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ATM signaling and 53BP1
Radiotherapy and Oncology 76 (2005) 119–122 www.thegreenjournal.com
Review
ATM signaling and 53BP1 Omar Zgheiba,b, Yentram Huyena,b, Richard A. DiTullio Jra,b, Andrew Snydera, Monica Venerea,b, Elena S. Stavridia, Thanos D. Halazonetisa,c,* a
The Wistar Institute, USA, bBiomedical Graduate Studies, and cDepartment of Pathology and Laboratory Medicine, University of Pennsylvania, USA
Abstract The ATM (mutated in Ataxia-Telangiectasia) protein kinase is an important player in signaling the presence of DNA double strand breaks (DSBs) in higher eukaryotes. Recent studies suggest that ATM monitors the presence of DNA DSBs indirectly, through DNA DSB-induced changes in chromatin structure. One of the proteins that sense these chromatin structure changes is 53BP1, a DNA damage checkpoint protein conserved in all eukaryotes and the putative ortholog of the S. cerevisiae RAD9 protein. We review here the mechanisms by which ATM is activated in response to DNA DSBs, as well as key ATM substrates that control cell cycle progression, apoptosis and DNA repair. q 2005 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 76 (2005) 119–122. Keywords: ATM; 53BP1; NBS1; MRE11; RAD50; DNA damage checkpoint
Overview of checkpoint responses to DNA damage Of all the types of DNA damage, DNA double strand breaks (DSBs) represent the greatest threat to the integrity of the genome. To respond to this threat, eukaryotic cells have developed mechanisms that sense the presence of DNA DSBs and initiate a DNA damage response that is appropriate for the extent of the damage. This response includes DNA repair, but also a so-called ‘checkpoint’ response that leads to cell cycle delay or, in multicellular organisms, to programmed cell death or senescence [16,24,31]. The checkpoint response is critical for maintaining genomic integrity. The cell cycle delay provides time for DNA repair, as evidenced by rescue of the radiosensitive phenotype of yeast checkpoint mutants when cell cycle progression is experimentally slowed down [36]. In multicellular organisms genomic integrity is also maintained by programmed cell death or senescence, both of which prevent cells that might accumulate mutations from replicating and possibly developing into cancer [3,13]. Significant progress has been made in elucidating the DNA DSB checkpoint pathway. This pathway consists of sensors that sense the presence of DNA DSBs, signal transducers that generate and amplify the DNA damage signal and effectors that induce cell cycle delay, programmed cell death or senescence. The signal transducers and effectors have been characterized the most, although recently progress has been made in characterizing DNA DSB sensors. The signal transducers for DNA DSBs are the kinase ATM and its downstream kinase Chk2 [16,24,31]. These two kinases
constitute a kinase cascade that amplifies the DNA damage signal and phosphorylates multiple targets, which are substrates of either ATM or Chk2 or both. Most of these substrates are effectors of the DNA DSB checkpoint pathway. In what follows, we briefly describe the function and regulation of ATM, Chk2 and some of their best characterized substrates. We then conclude with recent insights regarding 53BP1, which turns out to be one of the sensors of DNA DSBs upstream of ATM.
ATM activation ATM belongs to a family of kinases that have sequence homology to phosphoinositide 3-kinase (PI3K) [31]. It is activated rapidly after formation of DNA DSBs in every phase of the cell cycle and phosphorylates serine or threonine residues that are followed by a glutamine. The mechanism by which ATM is activated in response to DNA DSBs is being elucidated (Fig. 1). In non-irradiated cells ATM exists as a dimer, is not phosphorylated and is present throughout the nucleus. After irradiation, which leads to formation of DNA DSBs, ATM becomes a monomer, it is phosphorylated on Ser1981 and some pool of it is present at sites of DNA DSBs [1]. It is well-established that ATM phosphorylation on Ser1981 represents autophosphorylation; however, precisely how ATM switches from its inactive (dimeric non-phosphorylated) to its active (monomeric phosphorylated) form is unclear. Studies in yeast suggest that recruitment of Tel1, the yeast ATM ortholog, to sites of DNA DSBs requires Nbs1, a protein that is rapidly recruited to sites of DNA DSBs
0167-8140/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2005.06.026
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Fig. 1. Current model of the ATM signaling pathway. ATM is activated by the Mre11–Rad50–Nbs1 complex (1) or 53BP1 (2). The former is thought to be recruited at the DNA double strand break (DSB), whereas the latter is recruited at chromatin regions flanking the DNA DSB and extending up to a few megabases from the DSB. ATM activated at sites of DNA DSBs may also phosphorylate and activate ATM in the nucleoplasm (3). A subset of ATM substrates (Chk2, p53, SMC1 and histone H2AX) and their function are also indicated.
and which exists in cells in complex with Rad50 and Mre11 [27]. Studies in yeast and human cells further indicate that suppressing Nbs1 or Mre11 function compromises significantly the conversion of ATM from its non-phosphorylated to phosphorylated form [7,25,32,33]. In addition, in vitro the ability of ATM to phosphorylate various substrates is enhanced in the presence of the Mre11–Rad50–Nbs1 (MRN) complex [20]. These observations suggest a model for ATM activation that involves recruitment of MRN to sites of DNA DSBs and subsequent recruitment and activation of ATM by MRN. However, other observations suggest that ATM activation may not occur exclusively at sites of DNA DSBs. Changes in chromatin structure can also activate ATM, even in the absence of DNA DSBs, suggesting a model whereby altered chromatin structures lead to phosphorylation of Ser1981 of ATM in trans and dissociation of inactive ATM dimers into active monomers [1]. A variation of this latter mechanism may also activate ATM molecules that are far away from the sites of DNA DSBs: an activated ATM molecule that has diffussed away from the sites of DNA DSBs may phosphorylate a dormant ATM dimer dissociating it into two active ATM monomers. Recent results suggest that 53BP1 is also involved in ATM activation, since suppression of 53BP1 leads to reduced ATM autophosphorylation [25]. A model on how 53BP1 could be involved in ATM activation is discussed below.
ATM substrates and role in the checkpoint response Once ATM is activated it phosphorylates multiple substrates [31]. Two of these, Chk2 and p53, mediate many of the cell cycle effects of ATM, while two others, SMC1 (structural maintainance of chromosomes 1) and histone H2AX, are important for cell survival after irradiation (Fig. 1). Chk2, is a protein kinase, that once activated amplifies the DNA damage signal of ATM. Two of the key substrates of Chk2 are Cdc25A and Cdc25C [5,12,23, 38]. Cdc25A and Cdc25C are protein phosphatases; Cdc25A activates Cdk2 and promotes progression through S phase, while Cdc25C activates Cdc2 and promotes progression from G2 into mitosis. When Cdc25A and Cdc25C become
phosphorylated by Chk2, their function is inhibited and cells delay progression through S phase or arrest in G2. Cell cycle arrest in G1 is mediated by p53, which is a substrate of both Chk2 and ATM [2,6,9,14,30]. p53 is a transcription factor that induces expression of p21/waf1, a Cdk2 inhibitor. p53 can also induce expression of genes that induce apoptosis; and in certain tissues, such as for example, the hematopoietic system, induction of p53 leads to apoptosis, rather than cell cycle arrest. It is not clear where in the cell Chk2 and p53 are phosphorylated by ATM. It is possible that activated ATM at sites of DNA DSBs phosphorylates pools of Chk2 and p53 that transiently associate with activated ATM. Alternatively, active ATM that is released from the sites of DNA DSBs or that is activated far away from the sites of DNA DSBs may phosphorylate Chk2 and p53 throughout the nucleus of the cell. What is clear, is that neither phosphorylated Chk2, nor p53, accumulate at sites of DNA DSBs [10,22]. Instead, activated Chk2 and p53 diffuse throughout the nucleus, so that Chk2 can phosphorylate its various substrates, while p53 can be targeted to the promoters of genes, whose expression is induced by p53. Unlike, Chk2 and p53, phosphorylated SMC1 and histone H2AX are found exclusively at sites of DNA DSBs [18,28,37]. Histone H2AX is part of chromatin and SMC1 is a chromatin-associated protein; thus, these two proteins do not diffuse freely in the cell, which may explain why their phosphorylated forms are found only at sites of DNA DSBs. SMC1 phosphorylation is critical for cells to survive after irradiation, as indicated by the observation that cells harboring a mutant form of SMC1 that cannot be phosphorylated by ATM are as radiosensitive as cells that lack ATM [19]. SMC1 phosphorylation is not required for cell cycle arrest, implying that it favors survival after irradiation by facilitating DNA repair. Similar to SMC1 phosphorylation, histone H2AX phosphorylation also appears to be important for DNA repair and is not required for cell cycle arrest [4,8]. The mechanisms by which SMC1 and histone H2AX phosphorylation facilitate DNA repair are not known, but recent evidence suggests that phosphorylated histone H2AX recruits chromatin remodeling complexes to sites of DNA DSBs [26,34].
O. Zgheib et al. / Radiotherapy and Oncology 76 (2005) 119–122
53BP1 as a sensor of DNA DSBs Recently, our laboratory has been interested in elucidating the mechanism by which 53BP1 is recruited to sites of DNA DSBs in an attempt to understand how cells sense the presence of DNA DSBs to then activate the DNA DSB checkpoint pathway. Two observations suggested that 53BP1 might be a sensor of DNA DSBs. First, 53BP1 recruitment to sites of DNA DSBs does not require ATM or other DNA damage checkpoint proteins; and, second, depletion of 53BP1 by siRNA leads to reduced ATM autophosphorylation [25,29]. Analysis of 53BP1 deletion mutants by several laboratories led to the identification of a 120 amino acid domain within 53BP1 that is sufficient and necessary for localization of 53BP1 to sites of DNA DSBs [17,35]. This domain is conserved in all eukaryotic 53BP1 orthologs, including the budding yeast Rad9 and fission yeast Rhp9/Crb2 DNA damage checkpoint proteins. To understand the mechanism by which this domain is recruited to sites of DNA DSBs, we solved its three-dimensional structure by X-ray crystallography. The domain was found to consist of two tandem tudor folds with a deep pocket at their interface formed by evolutionarily conserved hydrophobic residues. The presence of a deep hydrophobic pocket raised the possibility that 53BP1 binds to methylated lysine or arginine residues, because methylated lysine/arginine side chains are long and hydrophobic. In turn, this suggested that 53BP1 might bind to histones, because histones are often methylated. Indeed, in vitro the tandem tudor domain of 53BP1 bound histone H3 [15]. Mass spectrometry analysis of histone H3 bound to 53BP1 identified a single modification that correlated with 53BP1 binding: methylation of Lys79. Two observations suggested that the interaction of 53BP1 with histone H3 methylated on Lys79 mediates the recruitment of 53BP1 to sites of DNA DSBs. First, single amino acid substitutions of residues that form the walls of
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the 53BP1 hydrophobic pocket abrogated both binding to methylated histone H3 and also recruitment to sites of DNA DSBs. Second, depletion of Dot1L, the enzyme that methylates histone H3 on Lys79 in vivo, also inhibited recruitment of 53BP1 to sites of DNA DSBs. We considered two possibilities regarding how the interaction of 53BP1 with histone H3 may allow 53BP1 to sense DNA DSBs. Either methylation of Lys79 is enhanced at sites of DNA DSBs or DNA DSBs expose preexisting methylated lysines. In support of the second model, methylation of histone H3 Lys79 was not enhanced in response to DNA damage [15]. Further, Lys79 of histone H3 maps to the histone core (Fig. 2), which means that if nucleosomes stack to form higher order chromatin structures, then Lys79 would not be exposed [11,21]. However, a DNA DSB could lead to disruption of higher order chromatin structure, resulting in nucleosome unstacking and exposure of methylated Lys79 of histone H3. How does this model of DNA DSB sensing by 53BP1 fit into the existing models of ATM activation by DNA DSBs? Depletion of 53BP1 in normal and Nbs1-mutant cells suggests that 53BP1 and the MRN complex activate ATM via distinct pathways [25]. As stated above, activation of ATM by the MRN complex is dependent on the presence of DNA DSBs, which recruit MRN. A second pathway of ATM activation involves changes in chromatin structure [1]. It is likely, although not yet proven, that this pathway involves 53BP1. Changes in higher order chromatin structure that physiologically occur in response to DNA DSBs, but which experimentally can also be induced without DNA DSB formation, can result in recruitment of 53BP1 and subsequent ATM activation. Clearly, there are many unresolved questions regarding how eukaryotic cells respond to DNA DSBs. Yet, in the last 5 years there has been major progress in the field through the combined efforts of many research groups and collectively we may have sketched in broad terms the DNA DSB checkpoint pathway.
Fig. 2. Model for recognition of DNA double strand breaks (DSBs) by 53BP1. Ribbons representation of the structure of the nucleosome [21] showing the DNA (blue) and histones (yellow, except one histone H3 molecule in pink). The side chain of Lys79 (K79) of histone H3 is shown in red. Our model proposes that in the absence of a DNA DSB the nucleosomes are stacked and histone H3 Lys79 is not exposed. The DNA DSB leads to nucleosome unstacking, exposing Lys79 and resulting in recruitment of 53BP1.
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Acknowledgements The authors thank N. Pavletich, S. Berger, G. Dreyfuss, D. Speicher and R. Kaufman for support and helpful discussions. Work in the laboratory of T.D.H. is supported by the National Cancer Institute.
* Corresponding author. Thanos D. Halazonetis, The Wistar Institute, USA. E-mail address: [email protected] Received 9 May 2005; received in revised form 13 May 2005; accepted 18 June 2005; available online 15 July 2005
References [1] Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003;421:499–506. [2] Banin S, Moyal L, Shieh S, et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 1998;281:1674–7. [3] Bartkova J, Horejsi Z, Koed K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864–70. [4] Bassing CH, Chua KF, Sekiguchi J, et al. Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc Natl Acad Sci USA 2002;99:8173–8. [5] Boddy MN, Furnari B, Mondesert O, Russell P. Replication checkpoint enforced by kinases Cds1 and Chk1. Science 1998; 280:909–12. [6] Canman CE, Lim DS, Cimprich KA, et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998;281:1677–9. [7] Carson CT, Schwartz RA, Stracker TH, Lilley CE, Lee DV, Weitzman MD. The Mre11 complex is required for ATM activation and the G2/M checkpoint. Eur Mol Biol Org J 2003; 22:6610–20. [8] Celeste A, Petersen S, Romanienko PJ, et al. Genomic instability in mice lacking histone H2AX. Science 2002;296: 922–7. [9] Chehab NH, Malikzay A, Appel M, Halazonetis TD. Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev 2000;14:278–88. [10] DiTullio Jr RA, Mochan TA, Venere M, et al. 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat Cell Biol 2002;4:998–1002. [11] Dorigo B, Schalch T, Kulangara A, Duda S, Schroeder RR, Richmond TJ. Nucleosome arrays reveal the two-start organization of the chromatin fiber. Science 2004;306:1571–3. [12] Falck J, Mailand N, Syljuasen RG, Bartek J, Lukas J. The ATMChk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 2001;410:842–7. [13] Gorgoulis VG, Vassiliou LV, Karakaidos P, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907–13. [14] Hirao A, Kong YY, Matsuoka S, et al. DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 2000; 287:1824–7. [15] Huyen Y, Zgheib O, Ditullio Jr RA, et al. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 2004;432:406–11. [16] Iliakis G, Wang Y, Guan J, Wang H. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 2003; 22:5834–47. [17] Iwabuchi K, Basu BP, Kysela B, et al. Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA. J Biol Chem 2003;278:36487–95.
[18] Kim ST, Xu B, Kastan MB. Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev 2002;16:560–70. [19] Kitagawa R, Bakkenist CJ, McKinnon PJ, Kastan MB. Phosphorylation of SMC1 is a critical downstream event in the ATMNBS1-BRCA1 pathway. Genes Dev 2004;18:1423–38. [20] Lee JH, Paull TT. Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science 2004;304:93–6. [21] Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 Angstrom resolution. Nature 1997;389:251–60. [22] Lukas C, Melander F, Stucki M, et al. Mdc1 couples DNA doublestrand break recognition by Nbs1 with its H2AX-dependent chromatin retention. Eur Mol Biol Org J 2004;23:2674–83. [23] Matsuoka S, Huang M, Elledge SJ. Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 1998;282: 1893–7. [24] Melo J, Toczyski D. A unified view of the DNA-damage checkpoint. Curr Opin Cell Biol 2002;14:237–45. [25] Mochan TA, Venere M, DiTullio Jr RA, Halazonetis TD. 53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating ataxia-telangiectasia mutated (ATM) in response to DNA damage. Cancer Res 2003;63:8586–91. [26] Morrison AJ, Highland J, Krogan NJ, et al. INO80 and gammaH2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 2004;119:767–75. [27] Nakada D, Matsumoto K, Sugimoto K. ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev 2003;17:1957–62. [28] Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 1999;146:905–16. [29] Schultz LB, Chehab NH, Malikzay A, Halazonetis TD. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol 2000;151:1381–90. [30] Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev 2000;14:289–300. [31] Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155–68. [32] Theunissen JW, Kaplan MI, Hunt PA, et al. Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol Cell 2003;12:1511–23. [33] Uziel T, Lerenthal Y, Moyal L, Andegeko Y, Mittelman L, Shiloh Y. Requirement of the MRN complex for ATM activation by DNA damage. Eur Mol Biol Org J 2003;22:5612–21. [34] Van Attikum H, Fritsch O, Hohn B, Gasser SM. Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 2004;119:777–88. [35] Ward IM, Minn K, Jorda KG, Chen J. Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J Biol Chem 2003;278:19579–82. [36] Weinert TA, Hartwell LH. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science 1988;241:317–22. [37] Yazdi PT, Wang Y, Zhao S, Patel N, Lee EY, Qin J. SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint. Genes Dev 2002;16:571–82. [38] Zeng Y, Forbes KC, Wu Z, Moreno S, Piwnica-Worms H, Enoch T. Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature 1998; 395:507–10.
Review
ATM signaling and 53BP1 Omar Zgheiba,b, Yentram Huyena,b, Richard A. DiTullio Jra,b, Andrew Snydera, Monica Venerea,b, Elena S. Stavridia, Thanos D. Halazonetisa,c,* a
The Wistar Institute, USA, bBiomedical Graduate Studies, and cDepartment of Pathology and Laboratory Medicine, University of Pennsylvania, USA
Abstract The ATM (mutated in Ataxia-Telangiectasia) protein kinase is an important player in signaling the presence of DNA double strand breaks (DSBs) in higher eukaryotes. Recent studies suggest that ATM monitors the presence of DNA DSBs indirectly, through DNA DSB-induced changes in chromatin structure. One of the proteins that sense these chromatin structure changes is 53BP1, a DNA damage checkpoint protein conserved in all eukaryotes and the putative ortholog of the S. cerevisiae RAD9 protein. We review here the mechanisms by which ATM is activated in response to DNA DSBs, as well as key ATM substrates that control cell cycle progression, apoptosis and DNA repair. q 2005 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 76 (2005) 119–122. Keywords: ATM; 53BP1; NBS1; MRE11; RAD50; DNA damage checkpoint
Overview of checkpoint responses to DNA damage Of all the types of DNA damage, DNA double strand breaks (DSBs) represent the greatest threat to the integrity of the genome. To respond to this threat, eukaryotic cells have developed mechanisms that sense the presence of DNA DSBs and initiate a DNA damage response that is appropriate for the extent of the damage. This response includes DNA repair, but also a so-called ‘checkpoint’ response that leads to cell cycle delay or, in multicellular organisms, to programmed cell death or senescence [16,24,31]. The checkpoint response is critical for maintaining genomic integrity. The cell cycle delay provides time for DNA repair, as evidenced by rescue of the radiosensitive phenotype of yeast checkpoint mutants when cell cycle progression is experimentally slowed down [36]. In multicellular organisms genomic integrity is also maintained by programmed cell death or senescence, both of which prevent cells that might accumulate mutations from replicating and possibly developing into cancer [3,13]. Significant progress has been made in elucidating the DNA DSB checkpoint pathway. This pathway consists of sensors that sense the presence of DNA DSBs, signal transducers that generate and amplify the DNA damage signal and effectors that induce cell cycle delay, programmed cell death or senescence. The signal transducers and effectors have been characterized the most, although recently progress has been made in characterizing DNA DSB sensors. The signal transducers for DNA DSBs are the kinase ATM and its downstream kinase Chk2 [16,24,31]. These two kinases
constitute a kinase cascade that amplifies the DNA damage signal and phosphorylates multiple targets, which are substrates of either ATM or Chk2 or both. Most of these substrates are effectors of the DNA DSB checkpoint pathway. In what follows, we briefly describe the function and regulation of ATM, Chk2 and some of their best characterized substrates. We then conclude with recent insights regarding 53BP1, which turns out to be one of the sensors of DNA DSBs upstream of ATM.
ATM activation ATM belongs to a family of kinases that have sequence homology to phosphoinositide 3-kinase (PI3K) [31]. It is activated rapidly after formation of DNA DSBs in every phase of the cell cycle and phosphorylates serine or threonine residues that are followed by a glutamine. The mechanism by which ATM is activated in response to DNA DSBs is being elucidated (Fig. 1). In non-irradiated cells ATM exists as a dimer, is not phosphorylated and is present throughout the nucleus. After irradiation, which leads to formation of DNA DSBs, ATM becomes a monomer, it is phosphorylated on Ser1981 and some pool of it is present at sites of DNA DSBs [1]. It is well-established that ATM phosphorylation on Ser1981 represents autophosphorylation; however, precisely how ATM switches from its inactive (dimeric non-phosphorylated) to its active (monomeric phosphorylated) form is unclear. Studies in yeast suggest that recruitment of Tel1, the yeast ATM ortholog, to sites of DNA DSBs requires Nbs1, a protein that is rapidly recruited to sites of DNA DSBs
0167-8140/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2005.06.026
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Fig. 1. Current model of the ATM signaling pathway. ATM is activated by the Mre11–Rad50–Nbs1 complex (1) or 53BP1 (2). The former is thought to be recruited at the DNA double strand break (DSB), whereas the latter is recruited at chromatin regions flanking the DNA DSB and extending up to a few megabases from the DSB. ATM activated at sites of DNA DSBs may also phosphorylate and activate ATM in the nucleoplasm (3). A subset of ATM substrates (Chk2, p53, SMC1 and histone H2AX) and their function are also indicated.
and which exists in cells in complex with Rad50 and Mre11 [27]. Studies in yeast and human cells further indicate that suppressing Nbs1 or Mre11 function compromises significantly the conversion of ATM from its non-phosphorylated to phosphorylated form [7,25,32,33]. In addition, in vitro the ability of ATM to phosphorylate various substrates is enhanced in the presence of the Mre11–Rad50–Nbs1 (MRN) complex [20]. These observations suggest a model for ATM activation that involves recruitment of MRN to sites of DNA DSBs and subsequent recruitment and activation of ATM by MRN. However, other observations suggest that ATM activation may not occur exclusively at sites of DNA DSBs. Changes in chromatin structure can also activate ATM, even in the absence of DNA DSBs, suggesting a model whereby altered chromatin structures lead to phosphorylation of Ser1981 of ATM in trans and dissociation of inactive ATM dimers into active monomers [1]. A variation of this latter mechanism may also activate ATM molecules that are far away from the sites of DNA DSBs: an activated ATM molecule that has diffussed away from the sites of DNA DSBs may phosphorylate a dormant ATM dimer dissociating it into two active ATM monomers. Recent results suggest that 53BP1 is also involved in ATM activation, since suppression of 53BP1 leads to reduced ATM autophosphorylation [25]. A model on how 53BP1 could be involved in ATM activation is discussed below.
ATM substrates and role in the checkpoint response Once ATM is activated it phosphorylates multiple substrates [31]. Two of these, Chk2 and p53, mediate many of the cell cycle effects of ATM, while two others, SMC1 (structural maintainance of chromosomes 1) and histone H2AX, are important for cell survival after irradiation (Fig. 1). Chk2, is a protein kinase, that once activated amplifies the DNA damage signal of ATM. Two of the key substrates of Chk2 are Cdc25A and Cdc25C [5,12,23, 38]. Cdc25A and Cdc25C are protein phosphatases; Cdc25A activates Cdk2 and promotes progression through S phase, while Cdc25C activates Cdc2 and promotes progression from G2 into mitosis. When Cdc25A and Cdc25C become
phosphorylated by Chk2, their function is inhibited and cells delay progression through S phase or arrest in G2. Cell cycle arrest in G1 is mediated by p53, which is a substrate of both Chk2 and ATM [2,6,9,14,30]. p53 is a transcription factor that induces expression of p21/waf1, a Cdk2 inhibitor. p53 can also induce expression of genes that induce apoptosis; and in certain tissues, such as for example, the hematopoietic system, induction of p53 leads to apoptosis, rather than cell cycle arrest. It is not clear where in the cell Chk2 and p53 are phosphorylated by ATM. It is possible that activated ATM at sites of DNA DSBs phosphorylates pools of Chk2 and p53 that transiently associate with activated ATM. Alternatively, active ATM that is released from the sites of DNA DSBs or that is activated far away from the sites of DNA DSBs may phosphorylate Chk2 and p53 throughout the nucleus of the cell. What is clear, is that neither phosphorylated Chk2, nor p53, accumulate at sites of DNA DSBs [10,22]. Instead, activated Chk2 and p53 diffuse throughout the nucleus, so that Chk2 can phosphorylate its various substrates, while p53 can be targeted to the promoters of genes, whose expression is induced by p53. Unlike, Chk2 and p53, phosphorylated SMC1 and histone H2AX are found exclusively at sites of DNA DSBs [18,28,37]. Histone H2AX is part of chromatin and SMC1 is a chromatin-associated protein; thus, these two proteins do not diffuse freely in the cell, which may explain why their phosphorylated forms are found only at sites of DNA DSBs. SMC1 phosphorylation is critical for cells to survive after irradiation, as indicated by the observation that cells harboring a mutant form of SMC1 that cannot be phosphorylated by ATM are as radiosensitive as cells that lack ATM [19]. SMC1 phosphorylation is not required for cell cycle arrest, implying that it favors survival after irradiation by facilitating DNA repair. Similar to SMC1 phosphorylation, histone H2AX phosphorylation also appears to be important for DNA repair and is not required for cell cycle arrest [4,8]. The mechanisms by which SMC1 and histone H2AX phosphorylation facilitate DNA repair are not known, but recent evidence suggests that phosphorylated histone H2AX recruits chromatin remodeling complexes to sites of DNA DSBs [26,34].
O. Zgheib et al. / Radiotherapy and Oncology 76 (2005) 119–122
53BP1 as a sensor of DNA DSBs Recently, our laboratory has been interested in elucidating the mechanism by which 53BP1 is recruited to sites of DNA DSBs in an attempt to understand how cells sense the presence of DNA DSBs to then activate the DNA DSB checkpoint pathway. Two observations suggested that 53BP1 might be a sensor of DNA DSBs. First, 53BP1 recruitment to sites of DNA DSBs does not require ATM or other DNA damage checkpoint proteins; and, second, depletion of 53BP1 by siRNA leads to reduced ATM autophosphorylation [25,29]. Analysis of 53BP1 deletion mutants by several laboratories led to the identification of a 120 amino acid domain within 53BP1 that is sufficient and necessary for localization of 53BP1 to sites of DNA DSBs [17,35]. This domain is conserved in all eukaryotic 53BP1 orthologs, including the budding yeast Rad9 and fission yeast Rhp9/Crb2 DNA damage checkpoint proteins. To understand the mechanism by which this domain is recruited to sites of DNA DSBs, we solved its three-dimensional structure by X-ray crystallography. The domain was found to consist of two tandem tudor folds with a deep pocket at their interface formed by evolutionarily conserved hydrophobic residues. The presence of a deep hydrophobic pocket raised the possibility that 53BP1 binds to methylated lysine or arginine residues, because methylated lysine/arginine side chains are long and hydrophobic. In turn, this suggested that 53BP1 might bind to histones, because histones are often methylated. Indeed, in vitro the tandem tudor domain of 53BP1 bound histone H3 [15]. Mass spectrometry analysis of histone H3 bound to 53BP1 identified a single modification that correlated with 53BP1 binding: methylation of Lys79. Two observations suggested that the interaction of 53BP1 with histone H3 methylated on Lys79 mediates the recruitment of 53BP1 to sites of DNA DSBs. First, single amino acid substitutions of residues that form the walls of
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the 53BP1 hydrophobic pocket abrogated both binding to methylated histone H3 and also recruitment to sites of DNA DSBs. Second, depletion of Dot1L, the enzyme that methylates histone H3 on Lys79 in vivo, also inhibited recruitment of 53BP1 to sites of DNA DSBs. We considered two possibilities regarding how the interaction of 53BP1 with histone H3 may allow 53BP1 to sense DNA DSBs. Either methylation of Lys79 is enhanced at sites of DNA DSBs or DNA DSBs expose preexisting methylated lysines. In support of the second model, methylation of histone H3 Lys79 was not enhanced in response to DNA damage [15]. Further, Lys79 of histone H3 maps to the histone core (Fig. 2), which means that if nucleosomes stack to form higher order chromatin structures, then Lys79 would not be exposed [11,21]. However, a DNA DSB could lead to disruption of higher order chromatin structure, resulting in nucleosome unstacking and exposure of methylated Lys79 of histone H3. How does this model of DNA DSB sensing by 53BP1 fit into the existing models of ATM activation by DNA DSBs? Depletion of 53BP1 in normal and Nbs1-mutant cells suggests that 53BP1 and the MRN complex activate ATM via distinct pathways [25]. As stated above, activation of ATM by the MRN complex is dependent on the presence of DNA DSBs, which recruit MRN. A second pathway of ATM activation involves changes in chromatin structure [1]. It is likely, although not yet proven, that this pathway involves 53BP1. Changes in higher order chromatin structure that physiologically occur in response to DNA DSBs, but which experimentally can also be induced without DNA DSB formation, can result in recruitment of 53BP1 and subsequent ATM activation. Clearly, there are many unresolved questions regarding how eukaryotic cells respond to DNA DSBs. Yet, in the last 5 years there has been major progress in the field through the combined efforts of many research groups and collectively we may have sketched in broad terms the DNA DSB checkpoint pathway.
Fig. 2. Model for recognition of DNA double strand breaks (DSBs) by 53BP1. Ribbons representation of the structure of the nucleosome [21] showing the DNA (blue) and histones (yellow, except one histone H3 molecule in pink). The side chain of Lys79 (K79) of histone H3 is shown in red. Our model proposes that in the absence of a DNA DSB the nucleosomes are stacked and histone H3 Lys79 is not exposed. The DNA DSB leads to nucleosome unstacking, exposing Lys79 and resulting in recruitment of 53BP1.
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ATM signaling and 53BP1
Acknowledgements The authors thank N. Pavletich, S. Berger, G. Dreyfuss, D. Speicher and R. Kaufman for support and helpful discussions. Work in the laboratory of T.D.H. is supported by the National Cancer Institute.
* Corresponding author. Thanos D. Halazonetis, The Wistar Institute, USA. E-mail address: [email protected] Received 9 May 2005; received in revised form 13 May 2005; accepted 18 June 2005; available online 15 July 2005
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