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Query: UMLS:C0004135 (ATM)
 13,001 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.
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PMID:MDC1 is required for the intra-S-phase DNA damage checkpoint. 1260 3

Forkhead-homology-associated (FHA) domains function as protein-protein modules that recognize phosphorylated serine/threonine motifs. Interactions between FHA domains and phosphorylated proteins are thought to have essential roles in the transduction of DNA damage signals; however, it is unclear how FHA-domain-containing proteins participate in mammalian DNA damage responses. Here we report that a FHA-domain-containing protein-mediator of DNA damage checkpoint protein 1 (MDC1; previously known as KIAA0170)--is involved in DNA damage responses. MDC1 localizes to sites of DNA breaks and associates with CHK2 after DNA damage. This association is mediated by the MDC1 FHA domain and the phosphorylated Thr 68 of CHK2. Furthermore, MDC1 is phosphorylated in an ATM/CHK2-dependent manner after DNA damage, suggesting that MDC1 may function in the ATM-CHK2 pathway. Consistent with this hypothesis, suppression of MDC1 expression results in defective S-phase checkpoint and reduced apoptosis in response to DNA damage, which can be restored by the expression of wild-type MDC1 but not MDC1 with a deleted FHA domain. Suppression of MDC1 expression results in decreased p53 stabilization in response to DNA damage. These results suggest that MDC1 is recruited through its FHA domain to the activated CHK2, and has a critical role in CHK2-mediated DNA damage responses.
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PMID:MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways. 1260 4

Phosphorylation of NBS1, the product of the gene mutated in Nijmegen breakage syndrome (NBS), by ataxia telangiectasia mutated (ATM), the product of the gene mutated in ataxia telangiectasia, is required for activation of the S phase checkpoint in response to ionizing radiation (IR). However, NBS1 is also thought to play additional roles in the cellular response to DNA damage. To clarify these additional functions of NBS1, we generated NBS cell lines stably expressing various NBS1 mutants from retroviral vectors. The ATM-dependent activation of CHK2 by IR was defective in NBS cells but was restored by ectopic expression of wild-type NBS1. The defects in ATM-dependent activation of CHK2, S phase checkpoint control, IR-induced nuclear focus formation, and radiation sensitivity apparent in NBS cells were not corrected by expression of NBS1 mutants that lack an intact MRE11 binding domain, suggesting that formation of the NBS1-MRE11-RAD50 complex is required for the corresponding normal phenotypes. Expression of NBS1 proteins with mutated ATM-targeted phosphorylation sites (serines 278 or 343) did not restore S phase checkpoint control but did restore the ability of IR to activate CHK2 and to induce nuclear focus formation and normalized the radiation sensitivity of NBS cells. Expression of NBS1 containing mutations in the forkhead-associated or BRCA1 COOH terminus domains did not correct the defects in radiation sensitivity or nuclear focus formation but did restore S phase checkpoint control in NBS cells. Together, these data demonstrate that multiple functional domains of NBS1 are required for ATM-dependent activation of CHK2, nuclear focus formation, S phase checkpoint control, and cell survival after exposure to IR.
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PMID:Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage. 1286 Oct 53

Damage induced in the DNA after exposure of cells to ionizing radiation activates checkpoint pathways that inhibit progression of cells through the G1 and G2 phases and induce a transient delay in the progression through S phase. Checkpoints together with repair and apoptosis are integrated in a circuitry that determines the ultimate response of a cell to DNA damage. Checkpoint activation typically requires sensors and mediators of DNA damage, signal transducers and effectors. Here, we review the current state of knowledge regarding mechanisms of checkpoint activation and proteins involved in the different steps of the process. Emphasis is placed on the role of ATM and ATR, as well on CHK1 and CHK2 kinases in checkpoint response. The roles of downstream effectors, such as P53 and the CDC25 family of proteins, are also described, and connections between repair and checkpoint activation are attempted. The role of checkpoints in genomic stability and the potential of improving the treatment of cancer by DNA damage inducing agents through checkpoint abrogation are also briefly outlined.
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PMID:DNA damage checkpoint control in cells exposed to ionizing radiation. 1294 90

NFBD1/MDC1 (mediator of DNA damage checkpoint 1) is a nuclear factor with an amino-terminal FHA (forkhead-associated) domain and a tandem repeat of BRCT (breast cancer susceptibility gene-1 carboxyl terminus) domains. We have previously shown that NFBD1 is an early participant in DNA damage signaling pathways and that ionizing radiation-induced nuclear foci (IRIF) of NFBD1 colocalize with several DNA checkpoint signaling and repair factors. We report here that NFBD1 physically associates with ATM, p53, components of the MRE11-RAD50-NBS1 (MRN) complex, and gamma-H2AX. An overexpressed FHA domain-containing fragment of NFBD1 binds to endogenous NFBD1 and components of the MRN complex, but not to gamma-H2AX. This fragment interferes with IRIF formation by endogenous NFBD1, MRE11, or NBS1. A BRCT domain-containing fragment of NFBD1 binds to gamma-H2AX and 53BP1, but not to components of the MRN complex, and abolishes IRIF formation by NFBD1, MRE11, NBS1, 53BP1, CHK2 phospho-T68, gamma-H2AX, and possible ATM/ATR substrates recognized by anti-phospho-SQ/TQ antibody. These results suggest that NFBD1 is an ATM/ATR-dependent organizer that recruits DNA checkpoint signaling and repair proteins to the sites of DNA damage.
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PMID:NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors. 1451 63

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.
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PMID:Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. 1457 10

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.
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PMID:Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains. 1468 Dec 23

Many conventional anticancer treatments kill cells irrespective of whether they are normal or cancerous, so patients suffer from adverse side effects due to the loss of healthy cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G2 checkpoint during cell replication. Damaged DNA in humans is detected by sensor proteins (such as hHUS1, hRAD1, hRAD9, hRAD17, and hRAD26) that transmit a signal via ATR to CHK1, or by another sensor complex (that may include gammaH2AX, 53BP1, BRCA1, NBS1, hMRE11, and hRAD50), the signal of which is relayed by ATM to CHK2. Most of the damage signals originated by the sensor complexes for the G2 checkpoint are conducted to CDC25C, the activity of which is modulated by 14-3-3. There are also less extensively explored pathways involving p53, p38, PCNA, HDAC, PP2A, PLK1, WEE1, CDC25B, and CDC25A. This review will examine the available inhibitors of CHK1 (Staurosporin, UCN-01, Go6976, SB-218078, ICP-1, and CEP-3891), both CHK1 and CHK2 (TAT-S216A and debromohymenialdisine), CHK2 (CEP-6367), WEE1 (PD0166285), and PP2A (okadaic acid and fostriecin), as well as the unknown checkpoint inhibitors 13-hydroxy-15-ozoapathin and the isogranulatimides. Among these targets, CHK1 seems to be the most suitable target for therapeutic G2 abrogation to date, although an unexplored target such as 14-3-3 or the strategy of targeting multiple proteins at once may be of interest in the future.
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PMID:G2 checkpoint abrogators as anticancer drugs. 1507 95

Mammalian CHK1 is a Ser/Thr effector kinase that plays critical roles in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3-related protein kinase). This chapter is focused on describing an assay to measure CHK1 activity in vitro. The basic mechanism of this assay is to observe the phosphorylated levels of a fragment of CDC25C containing the site that can be phosphorylated by CHK1 in vitro. This assay includes five major steps: (1) preparing extracts from the control or treated cells; (2) preparing substrate; (3) immunoprecipitating CHK1 protein from the cells; (4) assembling the kinase assay; and (5) analyzing the phosphorylated level of the substrates by CHK1. Besides CHK1, CHK2 is another important checkpoint regulator that responds to DNA damage. Because CHK1 and CHK2 share some substrates such as CDC25C in vitro, this assay could also be used for CHK2 activity assay, except that the CHK2 antibody will replace the CHK1 antibody.
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PMID:CHK1 kinase activity assay. 1522 May 26

To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.
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PMID:Intra-S-phase checkpoint activation by direct CDK2 inhibition. 1522 29


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