Breaking down cell cycle checkpoints and DNA repair during ... - Nature

Oncogene (2007) 26, 7759–7764

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REVIEW

Breaking down cell cycle checkpoints and DNA repair during antigen receptor gene assembly E Calle´n1, MC Nussenzweig2,3 and A Nussenzweig1 1 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA and 3Howard Hughes Medical Institute, New York, NY, USA

Double-strand breaks (DSBs) are intermediates in several physiological processes including V(D)J and class switch recombination. They are also potent substrates for chromosomal translocations that arise as by-products of antigen receptor gene assembly in lymphocytes. ATM is one among several key proteins involved in the detection, signaling and repair of DNA breaks. Despite redundancies in DSB signaling pathways, it has recently been demonstrated that ATM deficient lymphocytes can survive and proliferate several generations in vitro and in vivo despite harboring terminally deleted chromosomes produced by V(D)J recombination. In this review, we discuss how two complementary genome maintenance functions mediated by ATM prevent lymphocytes from adapting to persistent DNA damage. Oncogene (2007) 26, 7759–7764; doi:10.1038/sj.onc.1210873 Keywords: DNA repair; cell cycle checkpoints; classswitching; V(D)J recombination; ATM; lymphoma

Physiological DNA damage in lymphocytes Lymphocytes develop a broad repertoire of antigen receptors able to recognize foreign antigens by reactions that require programmed DNA damage. Diversification is initiated by expression of RAG1/2 proteins which introduce DNA double-strand breaks (DSBs) at variable (V), diversity (D) and joining (J) gene segments, in a reaction called V(D)J recombination. Joining of the gene segments by non-homologous end-joining (NHEJ) leads to assembly of a wide variety of antigen receptor genes in both T and B lymphocytes. Four separate loci recombine in T cells to produce T-cell receptors TCRa/b or TCRg/d and three loci in B cells to produce IgH/k or IgH/l antibodies. Production of cell membrane-anchored antigen receptors regulates V(D)J recombination to ensure that each lymphocyte expresses only a single antigen receptor gene and that only cells expressing functional receptors can complete development Correspondence: Dr E Callen, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1360, USA. E-mail: [email protected]

(Nussenzweig et al., 1987, 1988). B cells further diversify antibody genes during immune responses to produce antibodies with higher affinity and a broad range of effector functions through two reactions, class-switch recombination (CSR) and somatic hypermutation. CSR is a region-specific DNA recombination reaction that replaces antibody constant region genes and somatic hypermutation introduces point mutations in antibody variable regions. Both of these reactions are initiated by AID (activation-induced deaminase), an enzyme that introduces uracil–guanine mismatches in DNA (Jankovic et al., 2007). AID-induced mismatches can be processed by error-prone repair pathways to produce somatic mutation or excised to create the obligate DSB intermediates for CSR. CSR-induced breaks in the switch region are joined together by core components of the NHEJ machinery, but alternative end-joining pathways may also contribute to this reaction (Nussenzweig and Nussenzweig, 2007; Yan et al., 2007). DSBs are particularly dangerous lesions because they can lead to chromosomal abnormalities, cell death and neoplastic transformation. These lesions are initially recognized by sensors that activate phosphatidyl-inositol 3 kinase-like protein kinases, which amplify the initial damage response and activate effectors such as p53, which regulate cell cycle, apoptosis and DNA repair pathways. At least three phosphatidyl-inositol 3 kinase-like protein kinase family members are involved in coordinating DSB responses in mammalian cells: ataxia telangiectasia mutated (ATM) is recruited to and activated by DSBs via an interaction with the Mre11/ Rad50/Nbs1 (MRN) sensor complex; DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to DSBs by Ku; and the ATM and Rad-3 related kinase (ATR) is recruited to RPA-coated single strand lesions generated at stalled replication forks or following the resection of DSBs. During V(D)J and class switch recombination, DNA damage response proteins such as Nbs1, 53BP1, phosphorylated H2AX and ATM localize to DSBs and the chromatin region surrounding the recombining loci, forming nuclear foci (Perkins et al., 2002; Jankovic et al., 2007). Foci formation at antigen-receptor loci requires RAG-mediated cleavage in developing lymphocytes and is dependent on AID in cells undergoing CSR (Chen et al., 2000; Petersen et al., 2001). Loss of ATM

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Figure 1 Various roles of ataxia telangiectasia mutated (ATM) in response to RAG- or AID-induced breaks. From top to bottom: (1) ATM promotes the repair of V(D)J recombination-induced breaks by the non-homologous end-joining (NHEJ) pathway. (2) ATM facilitates the resolution of class-switch recombination (CSR) breaks by a Ku-dependent end-joining pathway. (3) ATM prevents double-strand breaks from being channeled into a Ku-independent alternative NHEJ (A-NHEJ) pathway that creates aberrant translocations. (4) Deregulated expression of an oncogene after translocation can evoke an ‘oncogenic stress’ response that induces an ATM-dependent checkpoint that induces arrest/apoptosis. (5) The ATM checkpoint also prevents the persistence and propagation of chromosome breaks.

does not abrogate V(D)J recombination, but significantly impairs the ability to repair coding ends, resulting in the loss of chromosome integrity (Bredemeyer et al., 2006; Matei et al., 2006; Huang et al., 2007; Vacchio et al., 2007). Approximately 10% of primary ATM / T cells harbor chromosomal aberrations associated with TCRa loci (Liyanage et al., 2000; Callen et al., 2007; Huang et al., 2007), and these lesions are dependent on RAG-mediated cleavage (Callen et al., 2007). Consistent with this, RAG is essential for the rapid generation of thymomas in ATM / mice that carry TCRa translocations (Petiniot et al., 2000). It has been proposed that ATM activity facilitates NHEJ repair of V(D)J recombination associated breaks by stabilizing DNA ends in the RAG post-synaptic cleavage complex (Bredemeyer et al., 2006; Huang et al., 2007) (Figure 1). Deficiency in Nbs1, H2AX and 53BP1 also increases the frequency of TCRa translocations (Celeste et al., 2003; Difilippantonio et al., 2005; Ward et al., 2005; Morales et al., 2006). However, the defect is less severe than in ATM / mice, and Nbs1, H2AX and 53BP1-deficient mice are not highly prone to spontaneous lymphomas (Bassing et al., 2003; Celeste et al., 2003; Difilippantonio et al., 2005; Ward et al., 2005; Morales et al., 2006). Moreover, in contrast to ATM knockouts, no impairment in the joining of V(D)J recombination associated breaks have been detected in H2AX / and 53BP1 / mice (Bassing et al., 2002; Manis et al., 2004; Ward et al., 2004). In contrast to V(D)J recombination, CSR is nearly completely dependent on 53BP1 (Manis et al., 2004; Ward Oncogene

et al., 2004; Reina-San-Martin et al., 2007), but only mildly defective in mice lacking Nbs1, ATM or H2AX (Celeste et al., 2002; Reina-San-Martin et al., 2003, 2004, 2005; Lumsden et al., 2004; Kracker et al., 2005). Although the precise function of ATM, 53BP1, H2AX and Nbs1 in the repair of CSR breaks remains to be defined, we have proposed that these proteins stabilize the synapsis of distal switch regions, potentially by modification of chromatin structure at sites of AID-dependent damage (Jankovic et al., 2007). In addition to facilitating the repair of V(D)J and CSR induced breaks, the ATM checkpoint protects against V(D)J- and CSR-associated translocations (Figure 1). As a result, AID-dependent c-myc/IgH translocations accumulate at a high frequency in B cells deficient for ATM (Ramiro et al., 2004; Callen et al., 2007). Similarly, p53 has been linked to the surveillance pathway that suppresses c-myc/IgH translocations (Ramiro et al., 2006a, b). We speculate that the cells that fail CSR and harbor free broken ends could be eliminated before the translocation by an ATM/p53 DNA damage response pathway. If DSBs are channeled into a translocation, this may induce oncogenic stress. For example, deregulation of c-myc caused by juxtaposition to the Ig enhancer leads to abnormal mitogenic signals. This form of stress can in turn activate p53 through the p19(ARF) tumor suppressor pathway, or may also induce an ATM-dependent DNA damage response (Shreeram et al., 2006). Thus, the ATM-dependent checkpoint may eliminate cells before or after they suffer an oncogenic translocation (Ramiro et al., 2006a) (Figure 1).

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Figure 2 The critical combination and synergy between two ataxia telangiectasia mutated functions in double-strand break (DSB) repair and checkpoint control prevents the persistence/survival of cells harboring RAG-induced DSBs through several rounds of replication during lymphocyte development.

We recently discovered an additional role for the ATM checkpoint, in preventing the persistence and transmission of RAG-dependent DSBs (Callen et al., 2007) (Figure 1). We found that in the absence of ATM, telomere-deleted chromosomes produced by RAG cleavage in B- and T-cell precursors could survive through several rounds of replication in vivo and in vitro, apparently without triggering the DNA damage alarm (Figure 2). As a result, translocations could form between RAG-dependent breaks and chromosomes subsequently broken by other means (for example irradiation, CSR, DNA replication) in a daughter cell.

Long-term persistence of chromosomal damage Classic cytogenetic studies of irradiated cells revealed that certain types of structural chromosomal aberrations persist, whereas others are selected against in proliferating cells (Kano and Little, 1984). For example, symmetric exchanges such as reciprocal chromosomal translocations, associated with lymphomagenesis, have been observed to persist for many cell doublings in vitro (Kano and Little, 1984), for decades in peripheral blood lymphocytes from atomic bomb survivors (Awa et al., 1978) and in rapidly dividing mouse bone marrow cells (Spruill et al., 1996). These lesions are thought to be stable because they are not associated with any loss of genetic material, and the derivative chromosome, which carries a single centromere and ends with telomeric DNA, remains structurally intact. In contrast, cells bearing asymmetric aberrations such as monocentric, acentric and dicentric chromosomes rapidly disappear

from populations of dividing cells (Kano and Little, 1984). These aberrations are either processed into stable translocations, or the mutant cells are deleted from the population due to loss of genetic material, induction of a DSB checkpoint, or mitotic catastrophe. Even in WT cells, unstable aberrations can occasionally escape recognition by the DNA damage response; for example, the probability that an irradiated lymphocyte containing an unstable chromosome aberration will survive the next mitosis has been estimated to be 35% (Sasaki and Norman, 1967). Norman et al. (1966) detected dicentric chromosome aberrations in peripheral lymphocytes from cervical cancer patients 800 days after radiation therapy. Thus, asymmetric aberrations, though less stable than symmetric aberrations, may nonetheless persist for a few cell divisions or even years in vivo. Finally, it has been demonstrated that the G2/M checkpoint has a threshold of 10–20 DSBs, and therefore cells from normal individuals can enter mitosis with chromosome breaks (Rothkamm and Lobrich, 2003; Deckbar et al., 2007) Although some breaks may escape detection, persistent breaks are rare under physiologic conditions. Low numbers of DSBs are constantly being generated in developing lymphocytes, and if there were no fail-safe mechanism to trigger a checkpoint when these lesions are not rapidly resolved, malignant transformation might be expected to be much more frequent. Studies by Guidos et al. (1996) showed that RAG-dependent breaks that are not resolved by V(D)J recombination trigger a DNA damage checkpoint mediated by the tumor suppressor p53. This may explain why mice deficient in NHEJ are not tumor-prone, and indeed combined absence of p53 and proteins involved in NHEJ (for example Ku80, Oncogene

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Ku70, DNA-PKcs, Artemis) or factors that survey V(D)J DSB intermediates (that is H2AX, 53BP1, Nbs1) leads to the rapid development of lymphomas (Bassing et al., 2003; Celeste et al., 2003; Difilippantonio et al., 2005; Ward et al., 2005; Morales et al., 2006). Thus, we should consider that different cell types and/or cell cycle phases may have distinct thresholds for the DSB checkpoint, with the activation threshold in lymphocytes being exquisitely sensitive to a single DSB generated in the G0/G1 phase of the cell cycle. The study by Callen et al. (2007) demonstrated that ATM plays a unique role in triggering the checkpoint that averts the propagation of asymmetric chromosome aberrations. It was found that 20% of mature splenic ATM / B cells harbor Ig heavy chain associated broken chromosomes lacking telomeres. Approximately half of these lesions were generated earlier in development, in immature B cells as a result of failed V(D)J recombination, and these cells undergo several rounds of division during maturation. Furthermore, dye dilution experiments in ATM / B and T cells demonstrated that there was no selection against the propagation of DSBs, at least during five rounds of division. This perplexing persistence of DNA damage over several generations suggests that a telomere-free chromosome end is insufficient to trigger a DNA damage response in ATM / lymphocytes despite the presence of ATR and DNA-PKcs damage transducers. Nevertheless, the telomere-free end is detected by DNA damage sensors, evidenced by the finding that 53BP1 accumulates into foci near the IgH locus in ATM / AID / mice (Callen et al., 2007). Moreover, transiently restoring ATM after accumulation of IgH-associated breaks led to the reduction in the number of aberrations, indicating that these DNA ends are still capable of inducing an ATMdependent checkpoint. Finally, irradiation-induced breaks generated in mature B cells were shown to fuse with persistent RAG-dependent breaks created in immature cells. This suggests that there is no protective structure at the end of the broken chromosome that prevents recruitment of DSB sensors or translocation.

Checkpoint adaptation The arrest of cell-cycle progression allows time for repair, but when repair is incomplete or fails, damaged cells are permanently removed from the population, which prevents the propagation of the lesion. In yeast, the presence of a single irreparable DSB is not lethal: after initial checkpoint arrest, the broken chromosome can be replicated and persists for up to 10 cell divisions (Sandell and Zakian, 1993; Toczyski et al., 1997; Lee et al., 1998; Kaye et al., 2004). In checkpoint deficient strains (for example RAD9, RAD17 or MEC1 (similar to ATM/ATR)), cells do not even delay the first cycle and they continue to divide despite chromosome loss and genomic instability (Galgoczy and Toczyski, 2001). Adaptation to irreparable DSBs has also been observed in Xenopus oocytes (Yoo et al., 2004) and most recently Oncogene

in human cells after irradiation (Syljuasen et al., 2006). In these systems, and in some tumor cells mitotic reentry requires overexpression of the polo-like kinase Plk1 and the subsequent inhibition of the ATR substrate Chk1 (Bertoni et al., 1999; Strebhardt and Ullrich, 2006; Syljuasen et al., 2006). We speculate that inhibition of ATR activation might also explain our observation that loss of ATM allows the propagation of terminally deleted chromosomes. Until recently, ATM and ATR were considered to function largely in two distinct DNA damage signaling and repair pathways, but studies from several laboratories demonstrated that these pathways overlap (Cuadrado et al., 2006; Jazayeri et al., 2006; Myers and Cortez, 2006; Stiff et al., 2006). It was shown that ATM together with the MRN complex are necessary for the processing of irradiation induced DSBs and generation of RPA-coated singlestranded DNA (ssDNA) required for ATRIP-dependent ATR recruitment and activation, and subsequent Chk1 phosphorylation. Another mechanism by which ATM might impact on ATR-dependent activation of Chk1 is by enhancing the association between the ATR activator TopBP1 through ATM-dependent phosphorylation (Yoo et al., 2007). Similarly, the interdependence of ATM and ATR signaling pathways might in part account for the absence of ATR-induced cell-cycle arrest in ATM deficient lymphocytes. In this model, absence of ATM would thwart either the processing of antigen receptor DSBs into ssDNA lesions or the phosphorylation of TopBP1, which would prevent the loading and activation of ATR. This would abrogate Chk1 activation, which in turn would allow cells to adapt to the presence of the break. Notably, however neither Nbs1 nor ATM is required for the activation of Chk1 in lymphocytes in response to replication stress (Difilippantonio et al., 2005; Pellegrini et al., 2006). Adaptation mutants in yeast eventually die after 10 divisions due to loss of genetic material (Sandell and Zakian, 1993). Exonucleolytic processing of V(D)J recombination coding ends has been observed in ATM / lymphocytes (Bredemeyer et al., 2006), and we also noted a striking degradation of chromosomes in approximately 2/3 of the aberrant metaphases (Callen et al., 2007). This was evident by the loss of hybridization of a probe designed to be at least 100 kb downstream of the initial RAG-dependent break sites. Thus, in many of the ATM / metaphases there is extensive chromosome degradation, and we would expect that replicating cells with telomere-free ends would eventually die if one copy of the intact homologous chromosome were not sufficient to support viability. Nevertheless, cells with broken chromosomes were shown to persist for at least 2 weeks in vivo (Callen et al., 2007).

Conclusions ATM regulates both DSB repair and checkpoints in many physiological settings including meiosis, V(D)J and class-switch recombination. Nevertheless, it has been very difficult to determine the precise role of ATM

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in DSB repair. Although it has been suggested that ATM might function in a structural capacity, possibly by recruiting NHEJ components, studies using ATM kinase inhibitors indicate that the kinase activity is important for normal V(D)J recombination (Bredemeyer et al., 2006). Nevertheless, the critical downstream target(s) of ATM that might help stabilize DSB ends during V(D)J recombination has not been identified. When DSB repair fails, the ATM-dependent checkpoint seems to be uniquely important for rapidly eliminating damaged cells and preventing oncogenic translocations. Although it remains to be proven, p53 may be a critical target of this ATM-dependent checkpoint. When both ATM-dependent repair and checkpoints are abrogated,

lymphocytes have the remarkable ability to neglect broken chromosomes without complete loss of reproductive capacity. Antigen receptor breaks may persist throughout B/T cell development in the bone marrow/ thymus while antigen receptor genes on other chromosomes are undergoing additional programmed cleavage and joining reactions, followed by several rounds of DNA replication. During this developmental window, persistent DSBs may be able to sample different chromosome territories and eventually recombine with a broken chromosome in a progeny cell. We speculate that this form of delayed genomic instability may account for the etiology of some mature B- and T-cell lymphomas that harbor antigen receptor translocations.

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