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• W2111420919 abstract "ATM and ATR are two master checkpoint kinases activated by double-stranded DNA breaks (DSBs). ATM is critical for the initial response and the subsequent ATR activation. Here we show that ATR activation is coupled with loss of ATM activation, an unexpected ATM-to-ATR switch during the biphasic DSB response. ATM is activated by DSBs with blunt ends or short single-stranded overhangs (SSOs). Surprisingly, the activation of ATM in the presence of SSOs, like that 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-damage sensors. Progressive resection of DSBs directly promotes the ATM-to-ATR switch in vitro. In cells, the ATM-to-ATR switch is driven by both ATM and the nucleases participating in DSB resection. Thus, single-stranded DNA orchestrates ATM and ATR to function in an orderly and reciprocal manner in two distinct phases of DSB response. ATM and ATR are two master checkpoint kinases activated by double-stranded DNA breaks (DSBs). ATM is critical for the initial response and the subsequent ATR activation. Here we show that ATR activation is coupled with loss of ATM activation, an unexpected ATM-to-ATR switch during the biphasic DSB response. ATM is activated by DSBs with blunt ends or short single-stranded overhangs (SSOs). Surprisingly, the activation of ATM in the presence of SSOs, like that 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-damage sensors. Progressive resection of DSBs directly promotes the ATM-to-ATR switch in vitro. In cells, the ATM-to-ATR switch is driven by both ATM and the nucleases participating in DSB resection. Thus, single-stranded DNA orchestrates ATM and ATR to function in an orderly and reciprocal manner in two distinct phases of DSB response. Double-stranded DNA breaks (DSBs) are among the most deleterious DNA lesions that threaten genomic integrity. DSBs are generated not only by exogenous DNA-damaging agents but also by normal cellular processes such as V(D)J recombination, meiosis, and DNA replication. Furthermore, increased amounts of DSBs are induced by oncogenic stresses during the early stage of tumorigenesis (Bartkova et al., 2005Bartkova J. Horejsi Z. Koed K. Kramer A. Tort F. Zieger K. Guldberg P. Sehested M. Nesland J.M. Lukas C. et al.DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.Nature. 2005; 434: 864-870Crossref PubMed Scopus (2119) Google Scholar). In response to DSBs, the ATM kinase phosphorylates and regulates a large number of substrates involved in DNA repair, DNA replication, and other cellular processes important for genomic stability (Matsuoka et al., 2007Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. et al.ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2153) Google Scholar). In addition to DSBs, ATM also responds to other cellular stresses such as hypoxia and chromatin alterations (Bakkenist and Kastan, 2003Bakkenist C.J. Kastan M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Nature. 2003; 421: 499-506Crossref PubMed Scopus (2533) Google Scholar, Bencokova et al., 2008Bencokova Z. Kaufmann M.R. Pires I.M. Lecane P.S. Giaccia A.J. Hammond E.M. ATM activation and signalling under hypoxic conditions.Mol. Cell. Biol. 2008; 29: 526-537Crossref PubMed Scopus (157) Google Scholar, Gibson et al., 2005Gibson S.L. Bindra R.S. Glazer P.M. Hypoxia-induced phosphorylation of Chk2 in an ataxia telangiectasia mutated-dependent manner.Cancer Res. 2005; 65: 10734-10741Crossref PubMed Scopus (69) Google Scholar). Mutations of ATM in humans result in ataxia-telangiectasia (AT), a genetic disorder associated with radiation sensitivity, neuron degeneration, immune deficiencies, premature aging, and predisposition to cancers (Shiloh and Kastan, 2001Shiloh Y. Kastan M.B. ATM: genome stability, neuronal development, and cancer cross paths.Adv. Cancer Res. 2001; 83: 209-254Crossref PubMed Scopus (246) Google Scholar). ATM is also one of the most frequently mutated kinases in human cancers (Greenman et al., 2007Greenman C. Stephens P. Smith R. Dalgliesh G.L. Hunter C. Bignell G. Davies H. Teague J. Butler A. Stevens C. et al.Patterns of somatic mutation in human cancer genomes.Nature. 2007; 446: 153-158Crossref PubMed Scopus (2220) Google Scholar). All evidence indicates that ATM is a crucial guardian of genomic integrity. The mechanisms by which ATM is activated have been under intensive investigation (Harper and Elledge, 2007Harper J.W. Elledge S.J. The DNA damage response: ten years after.Mol. Cell. 2007; 28: 739-745Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar). The activation of ATM coincides with the autophosphorylation of ATM at Ser1981 and the conversion of ATM oligomers to monomers (Bakkenist and Kastan, 2003Bakkenist C.J. Kastan M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Nature. 2003; 421: 499-506Crossref PubMed Scopus (2533) Google Scholar). The Mre11-Rad50-Nbs1 (MRN) complex is a sensor of DSBs and a direct activator of the ATM kinase (Lee and Paull, 2005Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (992) Google Scholar). While ATM is not solely regulated by MRN in vivo (Kanu and Behrens, 2007Kanu N. Behrens A. ATMIN defines an NBS1-independent pathway of ATM signalling.EMBO J. 2007; 26: 2933-2941Crossref PubMed Scopus (62) Google Scholar), its activation at DSBs is primarily mediated by MRN (Berkovich et al., 2007Berkovich E. Monnat Jr., R.J. Kastan M.B. Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair.Nat. Cell Biol. 2007; 9: 683-690Crossref PubMed Scopus (354) Google Scholar, Falck et al., 2005Falck J. Coates J. Jackson S.P. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage.Nature. 2005; 434: 605-611Crossref PubMed Scopus (932) Google Scholar, Kitagawa et al., 2004Kitagawa R. Bakkenist C.J. McKinnon P.J. Kastan M.B. Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway.Genes Dev. 2004; 18: 1423-1438Crossref PubMed Scopus (338) Google Scholar, You et al., 2005You Z. Chahwan C. Bailis J. Hunter T. Russell P. ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.Mol. Cell. Biol. 2005; 25: 5363-5379Crossref PubMed Scopus (319) Google Scholar). After the initial ATM activation by DSBs, ATM executes specific functions around the breaks through a chromatin-mediated mechanism involving H2AX, Mdc1, and other proteins (Lou et al., 2006Lou Z. Minter-Dykhouse K. Franco S. Gostissa M. Rivera M.A. Celeste A. Manis J.P. van Deursen J. Nussenzweig A. Paull T.T. et al.MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals.Mol. Cell. 2006; 21: 187-200Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, Stewart et al., 2003Stewart G.S. Wang B. Bignell C.R. Taylor A.M. Elledge S.J. MDC1 is a mediator of the mammalian DNA damage checkpoint.Nature. 2003; 421: 961-966Crossref PubMed Scopus (667) Google Scholar, Stucki et al., 2005Stucki M. Clapperton J.A. Mohammad D. Yaffe M.B. Smerdon S.J. Jackson S.P. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks.Cell. 2005; 123: 1213-1226Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar). Direct tethering of a large number of ATM molecules or its regulators to an array of binding sites activates ATM even in the absence of DSBs (Soutoglou and Misteli, 2008Soutoglou E. Misteli T. Activation of the cellular DNA damage response in the absence of DNA lesions.Science. 2008; 320: 1507-1510Crossref PubMed Scopus (239) Google Scholar), indicating that a critical function of DSBs in ATM activation is to nucleate ATM and its regulators at sites of DNA damage. The activation of ATM at and around actual DSBs is a stepwise process initiated by the breaks. Despite the clear involvement of DSBs in ATM activation, the exact DNA structural determinants for ATM activation have not been clearly defined. Furthermore, how the 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 has overlapping substrate specificity with ATM. Like ATM, ATR is critical for the full checkpoint response to DSBs (Brown and Baltimore, 2003Brown E.J. Baltimore D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance.Genes Dev. 2003; 17: 615-628Crossref PubMed Scopus (387) Google Scholar, Cortez et al., 2001Cortez D. Guntuku S. Qin J. Elledge S.J. ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (709) Google Scholar), indicating that ATM and ATR have nonredundant functions in this process. Unlike ATM, however, ATR also responds to a broad spectrum of DNA damage besides DSBs, especially the damage interfering with DNA replication. The recruitment of ATR to DSBs requires RPA-coated single-stranded DNA (RPA-ssDNA), a structure generated by the nuclease-mediated resection of DSBs (Zou and Elledge, 2003Zou L. Elledge S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.Science. 2003; 300: 1542-1548Crossref PubMed Scopus (1901) Google Scholar). The junctions between single- and double-stranded DNA, another structure associated with resected DSBs, are also important for ATR activation (MacDougall et al., 2007MacDougall C.A. Byun T.S. Van C. Yee M.C. Cimprich K.A. The structural determinants of checkpoint activation.Genes Dev. 2007; 21: 898-903Crossref PubMed Scopus (171) Google Scholar, Zou, 2007Zou L. Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response.Genes Dev. 2007; 21: 879-885Crossref PubMed Scopus (85) Google Scholar). Several nucleases and helicases, including MRN, CtIP, Exo1, and BLM, have been implicated in the resection of DSBs (Gravel et al., 2008Gravel S. Chapman J.R. Magill C. Jackson S.P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.Genes Dev. 2008; 22: 2767-2772Crossref PubMed Scopus (426) Google Scholar, Lengsfeld et al., 2007Lengsfeld B.M. Rattray A.J. Bhaskara V. Ghirlando R. Paull T.T. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex.Mol. Cell. 2007; 28: 638-651Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, Limbo et al., 2007Limbo O. Chahwan C. Yamada Y. de Bruin R.A. Wittenberg C. Russell P. Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination.Mol. Cell. 2007; 28: 134-146Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, Mimitou and Symington, 2008Mimitou E.P. Symington L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.Nature. 2008; 455: 770-774Crossref PubMed Scopus (721) Google Scholar, Sartori et al., 2007Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (918) Google Scholar, Schaetzlein et al., 2007Schaetzlein S. Kodandaramireddy N.R. Ju Z. Lechel A. Stepczynska A. Lilli D.R. Clark A.B. Rudolph C. Kuhnel F. Wei K. et al.Exonuclease-1 deletion impairs DNA damage signaling and prolongs lifespan of telomere-dysfunctional mice.Cell. 2007; 130: 863-877Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, Zhu et al., 2008Zhu Z. Chung W.H. Shim E.Y. Lee S.E. Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.Cell. 2008; 134: 981-994Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). Interestingly, ATM is required for the efficient resection of DSBs and the activation of ATR by DSBs (Jazayeri et al., 2006Jazayeri A. Falck J. Lukas C. Bartek J. Smith G.C. Lukas J. Jackson S.P. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks.Nat. Cell Biol. 2006; 8: 37-45Crossref PubMed Scopus (825) Google Scholar, Myers and Cortez, 2006Myers J.S. Cortez D. Rapid activation of ATR by ionizing radiation requires ATM and Mre11.J. Biol. Chem. 2006; 281: 9346-9350Crossref PubMed Scopus (234) Google Scholar, Yoo et al., 2007Yoo H.Y. Kumagai A. Shevchenko A. Shevchenko A. Dunphy W.G. Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs through phosphorylation of TopBP1 by ATM.J. Biol. Chem. 2007; 282: 17501-17506Crossref PubMed Scopus (87) Google Scholar). The sequential activation of ATM and ATR by DSBs suggests that the checkpoint response to DSBs is biphasic. Although ATM is clearly critical for the initial response to DSBs, how ATM and ATR orchestrate the second phase of checkpoint response is unclear. A particularly interesting question is how ATM and ATR are coordinated at the DSBs undergoing resection, a dynamic structure that integrates checkpoint signaling with DNA repair. While the respective activation of ATM and ATR by DSBs has been extensively studied, these kinases and the DNA structures regulating them have rarely been characterized as a whole. Furthermore, the fundamental question of how exactly ATM and ATR distinguish DNA-damage structures remains to be addressed. In this study, using a newly developed ATM/ATR activation assay, we show that the activation of ATM is regulated by multiple DNA structural elements of DSBs. More importantly, we reveal that ATM and ATR are activated by similar yet distinct DNA structures 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 of single-stranded overhangs (SSOs). SSOs simultaneously attenuate ATM activation and potentiate ATR activation, thereby promoting an ATM-to-ATR switch during the process of DSB resection. These findings provide mechanistic insights into how the DNA-damage specificities of ATM and ATR are distinct from each other and, furthermore, how ATM and ATR function in concert to bring about the biphasic DSB response. Biochemical studies using purified proteins or Xenopus extracts have shown that ATM can be activated by DNA fragments in vitro (Dupre et al., 2006Dupre A. Boyer-Chatenet L. Gautier J. Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex.Nat. Struct. Mol. Biol. 2006; 13: 451-457Crossref PubMed Scopus (166) Google Scholar, Lee and Paull, 2005Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (992) Google Scholar, Yoo et al., 2004Yoo H.Y. Shevchenko A. Shevchenko A. Dunphy W.G. Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses.J. Biol. Chem. 2004; 279: 53353-53364Crossref PubMed Scopus (106) Google Scholar, You et al., 2007You Z. Bailis J.M. Johnson S.A. Dilworth S.M. Hunter T. Rapid activation of ATM on DNA flanking double-strand breaks.Nat. Cell Biol. 2007; 9: 1311-1318Crossref PubMed Scopus (77) Google Scholar). To reveal the DNA structural determinants for ATM activation, we devised an in vitro ATM activation assay using human cell extracts and defined DNA structures. A 70 bp dsDNA fragment with blunt ends was generated by using two complementary ssDNA oligomers. In HeLa cell nuclear extracts, dsDNA, but not ssDNA, induced the phosphorylation of ATM at Ser1981 in a concentration-dependent manner (Figure 1A). The phosphorylation of Chk2 at Thr68, a known ATM substrate site in cells, was also induced by dsDNA (Figure 1A). The dsDNA-induced phosphorylation of ATM and Chk2 was inhibited by KU-55933, a specific ATM inhibitor, suggesting that these phosphorylation events are ATM dependent (Figure 1B). The dsDNA-induced phosphorylation of ATM and Chk2 was not detected in AT cell extracts but was detected in the extracts of the AT cells complemented with ATM (Figure 1C), confirming that the phosphorylation of Chk2 is ATM dependent. To further assess if the dsDNA-induced phosphorylation of ATM and Chk2 indeed reflects the 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 the activation of DNA-PKcs, another kinase responsive to DSBs. We generated extracts from the HeLa cells in which Nbs1 or Ku70 was depleted by siRNA. The induction of ATM and Chk2 phosphorylation by dsDNA was significantly diminished in the Nbs1-depleted extracts compared to the controls (Figure 1D). In marked contrast, in the extracts with reduced levels of Ku70, ATM and Chk2 were substantially phosphorylated even when no dsDNA was added (Figure 1E). This phosphorylation of ATM and Chk2 may be due to the genomic instability in Ku70-depleted cells, or the binding of MRN to the residual genomic DNA in extracts when Ku70 was removed. Despite this basal phosphorylation, ATM and Chk2 were further phosphorylated when dsDNA was added to the Ku70-depleted extracts. These results suggest that the DSB-induced phosphorylation of ATM and Chk2 in extracts, like that in cells, is dependent on Nbs1, but not on DNA-PKcs. To directly determine if ATM is activated by dsDNA in extracts, we measured the kinase activity 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 (see Figure S1 available online). Similar elevations of ATM kinase activity were observed in cells treated with ionizing radiation (IR) (Pandita et al., 2000Pandita T.K. Lieberman H.B. Lim D.S. Dhar S. Zheng W. Taya Y. Kastan M.B. Ionizing radiation activates the ATM kinase throughout the cell cycle.Oncogene. 2000; 19: 1386-1391Crossref PubMed Scopus (129) Google Scholar). Collectively, these results suggest that the activation of ATM by dsDNA in extracts closely resembles the activation of ATM by DSBs in cells. Using the in vitro assay above, we sought to systematically characterize the DNA structural determinants for ATM activation. Studies using purified proteins or Xenopus extracts have shown that ATM is activated by dsDNA in a length-dependent manner (Lee and Paull, 2005Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (992) Google Scholar, You et al., 2007You Z. Bailis J.M. Johnson S.A. Dilworth S.M. Hunter T. Rapid activation of ATM on DNA flanking double-strand breaks.Nat. Cell Biol. 2007; 9: 1311-1318Crossref PubMed Scopus (77) Google Scholar). In these studies, only the DNA fragments longer than 200 bp efficiently activated ATM (Lee and Paull, 2005Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (992) Google Scholar, You et al., 2007You Z. Bailis J.M. Johnson S.A. Dilworth S.M. Hunter T. Rapid activation of ATM on DNA flanking double-strand breaks.Nat. Cell Biol. 2007; 9: 1311-1318Crossref PubMed Scopus (77) Google Scholar). In HeLa extracts, however, even 12.5 nM of 70 bp dsDNA (1.5 × 1010 DNA ends μl−1) induced substantial ATM phosphorylation (Figure 2A). The high sensitivity of this assay allowed us to analyze short dsDNA fragments with defined structural features. We first asked whether and how ATM is activated by dsDNA in a length-dependent manner in human cell extracts. When present at the same molar concentrations or the same DNA mass, dsDNA of 70, 40, or 20 bp induced ATM phosphorylation in a length-dependent manner (Figure 2A). Since these dsDNA fragments are much shorter than the DNA of a single nucleosome, a length-dependent mechanism for ATM activation may operate on the nucleosome-free dsDNA immediately flanking the breaks. To investigate how the length of dsDNA contributes to ATM activation, we generated a “bubble” DNA structure by converting an internal 30 bp region of the 70 bp dsDNA into a single-stranded region (Figure 2B). The ability of the bubble structure to induce ATM phosphorylation was between those of the 70 and the 40 bp dsDNA (Figure 2B), showing that the internal region of the 70 bp dsDNA contributes to the length-dependent activation of ATM. Using purified MRN complexes, we found that greater amounts of Nbs1 and Rad50 associated with 70 bp dsDNA than with 40 and 20 bp dsDNA (Figure 2C). These results suggest that the MRN complex associates with nucleosome-free dsDNA in a length-dependent manner, providing a possible mechanism for ATM activation along dsDNA. To assess if the ends of dsDNA are critical for ATM activation, we biotinylated all four DNA ends of the 20 and 70 bp fragments (5′ and 3′ ends of both strands). The biotinylated dsDNA efficiently induced ATM phosphorylation in the absence of streptavidin but lost this activity when the ends were blocked by streptavidin (Figure 2D). When only the 5′ or 3′ ends of 70 bp dsDNA were blocked, the ability of the fragment to activate ATM was substantially reduced (Figure S2). Since the streptavidin on one DNA strand may block access to both strands, it was not possible to resolve how 5′ or 3′ ends contribute to ATM activation. Nonetheless, blockage of DNA ends inhibited ATM activation regardless of the length of dsDNA, suggesting that the length-dependent mechanism for ATM activation needs 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 a fork-like DNA structure that possesses both paired and unpaired DNA ends (Figure 2E). The ability of the fork structure to activate ATM was lost when the paired ends were blocked but was unaffected when the unpaired ends were blocked (Figure 2E). Therefore, paired DNA ends are required for initiating ATM activation in extracts. These results suggest that ATM cannot be directly activated by unwound DNA ends or by the fork-like DNA structures associated with DNA replication or DNA repair. DSBs are not always blunt ended in cells. The DSBs generated by the HO or I-SceI endonuclease initially have 4 nt 3′ single-stranded overhangs (SSOs) (Colleaux et al., 1988Colleaux L. D'Auriol L. Galibert F. Dujon B. Recognition and cleavage site of the intron-encoded omega transposase.Proc. Natl. Acad. Sci. USA. 1988; 85: 6022-6026Crossref PubMed Scopus (219) Google Scholar, Kostriken et al., 1983Kostriken R. Strathern J.N. Klar A.J. Hicks J.B. Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae.Cell. 1983; 35: 167-174Abstract Full Text PDF PubMed Scopus (189) Google Scholar). V(D)J recombination and meiosis produce DSBs with 3′ and 5′ SSOs, respectively (Schlissel, 1998Schlissel M.S. Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination.Mol. Cell. Biol. 1998; 18: 2029-2037Crossref PubMed Scopus (66) Google Scholar, Xu and Kleckner, 1995Xu L. Kleckner N. Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast.EMBO J. 1995; 14: 5115-5128PubMed Google Scholar). The DSBs resulting from collapsed replication forks or broken ssDNA gaps may possess either 3′ or 5′ SSOs (Lopes et al., 2006Lopes M. Foiani M. Sogo J.M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.Mol. Cell. 2006; 21: 15-27Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). The “uncapped” telomeres resemble DSBs with 3′ SSOs (Celli and de Lange, 2005Celli G.B. de Lange T. DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion.Nat. Cell Biol. 2005; 7: 712-718Crossref PubMed Scopus (445) Google Scholar). When exposed in cells, DSBs can be resected by exo- or endonucleases in the 5′-to-3′ direction (Lee et al., 1998Lee S.E. Moore J.K. Holmes A. Umezu K. Kolodner R.D. Haber J.E. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage.Cell. 1998; 94: 399-409Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). ATM has been implicated in the response to the various types of DSBs above, indicating that it can be activated by DSBs with SSOs. In extracts, while the bulk of dsDNA appeared unaltered (Figure S3), a small fraction of it might be processed by nucleases. The in vivo functions of ATM in the response to SSO-bearing DSBs prompted us to investigate the role of SSOs in ATM activation. To directly assess the effects of SSOs on ATM activation, we analyzed the 20 and 70 bp dsDNA bearing either 5′ or 3′ SSOs of random sequences (Figures 3A and 3B). Both 5′ and 3′ SSOs of 5 nt slightly enhanced ATM phosphorylation. Interestingly, both 5′ and 3′ SSOs of 25 or 50 nt attenuated ATM and Chk2 phosphorylation (Figures 3A and 3B), suggesting that SSOs interfere with ATM activation in a length-dependent manner. SSOs of poly(A) also hindered ATM activation in a length-dependent manner (Figure 3C). SSOs not only attenuated the activation of ATM by 20 and 70 bp dsDNA but also that by linear plasmids (see Figure 5). Together, these results suggest that SSOs may interfere with a DNA end-dependent event in 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 1:5 molar ratios, a modest reduction of ATM activation was observed (Figure 3D). When the 25 nt ssDNA was linked to 70 bp dsDNA as overhangs, it interfered with ATM activation more effectively. 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 SSOs affect the binding of MRN to dsDNA. Indeed, 3′ SSOs of 25 nt substantially reduced the amounts of Nbs1 and Mre11 associated with 70 bp dsDNA in extracts (Figure 3E). However, purified MRN bound to dsDNA efficiently regardless of the presence or absence of SSOs (Figure 3E). Together, these results suggest that SSOs do not directly interfere with the binding of MRN to dsDNA, but they reduce MRN binding in the presence of other proteins. Although less potent than blunt-ended dsDNA, dsDNA bearing short SSOs retains some ability to associate with MRN and to active ATM in extracts. Our analysis of blunt-ended dsDNA suggests that ATM activation is dependent on DNA ends (Figure 2). Two types of DNA ends are present in the DNA fragments with SSOs: the ends of the dsDNA region (the junctions of dsDNA/ssDNA) and the ends of SSOs (Figure 4A). To assess how these DNA ends contribute to ATM activation, we tested three sets of DNA structures (20 bp dsDNA with 5′ or 3′ 25 nt SSOs and 70 bp dsDNA with 5′ 25 nt SSOs) in which either the junctions or the SSO ends were biotinylated (Figures 4A–4C). In the absence of streptavidin, all of the DNA structures with SSOs induced ATM phosphorylation at reduced levels compared to blunt-ended dsDNA (Figures 3A–3D). When the ends of the 5′ or 3′ SSOs were blocked by streptavidin, the ability of the DNA fragments to activate ATM and Chk2 was not affected (Figures 4A–4C). In striking contrast, when the 5′ or 3′ junctions of dsDNA/ssDNA were blocked by streptavidin, the DNA fragments failed to activate ATM and Chk2 (Figures 4A–4C). These results suggest that the junctions of dsDNA/ssDNA, but not the ends of SSOs, are critical for ATM activation. Furthermore, the junctions of dsDNA/ssDNA are required for ATM activation regardless of the length of dsDNA (Figures 4A–4C), suggesting that these ends are involved in an initiating event for ATM activation, possibly the DNA recognition by MRN. The junctions of dsDNA/ssDNA are present not only at DSBs but also at single-stranded DNA breaks, gaps, and DNA replication forks. To assess if dsDNA/ssDNA junctions are sufficient to activate ATM, we generated a plasmid carrying a single cleavage site of the nicking enzyme N. BbvCI (Figure S4A). Using the nicking enzyme or a restriction enzyme that cuts the plasmid in both DNA strands, we generated nicked plasmids and linear plasmids bearing blunt ends (Figure S4B). Like the short dsDNA fragments, linear plasmids induced ATM phosphorylation (Figure 4D). In contrast, nicked plasmids were unable to induce any ATM phosphorylation (Figure 4D). Moreover, when the DNA nicks were extended into ssDNA gaps by exonuclease III (Figure S4C), the gap-carrying plasmids were still unable to activate ATM (Figure 4D). Thus, while the junctions of dsDNA/ssDNA are required for ATM activation at DSBs, they are not sufficient to elicit ATM response when present internally on DNA. These internal junctions may be recognized by proteins that inhibit ATM activation. Alternatively, additional structural features of DSBs, such as the topological state of DNA (Figure S4B), may be involved in ATM activation. The involvement of dsDNA/ssDNA junctions in ATM activation is surprising, because these structures have been implicated in the activation of ATR (MacDougall et al., 2007MacDougall C.A. Byun T.S. Van C. Yee M.C. Cimprich K.A. The structural determinants of checkpoint activa" @default.
• W2111420919 created "2016-06-24" @default.
• W2111420919 creator A5026961383 @default.
• W2111420919 creator A5077786047 @default.
• W2111420919 date "2009-03-01" @default.
• W2111420919 modified "2023-10-15" @default.
• W2111420919 title "Single-Stranded DNA Orchestrates an ATM-to-ATR Switch at DNA Breaks" @default.
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