBs (Zou and Elledge, 2003). These findings led usto hypothesize that following the activation of ATM and the initiation of resection, SSOsmight promote an ATM-to-ATR switch at DSBs.

To directly investigate if the process of SSO generation can restrict ATM activation andinduce ATR activation, we used exonucleases to resect the DNA ends of a linear plasmid(Fig. S5A). In a time-dependent manner, T7 exonuclease progressively resects DNA ends inthe 5′-to-3′ direction, whereas Exonuclease III cleaves in the 3′-to-5′ direction (Fig. S5B).When the same amounts of processed or unprocessed plasmids were added to extracts, theprocessed plasmids exhibited a reduced ability to activate ATM compared to theunprocessed plasmids (Fig. 5A). These results confirm that the generation of SSOsprogressively interferes with ATM activation (Figs. 3A-C).

When generated at DSBs in cells, SSOs are recognized by RPA, leading to a DNA-proteinstructure recruiting ATR-ATRIP (Zou and Elledge, 2003). In extracts, SSOs of 50 ntassociated with both RPA and ATRIP (Fig. 5B). To assess if SSOs induce ATR activation,we monitored the phosphorylation of RPA32 at Ser33 (Olson et al., 2006). Thephosphorylation of RPA32 was induced by linear plasmids even in the absence ofexonuclease (Fig. 5C). This phosphorylation of RPA32 was partially inhibited by KU-55933alone, and virtually abolished by the combination of KU-55933 and NU7026, a specificinhibitor of DNA-PKcs, suggesting that the RPA32 phosphorylation induced by linearplasmids involves both ATM and DNA-PKcs. Interestingly, the phosphorylation of RPA32was progressively enhanced by the exonuclease-mediated resection of DNA ends (Figs. 5Aand 5D). This is in marked contrast to the decline of ATM phosphorylation when SSOs weregenerated (Fig. 5A). Furthermore, the phosphorylation of RPA32 induced by resected endswas not inhibited by the combination of KU-55933 and NU7026, but was inhibited byWortmannin, a pan-inhibitor of ATR, ATM, and DNA-PKcs (Fig. 5E). These results suggestthat the SSO-induced RPA32 phosphorylation is independent of ATM and DNA-PKcs, butmay be dependent on ATR.

To directly address if the increased phosphorylation of RPA32 reflects the activation ofATR, we used siRNA to knockdown ATR in HeLa cells and generated nuclear extracts fromthese cells. KU-55933 and NU7026 were added to the extracts to eliminate the potentialcontributions of DNA-PKcs and ATM to RPA32 phosphorylation. In the absence ofexonucleases, linear plasmids did not induce RPA32 phosphorylation in the ATR-depletedor the control extracts (Fig. 5F). Importantly, in the presence of exonucleases, thephosphorylation of RPA32 was efficiently induced in the control extracts, but not in theextracts lacking ATR (Fig. 5F). Together, these results demonstrate that the generation ofSSOs not only interferes with ATM activation, but also promotes ATR activation.



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Consecutive activation of ATM and ATR in cellsThe opposite effects of SSOs on ATM activation and ATR activation prompted us toinvestigate if ATM and ATR are reciprocally regulated in cells. We followed the IR-inducedphosphorylation of Chk2 and Chk1, two specific substrates of ATM and ATR, respectively.Within 5 min after IR, Chk2 but not Chk1 was strongly phosphorylated, showing that Chk2is phosphorylated more rapidly than Chk1 (Fig. 6A). Furthermore, Chk2 phosphorylationstarted to decline 30 min after IR, whereas Chk1 phosphorylation remained at high levelsuntil 2 hr (Fig. 6A). These results show that Chk2 is transiently phosphorylated during awindow that precedes the window of Chk1 phosphorylation. Nevertheless, there was a shortperiod (10 to 30 min) in which both Chk2 and Chk1 were strongly phosphorylated. Thismay be due to asynchronous resection of DSBs in individual cells (Barlow et al.,2008;Zierhut and Diffley, 2008), or asynchronous resection in different cell sub-populations(Jazayeri et al., 2006). It should be noted that IR not only induces DSBs but also other typesof DNA damage (Ward, 2000), some of which may interfere with DNA replication and leadto delayed ATR/ATM response. Overall, the decline of Chk2 phosphorylation coincidedwith strong Chk1 phosphorylation, which is consistent with an ATM-to-ATR switch in thesecells.

Chk2 is phosphorylated by ATM locally at DSBs and then moves throughout the nucleus(Lukas et al., 2003), prompting us to assess more directly if ATM is transiently activated atDSBs. The accumulation of phosphorylated ATM at DSBs marks one of the events duringthe process of ATM activation (Berkovich et al., 2007; You et al., 2005). PhosphorylatedATM appeared on chromatin within 5 min after IR and started to decline after 30 min (Fig.6B). Since the overall levels of phosphorylated ATM in cells remain high for many hoursafter IR (Fig. 6B; Bakkenist and Kastan, 2003), our data indicates that Chk2 is primarilytargeted by the phosphorylated ATM associated with DSBs. These results suggest that ATMis transiently activated at DSBs.

To monitor the resection of DSBs in cells, we analyzed the IR-induced RPA foci at differenttimes after irradiation (Figs. 6C and 6D). Few RPA foci were detected 5 min after IR. At 30min, RPA foci appeared in a significant fraction of cells. At 120 min, approximately half ofthe cells exhibited intense RPA foci. Thus, RPA gradually accumulated at DSBs as ATMactivation was attenuated. The numerous RPA foci at 120 min suggest that the attenuation ofATM activation was not due to the completion of DNA repair. These results provide furtherevidence that the activation of ATM is gradually attenuated by DSB resection in cells.Furthermore, because RPA-ssDNA is a key structure involved in ATR activation, theseresults also provide in vivo evidence linking the attenuation of ATM activation to ATRactivation.

Regulation of the ATM-to-ATR switch by ATM and nucleasesThe attenuation of ATM activation at resected DSBs could be attributed to the generation ofSSO or the consequent ATR activation. To distinguish these possibilities, we analyzed thephosphorylation of Chk1 and Chk2 in cells treated with ATR siRNA. While Chk1phosphorylation was abolished in cells lacking ATR, the kinetics of Chk2 phosphorylationwas not altered in these cells (Fig. 7A). These results strongly suggest that the process ofDSB resection, rather than the activation of ATR, is responsible of the loss of ATMactivation.

If the ATM-to-ATR switch is indeed driven by DSB resection in cells, one would expectthat this transition is controlled by the regulators of DSB resection. The resection of DSBs incells is regulated by ATM and exonucleases including MRN-CtIP and Exo1. To test if ATMregulates the ATM-to-ATR switch, we treated cells with KU-55933 prior to IR irradiation.



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In the presence of 5 μM of KU-55933, the phosphorylation of both Chk2 and Chk1 wasvirtually abolished (Fig. 7B). When ATM was partially inhibited by 1 μM of KU-55933,both Chk2 phosphorylation and Chk1 phosphorylation were reduced and delayed, and thetwo events became increasingly overlapped (Fig. 7C). Thus, a timely and synchronoustransition from ATM to ATR relies on ATM activity.

To more vigorously test if the ATM-to-ATR switch is driven by DSB resection in cells, weasked if it is possible to promote the switch by enhancing the functions of nucleases. CtIP,an activator of MRN (Sartori et al., 2007), and Exo1 were co-expressed in cells (Fig. S6). Inthe absence of IR, expression of CtIP and Exo1 did not induce significant ATM and Chk1phosphorylation (Fig. 7D). In response to IR, CtIP and Exo1 diminished Chk2phosphorylation but enhanced Chk1 phosphorylation (Fig. 7D), consistent with a moreefficient ATM-to-ATR switch. Although ATM phosphorylation was not compromised incells expressing CtIP and Exo1 (Fig. S6), its retention to chromatin was reduced (Fig. 7D).Interestingly, the IR-induced Chk1 phosphorylation was abolished by KU-55933 in cellsexpressing CtIP and Exo1 (Fig. 7E), suggesting that ATM may act before CtIP and Exo1during DSB resection. These results lend strong support to the conclusion that the ATM-to-ATR switch is driven by DSB resection in cells.

DISCUSSIONBoth ATM and ATR are key regulators of the cellular response to DSBs, yet how exactlythey function in concert is not well understood. Recent studies revealed that ATM isrequired for the resection of DSBs (Jazayeri et al., 2006; Myers and Cortez, 2006), a processnecessary for ATR activation as well as homology-directed DNA repair. While these studiesestablished a critical function of ATM in initiating DSB response, they have not resolvedhow ATM and ATR function during the dynamic process of DSB resection, a crucial periodfor both damage signaling and DNA repair. A major obstacle to understanding thecoordination of ATM and ATR is that the DNA structural elements regulating ATMactivation have not been clearly defined. In this study, we developed an extract-based invitro assay in which both ATM and ATR can be activated by dsDNA in a DNA structure-regulated manner. Using this assay, we systematically characterized the DNA structuraldeterminants for ATM activation, as well as the orchestration of ATM and ATR at DSBs.

The results of this study addressed two important issues with regard to the activation ofATM at resected DSBs; Can ATM be activated by resected DSBs? If so, is ATM activatedby the ends of SSOs or the junctions of single/double-stranded DNA? Our results clearlydemonstrated that ATM can be activated in the presence of short SSOs and, furthermore,that ATM activation relies on the junctions of single/double-stranded DNA but not the endsof SSOs (Fig. 4). Given that the activation of ATM by dsDNA requires Nbs1, it is plausiblethat the MRN complex directly recognizes the junctions of single/double-stranded DNAwhen SSOs are present. We also show that paired DNA ends are important for thisrecognition step (Fig. 2E). The MRN complex bound to DNA ends may directly activateATM and/or initiate the nucleation of ATM at DSBs that leads to its activation. Therecognition of DNA ends by MRN may be a prerequisite for its DNA unwinding, tethering,or nuclease activities implicated in ATM activation (Costanzo et al., 2004;Jazayeri et al.,2008;Lee and Paull, 2005;Uziel et al., 2003).

Our results also reveal that the activation of ATM is coordinately regulated by three distinctDNA structural elements of DSBs: (1) DNA ends, (2) dsDNA, and (3) ssDNA. Notably,ATM activation is oppositely regulated by the two DNA structures accompanying DNAends: it is enhanced by flanking dsDNA, but hindered by SSOs. Both of these regulatorymechanisms operate in a length-dependent manner and may function in concert to



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quantitatively regulate ATM activation at DNA ends. Interestingly, ATM is involved in theresection of DNA breaks (Jazayeri et al., 2006), suggesting that the activation of ATMelicits an inhibitory feedback loop through SSO formation. While these results present aclear picture of how ATM activation is regulated by the structures of DNA at DSBs, how theDNA- and chromatin-mediated regulatory mechanisms are integrated during ATMactivation remains to be determined (Bakkenist and Kastan, 2003; Lou et al., 2006; You etal., 2007). The in vitro ATM activation assay described here may provide a new basis forfuture biochemical studies to dissect the concerted action of DNA and chromatin in ATMactivation.

The involvement of dsDNA/ssDNA junctions in ATM activation reveals an unexpectedsimilarity between the DNA structural specificities of ATM and ATR, suggesting that thechoice between activating ATM or ATR at a resected DSB is made by another DNAstructure. We have previously shown that ssDNA coated by RPA is the key structure thatenables the ATR-ATRIP kinase complex to recognize DSBs (Zou and Elledge, 2003). Ourfinding that SSOs interfere with ATM activation immediately raised the possibility thatATR activation is coupled with loss of ATM activation through ssDNA. Consistent with thismodel, RPA gradually accumulates at DSBs (Fig. 6C), whereas Nbs1 associates with DSBsrapidly and transiently when it is unable to retain on the flanking chromatin (Celeste et al.,2003). The activation of yeast Tel1(ATM) is attenuated as DSBs are progressively resected(Mantiero et al., 2007). We find that Chk2, a specific substrate of ATM, is rapidly andtransiently phosphorylated after IR treatment. Furthermore, the ATR-dependent Chk1phosphorylation lags behind Chk2 phosphorylation, and Chk1 phosphorylation increases asChk2 phosphorylation declines (Fig. 6). Collectively, these results provide compellingevidence that an ATM-to-ATR switch indeed occurs in human cells in response to DSBs.

How does the ATM-to-ATR switch occur at DSBs? The progressive attenuation of ATMactivation could be attributed to the loss of DNA structures that activate ATM, or to thegeneration of DNA structures that interfere with ATM activation. Our finding that SSOs donot directly affect the binding of purified MRN to dsDNA, and that ssDNA interferes withATM activation in extracts both in cis and in trans support the latter possibility. Both therecruitment of ATR-ATRIP and the interference with ATM activation are dependent on thelength of SSOs. As DSBs are progressively resected by nucleases, SSOs are graduallylengthened, simultaneously enhancing the abilities of SSOs to interfere with ATM activationand to promote ATR activation (Fig. 7F). We show that SSOs attenuate the binding of MRNto dsDNA in extracts (Fig. 3E), but facilitate the recruitment of RPA and ATRIP (Fig. 5B).These results suggest that SSOs promote a swap of DNA damage sensors at DSBs, revealingthe underlying mechanism for the ATM-to-ATR switch. Interestingly, recent studiessuggested that the resection of DSBs by nucleases is also a biphasic process: it is initiated bythe MRN-CtIP complex and then extended by the Exo1- or BLM-dependent mechanisms(Gravel et al., 2008;Mimitou and Symington, 2008;Zhu et al., 2008). It is plausible that therelease of MRN from resected DNA ends may link the nuclease switch with the ATM-to-ATR switch during DSB resection.

We propose that the ATM-to-ATR switch driven by DSB resection is the key mechanismthrough which the functions of ATM and ATR are coordinated and integrated. The ATM-to-ATR switch is distinct from, but complementary with the sequential activation of ATM andATR reported previously (Jazayeri et al., 2006). Together, these two regulatory mechanismsensure that ATM launches DSB response, whereas ATR plays a primary role in the secondphase of this dynamic process. The associations of ATM and ATR with DSBs are not onlytemporally but also spatially distinct (Bekker-Jensen et al., 2006), and are differentiallyinfluenced by the cell cycle (Huertas et al., 2008; Ira et al., 2004; Jazayeri et al., 2006;Zierhut and Diffley, 2008). Moreover, the levels of ATM and ATR activation can be fine



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tuned by DSB resection (Mantiero et al., 2007; Vaze et al., 2002; Zierhut and Diffley, 2008).The ATM-to-ATR switch may enable the two kinases to target distinct sets of downstreameffectors in a regulated and coordinated manner. For instance, this switch may couple ATMand ATR with distinct events during DSB repair. The ATM-to-ATR switch driven by DSBresection may provide a unifying mechanism that brings together the temporal, spatial, andquantitative regulations of ATM and ATR, thus orchestrating the collective checkpointresponse in human cells. This study has set the stage for future investigations to reveal howthe ATM-to-ATR switch conducts the specific functions of ATM and ATR during thebiphasic DSB response.

EXPERIMENTAL PROCEDURESCell Culture and Transfection

HeLa cells were cultured in DMEM supplemented with 10% FBS. ATM-deficient (AT)fibroblasts (FT169) and a derivative line complemented with the wild-type ATM (AT+ATM) were cultured in DMEM with 15 % FBS. HeLa cells were transfected twice with100 nM siRNA using the X-treamGENE transfection reagent (Roche) and were analyzed 4days after the first transfection. The sequences of the siRNAs used in this study are listed inthe Supplemental Data. Plasmid transfections of HeLa cells were performed withLipofectamin 2000 (Invitrogen) and the transfected cells were analyzed after 48 hr.

Preparation of Cellular ExtractsThe nuclear extracts used in the ATM/ATR activation assay were prepared following theDignam’s protocol (Dignam et al., 1983), except that 600 mM KCl was used in Buffer C.Chromatin fractionation was performed as described previously with modifications (Yangand Zou, 2006). Cells were washed with phosphate buffered saline (PBS) and resuspendedin Solution A [10 mM Hepes (pH 7.9) 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10%glycerol, 1 mM DTT, 1 mM Na2VO3, protease inhibitors], and Triton X-100 wassubsequently added to a final concentration of 0.1%. After a 5-min incubation on ice, thesamples were spun at low speed (1300g for 4 min) to separate soluble proteins (Sol.) andpermeablized nuclei. The resulting nuclei were lysed with Solution B [3 mM EDTA, 0.2mM EGTA, 1 mM DTT] and a chromatin-enriched fraction was isolated by centrifugation(1700g for 4 min). These pellets were subsequently extracted with Solution C [50 mM Tris(pH 8.0), 600 mM NaCl, 0.5% TritonX-100, 0.5% sodium deoxycholate, 1 mM EDTA, 1mM PMSF, 1 mM Na3VO4, 1 mM NaF, protease inhibitors] to release the chromatin-boundproteins (Chr.).

DNA OligonucleotidesTo generate DNA structures with dsDNA regions, equal moles of the complimentary DNAoligonucleotides were annealed. Streptavidin was mixed with DNA at a 3:1 ratio to blockthe biotinylated ends. The sequences of the DNA oligonucleotides used in this study arelisted in the Supplemental Data.

Extract-Based ATM Activation AssayNuclear extracts were supplemented with the Reaction Buffer (Buffer R), which brings thefinal buffer compositions to 10 mM Hepes (pH 7.6), 50 mM KCl, 0.1 mM MgCl2, 1 mMPMSF, 0.5 mM DTT, 1 mM ATP, 10 μg/ml creatine kinase, and 5 mM phosphocreatine.Various DNA structures were incubated in the supplemented extracts for 30 min at 37 °C.The extracts were then subjected to Western blotting, immunoprecipitation, or the DNAbinding assay (see below).



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DNA Binding AssayThe biotinylated DNA structures were attached to streptavidin-coated magnetic beadsaccording to the manufacture’s instruction (Dynal). The beads coated with DNA wereincubated with purified MRN as described in (Lee and Paull, 2005), or with nuclear extractsin the binding buffer [10 mM Tris-HCl, (pH 7.5), 100 mM NaCl, 10% glycerol, 0.01 %NP-40, and 10 μg/ml bovine serum albumin]. After a 30-min incubation at roomtemperature, beads were retrieved and washed 3 times with the binding buffer. The proteinsbound to beads were denatured in the SDS sample buffer, separated on SDS-PAGE, andanalyzed by Western blotting. The MRN complex was purified from insect cells infectedwith the baculoviruses expressing Mre11, Rad50, and Nbs1. The baculoviruses were kindlyprovided by Drs. Stephen Elledge (Harvard) and Tanya Paull (U. of Texas Austin).

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Drs. R. Abraham, B. Chen, D. Chen, J. Chen, D. Cortez, G. Li, S. Elledge, T. Paull, and L. Rasmussenfor reagents, Drs. S. Elledge and N. Dyson for comments on the manuscript, and members of the Zou lab fordiscussions. L. Z. is a V Scholar and an Ellison New Scholar. This work is supported by grants from NIH(GM076388), the Susan G. Komen Foundation, and the Breast Cancer Alliance. B. S. was partly supported by afellowship from the Tosteson Foundation.

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Figure 1. Double-stranded DNA-induced ATM Activation in Human Cell Extracts(A) dsDNA but not ssDNA induces the phosphorylation of ATM and Chk2 in aconcentration-dependent manner. Increasing concentrations (1.25, 12.5, 125, and 1250 nM)of ssDNA (ss70) or dsDNA (ds70) was incubated in HeLa cell nuclear extracts. In all thepanels in this figure, the levels of phospho-ATM (Ser1981), ATM, phospho-Chk2 (Thr68),and Chk2 were analyzed by Western blotting. (B) dsDNA-induced phosphorylation of ATMand Chk2 is inhibited by ATM inhibitor. ds70 (125 nM) was incubated in extracts in thepresence or absence of 10 μM KU-55933. (C) dsDNA-induced Chk2 phosphorylationdepends on ATM in extracts. ds70 (125 nM) was incubated in the nuclear extracts derivedfrom AT cells or the AT cells complemented with ATM. (D) dsDNA-inducedphosphorylation of ATM and Chk2 is Nbs1-dependent. ds70 (125 nM) was incubated in theextracts derived from cells treated with Nbs1 siRNA or cells mock treated. (E) dsDNA-induced phosphorylation of ATM and Chk2 is Ku70-independent. ds70 (125 nM) wasincubated in the extracts derived from cells treated with Ku70 siRNA or cells mock treated.



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Figure 2. The Activation of ATM by dsDNA is Length- and End-Dependent(A) dsDNA activates ATM in a length-dependent manner. ds20, ds40, and ds70 wereincubated in HeLa cell nuclear extracts at the indicated concentrations. The three DNAfragments were used at either equal molar concentrations (the left half) or equal DNA mass(the right half). The three panels represent the experiments done with three different sets ofDNA concentrations, which increase by 10-fold from top to middle, and from middle tobottom panel. ATM and phospho-ATM were analyzed by Western blotting. (B) The dsDNAproximal to breaks contributes to ATM activation. ds40, bubble (30-nt ssDNA flanked by20-bp dsDNA on each side), and ds70 (all at 12.5 nM) were incubated in extracts andanalyzed as above. (C) The binding of MRN to dsDNA is length-dependent. Biotinylatedds20, ds40, and ds70 (all at 6.25 pmole) were attached to streptavidin-coated beads andincubated with purified MRN. The DNA-bound Nbs1 and Rad50 were detected by Westernblotting. (D) ATM activation depends on DNA ends. ds20 (left) and ds70 (right) wereunmodified or biotinylated at all 4 DNA ends. ds20 (1.25 μM) and ds70 (12.5 nM) wereincubated with or without streptavidin and added to extracts. (E) Paired DNA ends arerequired for ATM activation. The paired or unpaired ends of a fork structure (20-bp dsDNAand 50-nt ssDNA; 1.25 μM) were blocked with streptavidin as indicated, and were analyzedin extracts as above. The DNA structures used in each experiment are depicted below thecorresponding panel, with the dots representing biotin.



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Figure 3. Regulation of ATM Activation by SSOs(A–B) SSOs of random sequences interfere with ATM activation in a manner dependent onthe length of SSO, but independent of the length of dsDNA. (A) ds20 (1.25 μM) and (B)ds70 (12.5 nM) with 3′ or 5′ SSOs at the indicated length were incubated in extracts. ATM,phospho-ATM, Chk2, and phospho-Chk2 were analyzed by Western blotting. (C) Poly ASSOs interfere with ATM activation in a length-dependent manner. ds20 (1.25 μM) with 3′or 5′ poly A SSOs at the indicated length was analyzed as above. (D) ssDNA interferes withATM activation more efficiently in cis than in trans. ds70 (12.5 nM), either alone or incombination with free 25-nt random ssDNA (25 or 50 nM), and ds70 with 3′ or 5′ 25-ntSSOs of random sequences (12.5 nM) were incubated in extracts and analyzed as above. (E)SSOs attenuate the binding of MRN to dsDNA in extracts, but not in an assay using purifiedproteins. ds70 and ds70 with 25-nt 3′ SSOs were biotinylated at the 3′ end of one of the twostrands. The DNA structures (1.25 pmole) were incubated with extracts or purified MRNcomplex and retrieved with streptavidin-coated beads. The Nbs1 and Mre11 associated withDNA were analyzed by Western blotting.



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Figure 4. ATM Activation Requires the Junctions of ssDNA and dsDNA(A–C) ATM activation is dependent on ssDNA/dsDNA junctions but not SSO ends. ds20with 5′ (A) or 3′ (B) 25-nt SSOs, and ds70 with 5′ 25-nt SSOs (C) were unmodified orbiotinylated at the junctions or the SSO ends. These ds20- and ds70-derived DNA structures(1.25 μM and 12.5 nM, respectively) were incubated with or without streptavidin and addedto extracts. ATM, phospho-ATM, Chk2, and phospho-Chk2 were analyzed by Westernblotting. (D) ATM is not activated by DNA nicks and gaps. A plasmid was nicked by N.BbvCI or linearized by SmaI (Fig. S4A). The resulting nicked plasmid was further treatedwith Exonuclease III for the indicated periods to generate ssDNA gaps. Uncut, linear,nicked, and gapped plasmids (4 ng/μl) were incubated in extracts and analyzed as above.



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Figure 5. Resection of DNA Ends Promotes an ATM-to-ATR Switch in vitro(A) Inhibition of ATM activation by resection of DNA ends. A plasmid linearized with HpaIwas treated with T7 exonuclease (T7) or Exonuclease III (III) to generate SSOs. Theresulting DNA (12 ng/μl) was incubated in extracts. (B) Recruitment of RPA and ATRIP toSSOs. ds20 and ds20 with 5′ or 3′ 50-nt poly A SSOs (1.25 pmole) were attached to beadsand incubated in extracts. The RPA and ATRIP associated with DNA were detected byWestern blotting. (C) The phosphorylation of RPA32 induced by unprocessed linearplasmid is dependent on ATM and DNA-PKcs. Linear plasmid (4 ng/μl) was incubated inextracts in the presence of KU-55933 and NU7026 (10 μM). RPA and phospho-RPA(Ser33) were analyzed by Western blotting. (D) Induction of RPA phosphorylation (Ser33)by resection of DNA ends. Linear plasmid was resected as in (A) and incubated in extractsin the presence of KU-55933 and NU7026 (10 μM). (E) The SSO-induced RPA32phosphorylation is independent of ATM and DNA-PKcs. Linear plasmid was mock treatedor processed by T7 exonuclease (2 min) and Exonuclease III (5 min). The resulting DNA (4ng/μl) was incubated in extracts with KU-55933 and NU7026 (10 μM), or Wortmannin (20μM). (F) The SSO-induced RPA32 phosphorylation is ATR-dependent. Nuclear extractswere prepared from cells treated with ATR siRNA or cells mock treated. Linear plasmid wasresected as in (E) and incubated in both extracts.



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Figure 6. Consecutive Activation of ATM and ATR in Cells(A) Distinct kinetics of Chk2 and Chk1 phosphorylation after IR. HeLa cells were treatedwith 10 Gy of IR and were analyzed at the indicated time points. The levels of phospho-Chk2 (Thr68), Chk2, phospho-Chk1 (Ser345), Chk1, and Tubulin (a loading control) in thesoluble extracts (Sol., see Procedures) were analyzed by Western blotting. (B) Transientaccumulation of phosphorylated ATM on chromatin after IR. HeLa cells were treated with10 Gy of IR and the chromatin fractions (Chr., see Procedures) and whole cell extracts(WCE) were prepared at the indicated time points. The levels of phospho-ATM (Ser1981),ATM and Orc2 (a loading control) were analyzed by Western blotting. (C) Immunostainingof RPA in IR-treated cells. HeLa cells were treated with 10 Gy of IR, and RPA32 wasstained at the indicated time points. (D) Quantifications of the cells positive for IR-inducedRPA foci in the experiment shown in (C). More than 200 cells were counted at each timepoint.



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Figure 7. Regulation of the ATM-to-ATR switch by ATM, CtIP, and Exo1(A) ATR is not required for the attenuation of ATM activation by DSB resection. HeLa cellstransfected with control siRNA or ATR siRNA were irradiated with IR (10 Gy) andanalyzed at the indicated time points. The levels of ATR, phospho-Chk2 (Thr68), Chk2,phospho-Chk1 (Ser345), and Chk1 were monitored by Western blotting. (B) ATM isrequired for DSB-induced ATR activation. HeLa cells were treated with KU-55933 (5 μM)or mock treated with DMSO for 30 min prior to the IR treatment, and analyzed as above atthe indicated time points. (C) Partial inhibition of ATM leads to delayed and reducedactivation of Chk2 and Chk1. HeLa cells were treated with KU-55933 (1 μM) or DMSO for30 min prior to IR and analyzed at the indicated time points. (D) Expression of CtIP andExo1 promotes the ATM-to-ATR switch in cells. HeLa cells were transfected with theplasmids encoding CtIP and Exo1, and were treated with IR. Chromatin and solublefractions were prepared from cells collected at the indicated time points. The levels ofphospho-ATM (Ser1981), Orc2, phospho-Chk2, (Thr68), Chk2, phospho-Chk1 (Ser345),and Chk1 in the indicated fractions were analyzed as above. (E) Expression of CtIP andExo1 cannot bypass the requirement of ATM for IR-induced ATR activation. Cellsoverexpressing CtIP and Exo1 were treated with KU-55933 (5 μM) or mock treated withDMSO for 30 min prior to IR. The levels of phospho-Chk1 (Ser345), and Chk1 wereanalyzed at the indicated time points. (F) A model for an SSO-orchestrated ATM-to-ATRswitch at DSBs.



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Single-Stranded DNA Orchestrates an ATM-to-ATR Switch at DNA Breaks