ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses
Jun Yang1,2,
Yingnian Yu1,2,
Hope E. Hamrick3 and
Penelope J. Duerksen-Hughes4,5
1 Department of Pathology and Pathophysiology and 2 Department of Public Health, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310031, China, 3 Department of Psychology, Wellesley College, Wellesley, MA 02481, USA and 4 Center for Molecular Biology and Gene Therapy, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA
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Abstract
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Exposure to genotoxic agents is a major cause of human cancer, and cellular responses to genotoxic stress are important defense mechanisms. These responses are very complex, involving many cellular factors that form an extensive signal transduction network. This network includes a protein kinase cascade that connects the detection of DNA damage to the activation of transcription factors, which in turn regulate the expression of genes involved in DNA repair, cell cycle arrest and programmed cell death (apoptosis). The mitogen-activated protein kinases are the best-studied members of the kinase cascade with an acknowledged role in the genotoxic stress response. However, the initial activation of the protein kinase cascade is not fully understood, although several protein kinases, such as ataxia telangiectasia, mutated (ATM), ATM- and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) in humans, are increasingly recognized for their potential roles in the sensing of DNA damage and initiating the subsequent protein kinase cascade. In this review, the properties of these three kinases are discussed and their functions in the initiation of the genotoxic stress response are explored.
Abbreviations: ATM, ataxia telangiectasia, mutated; ATR, ATM- and Rad3-related; ATRIP, ATR-interacting protein; BRCA-1 breast cancer susceptibility gene 1; BRCT, BRCA-1 C-terminus; CPT, camptothecin; DNA-PK, DNA-dependent protein kinase; DSBs, double-strand breaks; FANCD2, Fanconi anaemia subtype D2 protein; FHA, forkhead-associated;
-H2AX, phosphorylated H2AX; HDAC, histone deacetylase; HR, homologous recombination; IR, ionizing irradiation; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; MAPKs, mitogen-activated protein kinases; MDC1, mediator of DNA damage checkpoint protein 1; NHEJ, non-homologous end-joining; PCNA, proliferating cell nuclear antigen; PI-3, phosphatidylinositol 3-kinase; PML, promyelocytic leukemia protein; PP1, protein phosphatase 1; RB, retinoblastoma protein; RPA, replication protein A; SAPK, stress-activated protein kinase; ssDNA, single-stranded DNA; TopBP1, DNA topoisomerase IIß binding protein 1; WRN, Werner's syndrome protein
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Introduction
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It has long been recognized that exposure to certain chemicals is associated with the development of human cancers. For example, this linkage has been made between aflatoxins and liver cancer, amine dyes and bladder cancer, benzene and leukemia, vinyl chloride and hepatic cancer and smoking and lung cancer. To date, many human carcinogens have been identified, most of which are categorized as genotoxic agents, meaning that these chemicals target DNA and produce alterations in the genetic material of the host (1,2). Such environmental agents are only one source of genotoxic stress, with other sources including UV and ionizing irradiation (IR), therapeutic agents and the products of normal metabolism. As a result, human cells are constantly exposed to a variety of genotoxic stresses. To cope with the resulting damage to cellular DNA, cells have developed a repertoire of responses that ensure the normal growth and survival of the organism. A logical consequence, therefore, is that alterations in these responses may form the basis for carcinogenesis. For this reason, a better understanding of the cellular responses to genotoxic stress is important in the prevention and treatment of human cancers.
Extensive studies from multiple disciplines have explored the mechanisms of the cellular genotoxic responses, leading to the identification of many of the cellular components. The genotoxic stress responses can be envisioned as a signal transduction cascade in which DNA lesions act as an initial signal that is detected by sensors and passed down through transducers. Eventually the effectors receive the signal and execute the various cellular functions. Over the years, much knowledge has been gained concerning the signal transducers, and a large group of serine-threonine protein kinases, named the mitogen-activated protein kinases (MAPKs), along with their upstream kinases, have been shown to play prominent roles in the cellular genotoxic responses (3). Three major classes of MAPKs, i.e. extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 (also known as SAPK2, RK, CSBP or Mxi2), can all be activated by various genotoxic stresses. The activated MAPKs then translocate to the nucleus and phosphorylate scores of target proteins, including many transcription factors. Among these transcription factors is the tumor suppressor p53 protein, which plays such an important role in the genotoxic stress responses that it has earned a reputation as the universal sensor for genotoxic stress (46).
Although the general picture of the MAPK cascade is relatively clear, the actual receptors or sensors for the DNA damage signal are still elusive. However, it is hypothesized that some multiprotein complexes that are involved in DNA maintenance or repair may also function as DNA damage sensors (79). For example, certain members of the Rad family, such as Rad1, Rad9, Rad17, Rad26 and Hus1, which can form protein complexes that function in cell checkpoint regulation, could act as potential sensors (7,8,1012). Others have suggested that members of the phosphatidylinositol 3-kinase (PI-3) superfamily, which are activated at very early stages of the DNA damage response, could serve as the sensors, as well as initiators, of the ensuing cellular genotoxic stress response (13,14). Members of this PI-3 family include Mec1p and Tel1p in the yeast Saccharomyces cerevisiae, Rad3 and Tel1 in Schizosaccharomyces pombe and ataxia telangiectasia, mutated (ATM), ATM- and Rad3-related (ATR), ATX/SMG-1, mTOR/FRAP and DNA-dependent protein kinase (DNA-PK) in humans. Although these proteins share the PI-3-like kinase domain, they do not function as lipid kinases, but rather as serine-threonine protein kinases (8,1517). In this review, we will focus on the three human kinases, namely ATM, ATR and DNA-PK, and discuss their role in the cellular genotoxic stress responses.
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ATM and ATR
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Signals that can activate ATM/ATR
Both ATM and ATR can be activated by DNA damage, although it is not known exactly how these two kinases sense such damage. However, ATM responds primarily to double-strand breaks (DSBs) induced by IR, while ATR also reacts to UV or stalled replication forks (7,8,1821). Nevertheless, there is no clean-cut differentiation between the signals for the two kinases, as ATM also functions in some UV responses, mediating the repair of UV-induced DNA damage and the phosphorylation of STAT3 (22,23). ATM is also involved in UVA-induced signaling and apoptosis, whereas ATR functions in UVC-induced signaling and apoptosis (24,25).
While DSBs have been regarded as the major signal to activate ATM, there is also evidence showing that ATM can be activated by signals other than DSBs under certain conditions. For example, chromium(VI) compounds, which cannot induce DSBs, can activate ATM (26). ATM is also involved in oxidative stress and it has been shown that ATM/ATR targets are phosphorylated by ATR in response to hypoxia and by ATM in response to reoxygenation (2729). Nitric oxide can induce the activation and phosphorylation of p53 through ATR/ATM during inflammation (30,31). Heat shock also induces phosphorylation of p53 at Ser15 in an ATM kinase-dependent fashion, which may contribute partially to heat-induced p53 accumulation (32). A human AMP-activated protein kinase family member, ARK5, when activated by Akt, can phosphorylate ATM under conditions of nutrient starvation (33). Interestingly, the integration of retrovial DNA (HIV-1 and avian sarcoma virus) triggers an ATR-dependent DNA damage response (34); the HIV-1 viral protein R, which is necessary and sufficient for the block of cellular proliferation, can activate ATR, resulting in Chk1 phosphorylation, just as does DNA damage (35).
Mechanism for the activation of ATM/ATR
Understanding how these two signaling kinases are activated may also help us understand how they function as damage sensors. It was previously thought that ATM and ATR might be activated both through interactions with DNA and with members of the repair complexes (36). For example, ATM can bind directly to DNA and pre-treatment of DNAcellulose matrix with IR or restriction enzymes stimulates ATM binding, suggesting that ATM binds to DNA ends (37,38). ATR also binds to DNA and with higher affinity to UV-damaged than to undamaged DNA. In addition, damaged DNA stimulates the kinase activity of ATR to a significantly higher level than does undamaged DNA (39,40). ATM and ATR also interact with many proteins that co-localize at the site of DNA damage to form foci (see following section for details).
However, recent findings have excluded a role of DNA in ATM activation. Kozlov and co-workers (41) showed that ATP can activate ATM by a mechanism involving autophosphorylation without the requirement for DNA. Furthermore, this process is very specific, as ATR and DNA-PK are not activated. In addition, cellular irradiation induces rapid intermolecular autophosphorylation, causes dimer dissociation and initiates cellular ATM kinase activity, and this process is not dependent on direct binding to DNA strand breaks (42). Therefore, ATM might be activated prior to binding to DNA and/or other proteins located in the foci.
Interaction with multiple proteins: the formation of foci
One striking feature following DNA damage is the aggregation of many protein factors at the damaged sites, thus forming foci. ATM/ATR can usually be found in these areas several minutes after the damage occurs, supporting a role for these two kinases in the damage sensing process. The other proteins involved include structural proteins of chromatin, proteins that function in chromosomal repair and maintenance and, on some occasions, transcription factors. Some of the better studied proteins present in foci are described briefly below.
Breast cancer susceptibility gene 1 (BRCA-1)
ATM forms part of a super-protein complex called the BRCA-1-associated genome surveillance complex, which is involved in the recognition and repair of aberrant DNA structures. This complex contains several other proteins, such as BRCA-1, the mismatch repair protein hRad50 and the BLM (Bloom's syndrome) helicase (43). It has been shown that BRCA-1 is required for the S and G2/M checkpoints and for the ATM- and ATR-dependent phosphorylation of p53, c-Jun, NBS1 (Nijimegen breakage syndrome) and Chk2 after treatment with IR or UV, respectively, as well as the ATM-dependent phosphorylation of CtIP, but not for the phosphorylation of Rad9, Hus1 and Rad17 or the re-localization of Rad9 and Hus1 (44). Based on these observations, it has been suggested that BRCA-1 might facilitate the ability of ATM and ATR to phosphorylate downstream substrates that directly influence cell cycle and apoptosis, but not necessarily the phosphorylation of DNA-associated ATM and ATR substrates (44).
BRCA-1 itself is probably a substrate of ATM/ATR. Phosphorylation of specific residues of BRCA-1 after DNA damage affects its localization and function (45). On the other hand, protein phosphatase 1 (PP1)
dephosphorylates BRCA-1. Furthermore, the two proteins co-immunoprecipitate and BRCA-1 regulates PP1
activity (46). It has also been shown that IR activates PP1
and PP1
by ATM-dependent dephosphorylation (47), thus forming a complex regulation network.
BRCA-1 also forms a constitutive complex with c-Abl. c-Abl has been shown to be involved in some functions of ATM, and ATM can phosphorylate Ser465 located within the kinase domain of c-Abl (4851). Following exposure to IR, the BRCA-1c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA-1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. Tyrosine phosphorylation of BRCA-1, however, is not required for disruption of the BRCA-1c-Abl complex. BRCA-1 mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. Therefore, BRCA-1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity (52).
H2AX
The histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage and this activity is phosphorylation dependent. ATR and DNA-PK are primarily responsible for the phosphorylation of H2AX (
-H2AX) at DSB.
-H2AX is required for MRN (Mre11Rad50NBS1) foci formation. (53). However, although the MRN complex co-localizes with proliferating cell nuclear antigen (PCNA) throughout S phase and the replication fork is a site of MRN complex chromatin loading, there is limited co-localization of the MRN complex with BRCA-1 and
-H2AX (54). 53BP1 also binds to
-H2AX at DNA breaks, though the phosphorylation of 53BP1 by ATM is not required for this activity (55).
Mediator of DNA damage checkpoint protein 1 (MDC1)
MDC1 (also referred to as NFBD1 or Kiaa0170), which contains the BRCA-1 C-terminus (BRCT) and forkhead-associated (FHA) domains, works with H2AX to form foci with
-H2AX. MDC1 also controls the formation of damage-induced 53BP1, BRCA-1 and MRN foci, in part by promoting efficient H2AX phosphorylation. Cells lacking MDC1 fail to activate the intra-S and G2/M checkpoints in response to IR and this lack was associated with an inability to regulate Chk1 properly (56,57). Furthermore, MDC1 regulates ATM-dependent phosphorylation events and down-regulation of MDC1 abolishes the relocalization and hyper-phosphorylation of BRCA-1 (58). MDC1 is phosphorylated in an ATM/Chk2-dependent manner and associates with Chk2, which requires its FHA domain and the phosphorylated Thr68 on Chk2 (59). However, down-regulation of MDC1 does not abolish IR-induced phosphorylation of NBS1, Chk2 and Smc1 or the degradation of Cdc25A (60).
Werner's syndrome protein (WRN)
WRN has 3'
5' exonuclease, DNA helicase and DNA-dependent ATPase activities. It is phosphorylated through an ATM/ATR-dependent pathway in response to replication blockage, although phosphorylation is not required for its subnuclear re-localization. WRN and ATR co-localize, suggesting that WRN and ATR collaborate to prevent genome instability during S phase (61). WRN also interacts with and increases the activity of Rad52, a recombination mediator protein (62). Subnuclear re-localization of WRN may require acetylation and it partially co-localizes with promyelocytic leukemia protein (PML) nuclear bodies (63). The PML tumor suppressor gene is consistently disrupted in patients with acute promyelocytic leukemia. It is involved in cell growth regulation, apoptosis, transcription regulation and genome stability. PML co-localizes with the DNA damage response protein DNA topoisomerase IIß binding protein 1 (TopBP1) in response to IR and regulates TopBP1 by stabilization of the protein. PML also co-localizes with Rad50, BRCA-1, ATM, Rad9 and BLM (64).
53BP1
The BRCT protein 53BP1, which associates with Mre11, BRCA-1 and
-H2AX, is important in the DNA damage responses and plays an integral role in maintaining genomic stability (65,66). 53BP1 binds to
-H2AX at DNA breaks, but the phosphorylation of 53BP1 by ATM is not required for this activity (55). 53BP1-/- cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression (67). This is further supported by a report showing that 53BP1 was localized to distinct nuclear foci in cancer cell lines expressing mutant p53 (68).
E2F1
Transcription factor E2F1 can be selectively stabilized through ATM-dependent phosphorylation in response to DNA damage. TopBP1, which contains eight BRCT motifs, interacts with the N-terminus of E2F1 via its BRCT motifs. This interaction is induced by DNA damage and the consequent phosphorylation of E2F1 by ATM, resulting in the suppression of E2F1 activity and its co-localization with TopBP1 and BRCA-1. These results suggest a direct role for E2F1 in DNA damage checkpoint/repair at stalled replication forks (69).
On the other hand, E2F1 elevates the ATM promoter activity and induces an increase in ATM at both the RNA and protein levels that is accompanied by an E2F-induced increase in p53 phosphorylation. Since E2F-1 is a critical downstream target of the tumor suppressor retinoblastoma protein (RB), ATM may thus provide a link between RB/E2F and p53 (70).
Other direct and indirect ATM/ATR interacting proteins
Replication protein A (RPA) associates with single-stranded DNA (ssDNA). RPA stimulates the binding of ATR-interacting protein (ATRIP) to ssDNA and this binding of ATRIP to RPA-coated ssDNA enables the ATRATRIP complex to associate with DNA and stimulate phosphorylation of Rad17 that is bound to DNA (71). ssDNA generated either by etoposide (a DNA topoisomerase II inhibitor) or exonuclease treatment can activate an ATRCdc7/Dbf4-dependent pathway, which also requires the loading of RPA onto chromatin (72). These data suggested that RPA-coated ssDNA is the critical structure at the sites of DNA damage that recruits the ATRATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.
ATM can bind to the histone deacetylase (HDAC)1 both in vitro and in vivo (73), while ATR associates with HDAC2 (74). It has been shown that HDAC inhibitors activate p21WAF1 expression, which requires ATM activity, suggesting that ATM is involved in histone acetylation-mediated gene regulation (75).
Rad51 is required for recombinational repair. IR-induced Rad51 nuclear focus formation is cell cycle-dependent and requires both ATM and c-Abl (76). Abl-related gene product interacts with and phosphorylates Rad51, suggesting that it plays a role in homologous recombination (HR) DNA repair by phosphorylating Rad51 (77).
Claspin is a newly identified protein that regulates Chk1 activation in Xenopus. It associates with Chk1 in response to DNA damage and phosphorylation of claspin appears to be required for its association with Chk1. Furthermore, claspin interacts with the checkpoint proteins ATR and Rad9. Since both ATR and Rad9Rad1Hus1 complexes (9-1-1 complex) are involved in Chk1 activation, it is possible that claspin may work as an adaptor molecule to bring these molecules together (78).
Chk1 and Chk2: the checkpoint kinases
Activation of ATM or ATR can lead to cell growth arrest, which may occur at the G1, S or G2 stages of the cell cycle. This process can be mediated through the action of Chk1 and Chk2, two checkpoint kinases (Figure 1). The major player in the G1 checkpoint is the p53 protein. Chk2 can phosphorylate Ser20 on p53, thereby activating the G1 checkpoint (79,80). This process is ATM-dependent, as ATM phosphorylates Chk2 at Thr68 near its N-terminus (81,82). Any involvement of the ATRChk1 pathway in the G1 checkpoint remains unclear.

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Fig. 1. The signaling pathways of ATM, ATR and DNA-PK following DNA damage. Once activated, these kinases can phosphorylate several downstream effectors to execute cellular responses that can lead either to growth arrest at the various cell cycle stages or to apoptosis. While ATM and ATR play prominent roles in mediating cell cycle checkpoints through the action of Chk1/2 and p53, DNA-PK usually activates the p53-mediated apoptosis pathway. Furthermore, a negative feedback regulation loop allows Mdm2 to down-regulate p53.
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Recent studies have shown that the ATMChk2 pathway does not function only in G1 arrest. IR exposure during S phase activates the same pathway as in G1, except that in this case an important outcome is the degradation of Cdc25A (83). Similarly, Chk1 also mediates the degradation of Cdc25A to induce the S phase checkpoint (84). In addition to Chk1 and Chk2, several other proteins have also been implicated in the ATM- or ATR-mediated S phase checkpoint, including NBS1, BRCA-1 and 53BP1, which can be phosphorylated by either ATM, ATR or both, in response to various DNA-damaging agents (44,8589). For example, IR activates ATM- and NBS1-dependent phosphorylation of Fanconi anaemia subtype D2 protein (FANCD2), resulting in an S phase checkpoint. Furthermore, NBS1 and FANCD2 co-localize in subnuclear foci (90). In addition, NBS1 functions upstream of Chk2 and Smc1 (91). NBS1 and Chk2 also behave differently in ATM-induced checkpoints: NBS1 rapidly forms foci, while Chk2 continues to move throughout the entire nucleus, although phosphorylation of Chk2 by ATM occurred exclusively at the DSB sites (92). Recently, the Ku subunit of DNA-PK has also been suggested to participate in both the ATM- and ATR-mediated S checkpoints (93,94).
Unlike the case for G1 and S phase checkpoints, the ATRChk1 pathway plays a more prominent role in the G2 checkpoint than does the ATMChk2 pathway (95,96). Recently, BRCA-1 was shown to be essential for activating the Chk1 kinase that regulates DNA damage-induced G2/M arrest (97). In addition, the phosphorylation of Chk1 by ATR, chromatin association and 14-3-3 binding all contribute to the regulation of Chk2 activity (98,99). Although the ATRChk1 pathway functions effectively at the G2 checkpoint when DNA damage occurs during G1 or S phase, it fails to arrest cells if the damage occurs during the G2 phase. In this case, the ATMChk2 pathway switches on and does so through the phosphorylation of Cdc25C (100).
The ATRChk1 and ATMChk2 pathways are not parallel branches of the DNA damage response pathway, but rather show a high degree of cross-talk and connectivity. For instance, ATR and ATM collaborate in the IR-induced G2/M checkpoint, but incomplete DNA replication in mammalian cells can prevent M phase entry independent of ATR (101). In irradiated AT cells an over-activation of ATRChk1 is responsible for the prolonged G2 accumulation (102). ATM does in fact signal to Chk1 in response to IR and phosphorylation of Chk1 on Ser317 in response to IR is ATM-dependent (103). In addition, functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the site of DNA damage (103,104).
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DNA-PK
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Like ATM/ATR, DNA-PK is a nuclear serine/threonine kinase composed of a catalytic subunit (DNA-PKcs) and a DNA binding Ku70/80 subunit. It can be activated by DNA damage induced by IR, UV or even V(D)J recombination, a vital process during maturation of the vertebrate immune system, in which each mature, immunocompetent B cell develops the ability to produce a specific antibody with a binding site encoded by the particular and unique sequence of its rearranged V gene (105). It is also the key player in non-homologous end-joining repair (NHEJ), also known as illegitimate recombination, the mechanism by which the majority of DSBs in mammalian cells are repaired. Compared with HR repair, in which the lost sequence information is accurately replaced by physical exchange of a segment from a homologous intact DNA, NHEJ is regarded as largely inaccurate because it involves end joining with junctions containing deletions back to regions of microhomology of 110 bases within 20 bp of the ends (106).
Activation of DNA-PK
Like ATM and ATR, DNA-PK is regarded more as a sensor of primary DNA damage than an inducible downstream effector of DNA damage signaling. Consistent with this model, DNA-PK is constitutively present at a relatively high level in the nucleus, with the protein level not significantly affected by genotoxic agents, while the activity of DNA-PK is regulated throughout the cell cycle (107). The activation of DNA-PK may involve interactions with DNA and other proteins. It has been proposed that Ku recruits DNA-PKcs to DNA, which in turn facilitates DNA-PKcsDNA interactions, thus inducing conformation changes in DNA-PKcs and releasing the catalytic potential of the DNA-PK complex (108). The DNA-PKcsDNA interaction may be very important, as this intimate contact may lead to conformational change. This idea is supported by the finding that DNA-PKcs can be activated to a certain degree by DNA in the absence of Ku (109,110).
Proteinprotein interactions may also help to regulate DNA-PK activity. One such example is the C1D protein, as DNA-bound C1D can activate DNA-PK in a DNA end-independent manner, probably by altering the structure of the DNA double helix (111). Since C1D is a component of the nuclear matrix and is induced by IR, it may help to target DNA-PK to DNA damage sites caused by genotoxic agents. Similar to C1D, the high mobility group proteins 1 and 2 can also activate DNA-PK, further supporting the notion that DNA-PK activation is influenced by chromatin context (112). On the other hand, heat shock transcription factor 1 has been shown to stimulate DNA-PK activity through direct interaction with Ku and a weaker interaction with DNA-PKcs, thus stabilizing the DNA-PKDNA interaction (113).
DNA-PK and NHEJ
As mentioned earlier, NHEJ is carried out in large part by DNA-PK and inhibition of DNA-PK activity enhances DNA damage induced by genotoxic agents (114). One suggestion is that the process may begin when Ku binds to both DNA ends of the DSB. Ku would then recruit DNA-PKcs to the site, likely initiating end joining by tethering the two DNA molecules together (115). However, a recent study showed that DNA-PKcs might be able to bring the two DNA ends together through the formation of a synaptic complex containing two DNA molecules. Electron microscopy showed complexes of two DNA ends brought together by two DNA-PKcs molecules. Furthermore, DNA-PKcs activity was cooperative with respect to DNA concentration, suggesting that activation of the kinase occurs only after DNA synapsis (116). In any case, the DNA-associated DNA-PK may recruit other proteins or factors that are involved in NHEJ to form a repair complex (117). One example is the X-ray cross-complementing proteinDNA ligase IV complex, which helps complete the DNA repair pathway (118,119). Another example is the MRN complex, whose nuclease activity may be critical in cleaning up the damaged DNA termini before they can be ligated together (120).
Another factor that influences the activity of DNA-PK in NHEJ is the autophosphorylation of DNA-PKcs. DNA-PKcs is autophosphorylated at multiple residues (Thr2609, Ser2612, Thr2638, Thr2647 and, possibly, additional residues not currently identified) in response to IR in vivo and this process occurs in a Ku-dependent manner. Mutation of Thr2609 to Ala leads to radiation sensitivity and impaired DSB rejoining. Thus, these findings establish that Ku-dependent phosphorylation of DNA-PKcs is required for the repair of DSBs by NHEJ (121,122).
Other proteins may also interact with DNA-PK to regulate its activity in NHEJ. For example, Ku70/80 can interact with WRN and stimulate WRN exonuclease activity (123). Furthermore, WRN forms a complex with DNA-PKcs and Ku in solution and WRN can be phosphorylated by DNA-PK (123). However, addition of WRN to a KuDNA-PKcsDNA complex results in the displacement of DNA-PKcs from the DNA, indicating that the tri-protein complex WRNKuDNA-PKcs cannot form on DNA ends. It was further shown that the displacement of DNA-PKcs from DNA requires the N- and C-terminal regions of WRN, both of which make direct contact with the Ku70/80 heterodimer. These results point to a potential role of WRN in influencing how DNA ends are processed during NHEJ (124).
DNA-PK and p53: the intimate interaction
The accumulated data point to an important role for p53 in the DNA-PK-mediated DNA damage signaling pathway, starting from the sensing of damage to the execution of various cellular responses (Figure 1). For example, p53 could participate in the detection of DNA damage by DNA-PK. It has been reported that DNA-PK and p53 can form a protein complex that preferentially binds to abnormal DNA structures, thus acting as a sensor complex that detects the disruption of DNA replication caused by nucleoside analog incorporation (125).
In addition, p53 also acts as an effector for the DNA-PK-mediated signaling pathway in response to DNA damage. Originally it was believed that DNA-PK might be involved in cell cycle checkpoint regulation through p53 (126,127). However, the current perception is that this is not the case (128130). Rather, DNA-PK selectively regulates the p53-dependent apoptosis pathway. Mice defective in DNA-PKcs showed a decreased apoptotic response and lowered Bax expression following exposure to IR, suggesting that DNA-PKcs serves as an upstream regulator of the p53-mediated apoptosis pathway (131). It was further shown that immediately after
-irradiation, latent p53 formed a complex with DNA-PKcs in mouse embryo fibroblasts. This complex was DNase-sensitive, suggesting that the proteins were also associated with DNA, most likely at strand breaks. This association was accompanied by phosphorylation of pre-existing latent p53 at Ser18 (corresponding to Ser15 in human p53) that was not found in DNA-PKcs-/- cells. More importantly, DNA damage-induced apoptosis was abolished in both DNA-PKcs-/- and p53-/- cells. Blocking synthesis of inducible p53 by cycloheximide did not abrogate apoptosis, suggesting that the latent population of p53 is sufficient to execute the apoptotic program. In addition, substituting Ala for Ser18 led to a decreased apoptotic response, indicating that phosphorylation of p53 is important for DNA-PK-mediated apoptosis (132).
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Cross-talk between ATM/ATR and DNA-PK
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It has been suggested that there might be a common underlying mechanism by which ATM, ATR and DNA-PK detect and transduce various DNA damage signals, although they may signal different but partially overlapping types of DNA damage, and that this underlying mechanism would involve interactions with other DNA repair or maintenance proteins (14,108). They may also function through a common effector and, if this is so, the most likely candidate is the p53 protein.
Although the significance is still unclear, it has been shown that these three DNA damage sensors can interact with each other directly or indirectly and thus regulate the activities of each other. For instance, it has been shown that the S phase checkpoint response in IR-irradiated Ku80-/- cells is stronger than in their wild-type counterparts. While this particular checkpoint is not related to the kinase activity of DNA-PK, it does correlate with a higher ATM activity and with more ATM bound to chromatin DNA in such cells. Wortmannin, a non-specific inhibitor of ATM, can reduce the higher activity of ATM kinase as well as the stronger S phase checkpoint response in Ku80-/- cells. In addition, inhibition of ATM activity by a specific ATM antisense oligonucleotide abolishes the stronger S checkpoint response in Ku80-/- cells and renders these cells practically indistinguishable from Ku80+/+ cells for this checkpoint (93).
Similarly, Ku may also affect the activity of ATR. Camptothecin (CPT), a broad-spectrum anticancer drug, induces DNA damage and activates an S checkpoint response in mammalian cells which is mediated by the ATRChk1 pathway. Similar to IR-treated Ku80-/- cells, CPT-treated Ku80-/- cells showed a stronger S checkpoint response. Furthermore, this stronger response was not directly related to the DNA-PK kinase activity, but did correlate with higher activities of ATR and Chk1. Caffeine (a non-specific inhibitor of ATR), UCN-01 (a non-specific inhibitor of Chk1) and a specific Chk1 antisense oligonucleotide can all abolish the stronger S checkpoint response in CPT-treated Ku80-/- cells (94). Together, these data lead to the hypothesis that ATM or ATR may recognize and bind to the damaged DNA site before DNA-PK in normal cells. Ku would then help to remove ATM/ATR from DNA in order to let DNA-PK bind to the site for repair.
A link between DNA-PK and ATM has been established, likely through the action of c-Abl. c-Abl is able to down-regulate DNA-PK activity (50,133). Blocking c-Abl activity through an inhibitor in normal cells or expression of an inactive form of c-Abl results in a failure of DNA-PK down-regulation and the expression of a constitutively active c-Abl mutant leads to low DNA-PK activity. Ataxia telangiectasia cells, which contain homozygous mutations in the ATM gene, display normal activation of DNA-PK but not c-Abl in response to IR, suggesting that ATM is responsible for the activation of c-Abl, while the activation of DNA-PK is ATM-independent. However, the activated DNA-PK fails to be down-regulated in ataxia telangiectasia cells as compared with normal cells. Therefore, the down-regulation of DNA-PK by c-Abl is an ATM-dependent process (50).
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Conclusions
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Although the mechanisms for DNA damage sensing require further clarification, recent studies regarding the ATM, ATR and DNA-PK proteins are providing a better understanding of this process. Although we have focused on these three kinases, the current consensus is that they cannot fulfill the DNA damage sensing function alone. Rather, they probably do so through interactions with other proteins, most likely the DNA repair complexes that also bind to the damaged DNA sites to form foci. This focus formation may then recruit additional factors that can then carry out the sensor function and initiate subsequent cellular events. Recent reports supply further support for this model. For instance, the replication factor C-related Rad17 protein binds to chromatin prior to damage. Following DNA damage, this protein is phosphorylated by ATR on chromatin, and then helps to recruit the PCNA-like Rad9Hus1Rad1 (9-1-1) protein complex, another potential DNA damage sensor candidate (12), onto the chromatin. The chromatin associations of Rad17 and ATR are largely independent, suggesting that they localize to DNA damage sites independently. Furthermore, the phosphorylation of Rad17 requires Hus1, suggesting that the 9-1-1 complex recruited by Rad17 enables ATR to recognize its substrates (134).
Similarly, the Bloom's syndrome protein BLM is specifically required for the proper re-localization of the MRN complex to sites of replication arrest. BLM is also phosphorylated after replication arrest in an ATR-dependent manner, although the phosphorylation is not required for subnuclear re-localization. Therefore, in ATR dominant negative mutant cells, assembly of the MRN complex in nuclear foci after replication blockage is almost completely abolished (135). These data are consistent with a model in which multiple checkpoint protein complexes localize to sites of DNA damage independently and interact to trigger the checkpoint signaling cascade (Figure 2).

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Fig. 2. A proposed model for how sensors detect DNA damage. First, repair proteins (circles), such as members of the Rad family, bind to the double-strand breaks or to other abnormal DNA structures induced by genotoxic agents, while PI-3 family members (such as ATM, ATR or DNA-PK, represented by squares) bind to the damaged site either simultaneously or subsequently. Their interaction then further recruits other factors (triangles) involved in the DNA damage detection process and initiates the subsequent cellular events.
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Many questions remain unanswered regarding the cellular response to genotoxic stress. One such question is how the cell decides to choose between the repair of damaged DNA and the initiation of apoptosis. The general perception is that the extent of DNA damage may be a major factor in this decision. For example, it has been shown that low doses of
-irradiation or cisplatin treatment resulted in an immediate enhancement of p53-dependent DNA repair, as measured by base excision repair activity (136). However, treatment of cells with high doses of the same DNA-damaging agents resulted in a reduction in p53-dependent DNA repair and the induction of pP53-dependent apoptosis (136). However, it is unknown how the extent of DNA damage is assessed. This has led to the suggestion that the DNA damage sensors may fulfill this role. The identification of the mechanisms involved in this process will certainly be an exciting area for future research.
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Notes
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5 To whom correspondence should be addressed Email: pdhughes{at}som.llu.edu 
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Acknowledgments
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This work was supported in part by the National Key Basic Research and Development Program, China (no. 2002CB512901, Y.Y. and J.Y.), the Initiative Funds for Young Scientists, Zhejiang University (J.Y.), the Initiative Funds for Returned Oversea Chinese Scholars, Zhejiang Province (J.Y.) and the Initiative Funds for Returned Oversea Chinese Scholars, Ministry of Education, China (J.Y.).
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Received February 4, 2003;
revised July 17, 2003;
accepted July 29, 2003.