The function of ATM and ATR at telomeres has been examined in Arabidopsis. Although plants lacking ATM or ATR display wild-type telomere length homeostasis, chromosome end protection is compromised in atm atr mutants. Moreover, atm tert Arabidopsis (TERT is the catalytic subunit of telomerase) experience an abrupt, early onset of genome instability, arguing that ATM is required for protection of short telomeres. The rate of telomere shortening is indistinguishable between atm tert and tert mutants, with telomeres declining by ~500 bp per plant generation in both settings. ATR, by contrast, is required for maintenance of telomeric DNA; telomere shortening is dramatically accelerated in atr tert mutants relative to tert plants. Thus, ATM and ATR make essential and distinct contributions to chromosome end protection and telomere maintenance in higher eukaryotes (Vespa, 2005).
Mutants of the Saccharomyces cerevisiae ataxia telangiectasia mutated (ATM) homolog MEC1/SAD3/ESR1 were identified that could live only if the RAD53/SAD1 checkpoint kinase was overproduced. MEC1 and a structurally related gene, TEL1, have overlapping functions in response to DNA damage and replication blocks that in mutants can be provided by overproduction of RAD53. Both MEC1 and TEL1 were found to control phosphorylation of Rad53p in response to DNA damage. These results indicate that RAD53 is a signal transducer in the DNA damage and replication checkpoint pathways and functions downstream of two members of the ATM lipid kinase family. Because several members of this pathway are conserved among eukaryotes, it is likely that a RAD53-related kinase will function downstream of the human ATM gene product and play an important role in the mammalian response to DNA damage (Sanchez, 1996).
DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. The Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle- and DNA damage-dependent Sae2 (SUMO-1-activating enzyme 2) phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions (Baroni, 2004).
The Schizosaccharomyces pombe checkpoint gene named rad3+ encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. A novel role is described for Rad3 in the control of telomere integrity. Mutations in the rad3+ gene alleviate telomeric silencing and produce shortened lengths in the telomere repeat tracts. Genetic analysis has revealed that the other checkpoint rad mutations (rad1, rad17, and rad26) belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1+, another ATM homolog in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1+ causes no obvious changes in the telomeric DNA structure. These results demonstrate a novel role of the S. pombe ATM homologs that is independent of chk1+ and cds1+ (Matsuura, 1999).
Yeast strains with a mutation in the MEC1 gene (an ATM homolog) are deficient in the cellular checkpoint response to DNA-damaging agents and have short telomeres. In wild-type yeast cells, genes inserted near the telomeres are transcriptionally silenced. mec1 strains have reduced ability to silence gene expression near the telomere. This deficiency was alleviated by the sml1 mutation. Overexpression of Mec1p also results in a silencing defect, although this overexpression does not affect the checkpoint function of Mec1p. Telomeric silencing is not affected by mutations in several other genes in the Mec1p checkpoint pathway (null mutations in RAD9 and CHK1 or in several hypomorphic rad53 alleles) but is reduced by a null mutation of DUN1. In addition, the loss of telomeric silencing in mec1 strains was not a consequence of the slightly shortened telomeres observed in these strains (Craven, 2000).
The Saccharomyces Mre11p, Rad50p, and Xrs2p proteins form a complex, called the MRX complex, that is required to maintain telomere length. Cells lacking any one of the three MRX proteins and Mec1p, an ATM-like protein kinase, undergo telomere shortening and ultimately die, phenotypes characteristic of cells lacking telomerase. The other ATM-like yeast kinase, Tel1p, appears to act in the same pathway as MRX: mec1 tel1 cells have telomere phenotypes similar to those of telomerase-deficient cells, whereas the phenotypes of tel1 cells are not exacerbated by the loss of a MRX protein. The nuclease activity of Mre11p was found to be dispensable for the telomerase-promoting activity of the MRX complex. The association of the single-stranded TG1-3 DNA binding protein Cdc13p with yeast telomeres occurs efficiently in the absence of Tel1p, Mre11p, Rad50p, or Xrs2p. Targeting of catalytically active telomerase to the telomere suppresses the senescence phenotype of mec1 mrx or mec1 tel1 cells. Moreover, when telomerase is targeted to telomeres, telomere lengthening is robust in mec1 mrx and mec1 tel1 cells. These data rule out models in which the MRX complex is necessary for Cdc13p binding to telomeres or in which the MRX complex is necessary for the catalytic activity of telomerase. Rather, the data suggest that the MRX complex is involved in recruiting telomerase activity to yeast telomeres (Tsukamoto, 2001).
Telomerase is a ribonucleoprotein that copies a short RNA template into telomeric DNA, maintaining eukaryotic chromosome ends and preventing replicative senescence. Telomeres differentiate chromosome ends from DNA double-stranded breaks. Nevertheless, the DNA damage-responsive ATM kinases Tel1p and Mec1p are required for normal telomere maintenance in Saccharomyces cerevisiae. Tests were performed to see whether the ATM kinases are required for telomerase enzyme activity or whether it is their action on the telomere that allows telomeric DNA synthesis. Cells lacking Tel1p and Mec1p had wild-type levels of telomerase activity in vitro. Furthermore, altering telomere structure in three different ways showed that telomerase can function in ATM kinase-deleted cells: tel1 mec1 cells senesced more slowly than tel1 mec1 cells that also lacked TLC1, which encodes telomerase RNA, suggesting that tel1 mec1 cells have residual telomerase function; deleting the telomere-associated proteins Rif1p and Rif2p in tel1 mec1 cells prevented senescence; a point mutation was isolated in the telomerase RNA template domain (tlc1-476A) that altered telomeric DNA sequences, causing uncontrolled telomeric DNA elongation and increasing single strandedness. In tel1 mec1 cells, tlc1-476A telomerase was also capable of uncontrolled synthesis, but only after telomeres had shortened for >30 generations. These results show that, without Tel1p and Mec1p, telomerase is still active and can act in vivo when the telomere structure is disrupted by various means. Hence, a primary function of the ATM-family kinases in telomere maintenance is to act on the substrate of telomerase, the telomere, rather than to activate the enzymatic activity of telomerase (Chan, 2001).
Telomeres protect chromosome ends from fusing to double-stranded breaks (DSBs). Using a quantitative real-time PCR assay, it has been shown that nonhomologous end joining between a telomere and an inducible DSB is undetectable in wild-type cells, but occurs within a few hours of DSB induction in approximately 1/2000 genomes in telomerase-deficient cells and in >1/1000 genomes in telomerase-deficient cells also lacking the ATM homolog Tel1p. The fused telomeres contained very little telomeric DNA, suggesting that catastrophic telomere shortening preceded fusion. Lengthening of telomeres did not prevent such catastrophic telomere shortening and fusion events. Telomere-DSB fusion also occurred in cells containing a catalytically inactive telomerase and in tel1 mec1 cells where telomerase cannot elongate telomeres. Thus, telomerase and Tel1p function in telomere protection as well as in telomere elongation (Chan, 2003).
The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae. However, the mechanism of PIKK-mediated telomere length control remains unclear. HereTel1p and Mec1p are shown to be recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner. In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres. A model is proposed in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres. This study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs (Takata, 2004).
The ATM protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia, mediates responses to ionizing radiation in mammalian cells. ATM is held inactive in unirradiated cells as a dimer or higher-order multimer, with the kinase domain bound to a region surrounding serine 1981 that is contained within the previously described 'FAT' domain. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. Most ATM molecules in the cell are rapidly phosphorylated on this site after doses of radiation as low as 0.5 Gy, and binding of a phosphospecific antibody is detectable after the introduction of only a few DNA double-strand breaks in the cell. Activation of the ATM kinase seems to be an initiating event in cellular responses to irradiation, and the data indicate that ATM activation is not dependent on direct binding to DNA strand breaks, but may result from changes in the structure of chromatin (Bakkenist, 2003).
The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogates the cell cycle arrest after ionizing radiation and diminishes several other readouts of the DNA damage response, including phosphorylation of Nbs1 (Drosophila homolog Nbs), induction of p53, and upregulation of p53 targets. TRF2 inhibits autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 directly interacts with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. It is proposed that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage (Karlseder, 2004).
The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM. MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. MRN makes multiple contacts with ATM and appears to stimulate ATM activity by facilitating the stable binding of substrates. Phosphorylation of Nbs1 is critical for MRN stimulation of ATM activity toward Chk2, but not p53. Kinase-deficient ATM inhibits wild-type ATM phosphorylation of Chk2, consistent with the dominant-negative effect of kinase-deficient ATM in vivo (Lee, 2004).
The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. The Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation is not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN is essential for ATM stimulation, consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage (Lee, 2005).
ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. It is concluded that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. It is proposed that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage (You, 2005).
The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. A C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, is sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, an Mre11 transgene with a C-terminal NLS sequence was expressed in NBS fibroblasts. The Mre11-NLS protein complexes with Rad50, localizes to the nucleus in NBS fibroblasts, and associates with chromatin. However, Atm autophosphorylation is not stimulated in cells expressing Mre11-NLS, nor are downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, nibrin transgenes lacking the Atm binding domain were expressed in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation is dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction (Cerosaletti, 2006).
DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. It is proposed that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome (Dupre, 2006).
The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogates the S-phase checkpoint after ionizing irradiation and enhances radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complements radiosensitivity and the S-phase checkpoint and reduces chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM (Morgan, 1997).
ATM phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR (ATM and Rad3 related) and DNA-PK, in vitro kinase assays were optimized and a rapid peptide screening method was developed to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs require Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, it was found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. A general phosphorylation consensus sequence for ATM was determined and putative in vitro targets were identified by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK (Kim, 1999).
The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ATM dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. ATM phosphorylates in vitro the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduces the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocks all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD (Matsuoka, 2000).
ATM is necessary for phosphorylation and activation of Cds1/Chk2 in vivo and can phosphorylate Cds1 in vitro, although evidence is lacking that the sites phosphorylated by ATM are required for activation. This study shows that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionizing radiation (Melchionna, 2000).
In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ATM gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, gene-targeted Chk2-deficient mice were generated. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggests that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis is restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3+ related (ATR) are mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis (Hirao, 2002).
In mammals, the ATM and ATR protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. The ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. Functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference results in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 fails to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also fails to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 functions downstream from ATR only and does indicate that ATM signals to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint (Gatei, 2003).
Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. The ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells (Ahn, 2000).
When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. A functional link is reported between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and this mechanism is implicated in controlling the S-phase checkpoint. IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. Tumor-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and cells expressing these Chk2 alleles or elevated Cdc25A, or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) all fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumor suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis (Falck, 2001).
The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site. The Chk2 FHA domain mediates ATM-dependent Chk2 phosphorylation and targeting of Chk2 to in vivo binding partners such as BRCA1 through either or both of two structurally distinct mechanisms. Although phospho-dependent binding is important for Chk2 activity, previously uncharacterized phospho-independent FHA domain interactions appear to be the primary target of oncogenic lesions (Li, 2002).
Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs. Modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promotes rapid and synchronous origin firing; conversely, inhibition of Cdc25A slows DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. It is proposed that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. These observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage (Shechter, 2004).
ATM is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homolog of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, the modification of hRad9 in response to DNA damage was investigated. hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increases cellular sensitivity to IR. Together, these results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation (Chen, 2001).
To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult, the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9 was investigated. C-terminal T412/S423 phosphorylation of Rad9 by Rad3ATR occurs in S phase without replication stress. Rad3ATR and Tel1ATM phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4TOPBP1 protein. Rad9-Rad4TOPBP1 interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4TOPBP1 coprecipitates with Rad3ATR, suggesting that phosphorylation coordinates formation of an active checkpoint complex (Furuya, 2004).
Most of the proteins involved in the DNA damage and DNA replication checkpoint have been identified, and the majority are highly conserved through evolution. Many features of the checkpoint pathways remain unexplained, including their apparent complexity and the fact that many of the same proteins participate in both the DNA damage and DNA replication responses. The data presented in this study begin to uncover the molecular organization of the checkpoint proteins following their recruitment to sites of DNA damage or collapsed DNA replication forks. It is suggested that one of the reasons for the apparent complexity of the system is because it allows cells to distinguish between similar biochemical consequences of DNA damage (such as ssDNA-RPA complexes) that occurs in distinct circumstances (such as induced damage in G2 and collapsed replication forks). It is important to make these distinctions because different signaling responses to the cell cycle and the DNA repair apparatus will be appropriate in each case. The data suggest that the Rad3ATR-dependent phosphorylation of Rad9 promotes association between Rad9 and Rad4TOPBP1 through phospho-specific BRCT-domain interactions during unperturbed S phase, and that this helps cells distinguish collapsed forks from DNA damage in G2 cells. It is intriguing that a phospho-specific BRCT-mediated interaction between BRCA1 and BACH1 in human cells is promoted by cyclin-dependent kinase activity against BACH1 in G2 and also by the G2 checkpoint. Together, these observations suggest that a combination of phosphorylation events can orchestrate the organization of the checkpoint apparatus before it is activated and that, upon activation, the consequent phospho-specific protein interactions dictate the downstream consequences of this activation (Furuya, 2004).
DNA replication stress triggers the activation of Checkpoint Kinase 1 (Chk1) in a pathway that requires the independent chromatin loading of the ATRIP-ATR (ATR-interacting protein/ATM [ataxia-telangiectasia mutated]-Rad3-related kinase) complex and the Rad9-Hus1-Rad1 (9-1-1) clamp (See Drosophila Hus1). Rad9ís role in Chk1 activation is to bind TopBP1, which stimulates ATR-mediated Chk1 phosphorylation via TopBP1ís activation domain (AD), a domain that binds and activates ATR. Notably, fusion of the AD to proliferating cell nuclear antigen (PCNA) or histone H2B bypasses the requirement for the 9-1-1 clamp, indicating that the 9-1-1 clampís primary role in activating Chk1 is to localize the AD to a stalled replication fork (Delacroix, 2007).
Genotoxic damage activates conserved checkpoint signaling pathways that maintain genomic stability by regulating cell cycle progression, triggering apoptosis, and influencing DNA repair. One pathway that is potently activated by replication stress leads to activation of Checkpoint Kinase 1 (Chk1), which promotes cell survival by blocking the firing of replication origins, preventing entry into mitosis, stabilizing stalled replication forks, and facilitating DNA repair. This pathway is initiated when the replicative DNA polymerases stall and large tracts of single-stranded DNA are created by the uncoupling of the replicative helicase from the advancing replication fork. The single-stranded DNA is then coated by replication protein A (RPA), which signals the independent recruitment of two checkpoint complexes: the ataxia-telangiectasia mutated (ATM)-Rad3-related kinase-ATR-interacting protein (ATR-ATRIP) complex and the Rad9-Hus1-Rad1 (9-1-1) complex. The ATRIP-ATR complex is bound to DNA by a direct interaction between ATRIP and RPA. In contrast, loading of the 9-1-1 complex requires several steps. First, DNA polymerase is recruited, which in turn recruits the clamp loader, Rad17-replication factor C (RFC). Second, the Rad17-RFC then loads the proliferating cell nuclear antigen (PCNA)-like 9-1-1 complex onto chromatin in a reaction that is analogous to the loading of PCNA onto sites of DNA replication. Although the binding of the ATRIP-ATR complex and the loading of the 9-1-1 complex occur independently of one another, both events are essential for optimal ATR-mediated Chk1 phosphorylation and activation (Delacroix, 2007 and references therein).
Despite the tremendous progress that has been made in deciphering the biochemical functions of the 9-1-1 complex and the in-depth understanding of the signals that lead to the loading of the 9-1-1 clamp, it has remained unclear how the chromatin-bound 9-1-1 complex initiates and propagates the Chk1-activating signal. Several studies have demonstrated that Rad9 orthologs in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans interact with their respective TopBP1 orthologs (Cut4, Dpb11, and TopBP1). However, the significance of the Rad9-TopBP1 interaction in 9-1-1 function has not been explored. This study shows that the role of the 9-1-1 clamp is to recruit TopBP1, which then triggers ATR-mediated Chk1 phosphorylation. Thus, TopBP1 is a molecular bridge that links the independently recruited 9-1-1 and ATRIP-ATR complexes, leading to checkpoint activation (Delacroix, 2007).
The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. Four lines of evidence are described that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication (Cortez, 2004).
In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. A cDNA has been isolated encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, the essential replication protein Mcm2 has been identified as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 undergoes phosphorylation on serine 92 (S92) in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involves both ATM and ATR, but the relative contribution of each kinase depends upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylate Mcm2 directly on S92 in cell-free kinase assays. Immunodepletion of both ATM and ATR from egg extracts abrogates the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts (Yoo, 2004).
The human genetic disorder ataxia-telangiectasia (AT) is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects and cancer predisposition. The gene mutated in this syndrome, ATM (for AT mutated), encodes a protein containing a phosphatidyl-inositol 3-kinase (PI-3 kinase)-like domain. ATM also contains a proline-rich region and a leucine zipper, both of which implicate this protein in signal transduction. The proline-rich region has been shown to bind to the SH3 domain of c-Abl, which facilitates its phosphorylation and activation by ATM. AT cells are defective in the G1/S checkpoint activated after radiation damage and this defect is attributable to a defective p53 signal transduction pathway. There is a direct interaction between ATM and p53 involving two regions in ATM, one at the amino terminus and the other at the carboxy terminus, corresponding to the PI-3 kinase domain. Recombinant ATM protein phosphorylates p53 on serine 15 near the N terminus. Furthermore, ectopic expression of ATM in AT cells restores normal ionizing radiation (IR)-induced phosphorylation of p53, whereas expression of ATM antisense RNA in control cells abrogates the rapid IR-induced phosphorylation of p53 on serine 15. These results demonstrate that ATM can bind p53 directly and is responsible for its serine 15 phosphorylation, thereby contributing to the activation and stabilization of p53 during the IR-induced DNA damage response (Khanna, 1998).
The p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at the centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from the centrosomes by treatments with spindle inhibitors, p53 remains phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, do not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at the centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint (Tritarelli, 2004).
Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response (Luo, 2004).
The related kinases ATM and ATR phosphorylate a limited number of downstream protein targets in response to DNA damage. A new pathway is described in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap (Demonacos, 2004).
The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Because of the similarities between AT and NBS, the functional interactions between ATM and p95/nbs1 were evaluated. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicates that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 is phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogates an S-phase checkpoint induced by ionizing radiation in normal cells and fails to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases (Lim, 2000).
Polo-like kinases play multiple roles in different phases of mitosis. The mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and this inhibition may lead to cell cycle arrests at multiple points in mitosis. The role of the checkpoint kinases ATM and ATR in DNA damage-induced inhibition of Plk1 has been investigated. Inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM(-/-) cells it has been shown that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, UV radiation also causes inhibition of Plk1, and evidence is presented that this inhibition is mediated by ATR. Taken together, these data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents (van Vugt, 2001).
Structural maintenance of chromosomes (SMC) proteins (SMC1, SMC3) are evolutionarily conserved chromosomal proteins that are components of the cohesin complex, necessary for sister chromatid cohesion. These proteins may also function in DNA repair. SMC1 is a component of the DNA damage response network that functions as an effector in the ATM/NBS1-dependent S-phase checkpoint pathway. SMC1 associates with BRCA1 and is phosphorylated in response to IR in an ATM- and NBS1-dependent manner. Using mass spectrometry, it has been established that ATM phosphorylates S957 and S966 of SMC1 in vivo. Phosphorylation of S957 and/or S966 of SMC1 is required for activation of the S-phase checkpoint in response to IR. The phosphorylation of NBS1 (Nijmegen breakage syndrome gene product is a part of the hMre11 complex, a central player associated with double-strand break repair) by ATM is required for the phosphorylation of SMC1, establishing the role of NBS1 as an adaptor in the ATM/NBS1/SMC1 pathway. The ATM/CHK2/CDC25A pathway is also involved in the S-phase checkpoint activation, but this pathway is intact in NBS cells. These results indicate that the ATM/NBS1/SMC1 pathway is a separate branch of the S-phase checkpoint pathway, distinct from the ATM/CHK2/CDC25A branch. Therefore, this work establishes the ATM/NBS1/SMC1 branch, and provides a molecular basis for the S-phase checkpoint defect in NBS cells (Yazdi, 2002).
Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. The protein kinase Atm phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway (Kim, 2002).
The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and this recruitment is required for the phosphorylation of structural maintenance of chromosome protein 1 (SMC1) by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation (Kitagawa, 2004).
BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. This study identifies a constitutive BRCA1-c-Abl complex and evidence is provided for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. The C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. A model is suggested in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity (Foray, 2002).
The function has been examined of the human ortholog of Saccharomyces cerevisiae Rif1 (Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates their length. In contrast, human Rif1 does not accumulate at functional telomeres, but localizes to dysfunctional telomeres and to telomeric DNA clusters in ALT cells, a pattern of telomere association typical of DNA-damage-response factors. After induction of double-strand breaks (DSBs), Rif1 forms foci that colocalize with other DNA-damage-response factors. This response is strictly dependent on ATM and 53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition results in radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase checkpoint phenotype is independent of Nbs1 status, arguing that Rif1 and Nbs1 act in different pathways to inhibit DNA replication after DNA damage. These data reveal that human Rif1 contributes to the ATM-mediated protection against DNA damage and point to a remarkable difference in the primary function of this protein in yeast and mammals (Silverman, 2004).
Ataxia-telangiectasia (A-T) is a syndrome of cancer susceptibility, immune dysfunction, and neurodegeneration that is caused by mutations in the A-T-mutated (ATM) gene. ATM has been implicated as a critical regulator of cellular responses to DNA damage, including the activation of cell cycle checkpoints and induction of apoptosis. Although defective cell cycle-checkpoint regulation and associated genomic instability presumably contribute to cancer susceptibility in A-T, the mechanism of neurodegeneration in A-T is not well understood. In addition, although ATM is required for the induction of the p53 transcriptional program in response to DNA damage, the identities of the relevant transcription factors that mediate ATM-dependent changes in gene expression remain largely undetermined. In this article, a signal transduction pathway is described linking ATM directly to the Ca(2+)/cAMP response element-binding protein, CREB, a transcription factor that regulates cell growth, homeostasis, and survival. ATM phosphorylates CREB in vitro and in vivo in response to ionizing radiation (IR) and H2O2 on a stress-inducible domain. IR-induced phosphorylation of CREB correlates with a decrease in CREB transactivation potential and reduced interaction between CREB and its transcriptional coactivator, CREB-binding protein (CBP). A CREB mutant containing Ala substitutions at ATM phosphorylation sites displayed enhanced transactivation potential, resistance to inhibition by IR, and increased binding to CBP. It is proposed that ATM-mediated phosphorylation of CREB in response to DNA damage modulates CREB-dependent gene expression and that dysregulation of the ATM-CREB pathway may contribute to neurodegeneration in A-T (Shi, 2004).
c-Abl and Atm have been implicated in cell responses to DNA damage and oxidative stress. However, the molecular mechanisms by which they regulate oxidative stress response remain unclear. In this report, deficiency of c-Abl and deficiency of ATM are shown to differentially alter cell responses to oxidative stress; these signaling proteins function by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which require protein kinase C (PKC) delta activation and are mediated by reactive oxygen species. c-abl-/- osteoblasts display enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which are reinstated by reconstitution of c-Abl; Atm-/- osteoblasts show the opposite. These phenotypes correlate with increased PKC delta expression in c-abl-/- osteoblasts and decreased PKC delta expression in Atm-/- cells, respectively. The enhanced responses of c-abl-/- osteoblasts can be mimicked by overexpression of PKC delta in normal cells and impeded by inhibition of PKC delta, and diminished responses of Atm-/- cells can be rescued by PKC delta overexpression, indicating that PKC delta mediates the effects of c-Abl and ATM in oxidative stress response. Hence, these results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response (Li, 2004).
How does c-Abl or Atm regulate the protein level of PKC delta? c-Abl or Atm may affect the transcription of PKC delta gene, the stability of PKC delta mRNA or protein, or the translation efficiency of PKC delta mRNA. RT-PCR assays did not reveal any significant difference in the levels of PKC delta mRNA among wild-type and c-Abl-deficient osteoblasts, suggesting that c-Abl regulates PKC delta expression posttranscriptionally. It was found that c-Abl deficiency inhibits activation-induced degradation of PKC delta, but the molecular mechanism behind this warrants further investigation. Studies have indicated that in cells expressing activated Src (Y527F), PKC delta was down-regulated. This down-regulation is a result of phosphorylation-mediated degradation. It is speculated that c-Abl, a member of the Src family, may have a similar function in regulating the level of PKC delta. It has also been shown that c-Abl interacts with PKC delta in response to oxidative stress. c-Abl is able to phosphorylate PKC delta in fibroblasts. Unfortunately, PKC delta immunoprecipitated from c-Abl-deficient and control osteoblasts did not show significant difference in phosphorylation at tyrosine residues. One possible explanation is that PKC delta might have multiple sites for tyrosine phosphorylation that are carried out by several kinases. Hence, c-Abl deficiency would not make a detectable difference. The role for Atm in the regulation of PKC delta expression is even less clear. RT-PCR analysis revealed no significant difference in the levels of PKC delta mRNA, suggesting that the regulation, like that of c-Abl, occurs at posttranscriptional levels. Surprisingly, degradation of PKC delta was similar in Atm-/- osteoblasts and wild-type cells. One likely explanation is that the portion of degraded PKC delta molecules in Atm-/- osteoblasts may have a shortened lifespan, whereas the rest have a normal lifespan. Treatment of Atm-/- osteoblasts with MG132, a proteosome inhibitor, appeared to increase the PKC delta levels to that of control osteoblasts. The molecular mechanisms by which Atm regulates PKC delta protein levels need further investigation. Because Atm interacts with c-Abl and can activate it, it is possible that there exists a tertiary complex composed of PKC delta, c-Abl, and Atm in the cells, and that c-Abl may mediate the function of Atm in controlling PKC delta expression (Li, 2004).
Another layer of complexity is that Prx I/PAG is also a c-Abl interacting protein. c-Abl, a nonreceptor tyrosine kinase, plays a negative role in Prx I induction. Without c-Abl, osteoblasts show an enhanced induction of Prx I. On the basis of these facts, it is proposed that in normal osteoblasts, the induction of Prx I is suppressed, facilitating the activation of c-Abl. When c-Abl is deficient, the suppression is lifted and more Prx I is expressed. Therefore, a feedback circuit may exist that controls the activity of c-Abl in response to stress. Alternatively, interaction between c-Abl and Prx I may be involved in regulating the antioxidant activity of Prx I, for example, phosphorylation of Prx I by c-Abl. One such example is that Prx I could be phosphorylated by cdc2 and this phosphorylation reduces the activity of Prx I (Li, 2004).
Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. These interactions were examined in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affects the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, such interactions were examined in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene (Smilenov, 1999).
To examine the role of ataxia-telangiectasia mutated (Atm) in telomere function, Atm and telomerase null mice [Atm(-/-) mTR(-/-) iG6 mice] were generated. These mice exhibit increased germ cell death and chromosome fusions compared with either Atm(-/-) or mTR(-/-) iG6 mice. Furthermore, the Atm(-/-) mTR(--) iG6 mice have a delayed onset and reduced incidence of thymic lymphoma compared with Atm(-/-) mice. The tumors in the Atm(-/-) mTR(-/-) iG6 mice show increased apoptosis and anaphase bridges. Finally, lymphomas from Atm(-/-) mTR(-/-) iG6 mice were derived from CD8 immature, single-positive T cells, whereas Atm(-/-) lymphomas were from CD4(+)CD8(+) double-positive T cells. It is proposed that Atm protects short telomeres and that Atm deficiency cooperates with short telomeres, leading to increased cell death, decreased tumorigenesis, and increased overall survival (Qi. 2003)
Ataxia-telangiectasia results from the loss of ataxia-telangiectasia mutated (Atm) function and is characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, premature ageing and increased neoplasia incidence. The functional interaction of Atm and telomeres was examined in vivo. The impact of Atm deficiency was examined as a function of progressive telomere attrition at both the cellular and whole-organism level in mice doubly null for Atm and the telomerase RNA component (Terc). These compound mutants showed increased telomere erosion and genomic instability, yet they experienced a substantial elimination of T-cell lymphomas associated with Atm deficiency. A generalized proliferation defect was evident in all cell types and tissues examined, and this defect extended to tissue stem/progenitor cell compartments, thereby providing a basis for progressive multi-organ system compromise, accelerated ageing and premature death. Atm deficiency and telomere dysfunction act together to impair cellular and whole-organism viability, thus supporting the view that aspects of A-T pathophysiology are linked to the functional state of telomeres and its adverse effects on stem/progenitor cell reserves (Wong, 2003).
Centrosomes are the principal microtubule organising centers in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, cells were examined that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrest in G2 phase and form supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurs without an additional DNA replication round and is not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduces DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification is potentiated by Rad54 disruption. Gene targeting of ATM reduces, but does not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. These data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints (Dodson, 2004).
Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21, an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to gamma-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis were examined. p21 modifies the in vitro senescent response seen in AT fibroblasts. It is a downstream effector of ATM-mediated growth control. However, loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular (Wang, 1997).
Ataxia-telangiectasia is characterized by radiosensitivity, genome instability and predisposition to cancer. Heterozygous carriers of ATM, the gene defective in ataxia-telangiectasia, have a higher than normal risk of developing breast and other cancers. Atm 'knock-in' (Atm-Delta SRI) heterozygous mice harboring an in-frame deletion corresponding to the human 7636del9 mutation show an increased susceptibility to developing tumors. In contrast, no tumors are observed in Atm knockout [Atm(+/-)] heterozygous mice. In parallel, the appearance is reported of tumors in 6 humans from 12 families who are heterozygous for the 7636del9 mutation. Expression of ATM cDNA containing the 7636del9 mutation has a dominant-negative effect in control cells, inhibiting radiation-induced ATM kinase activity in vivo and in vitro. This reduces the survival of these cells after radiation exposure and enhances the level of radiation-induced chromosomal aberrations. These results show for the first time that mouse carriers of a mutated Atm that are capable of expressing Atm have a higher risk of cancer. This finding provides further support for cancer predisposition in human ataxia-telangiectasia carriers (Spring, 2002).
High NaCl activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), resulting in increased transcription of several protective genes, including the glycine betaine/gamma-aminobutyric acid transporter (BGT1). High NaCl damages DNA, and DNA damage activates ataxia telangiectasia mutated (ATM) kinase through autophosphorylation on Ser-1981. TonEBP/OREBP contains ATM consensus phosphorylation sites at Ser-1197, Ser-1247, and Ser-1367. The present studies test whether ATM is involved in activation of TonEBP/OREBP by high NaCl. Raising osmolality from 300 to 500 mosmol/kg by adding NaCl activates ATM, as indicated by phosphorylation at Ser-1981. High urea and radiation also activate ATM, but they do not increase TonEBP/OREBP transcriptional activity, as does high NaCl. Wortmannin, which inhibits ATM, reduces NaCl-induced TonEBP/OREBP transcriptional activation and BGT1 mRNA increase. Overexpression of wild-type TonEBP/OREBP increases ORE/TonE reporter activity much more than does overexpression of TonEBP/OREBP S1197A, S1247A, or S1367A. In AT cells (which express nonfunctional ATM), TonEBP/OREBP transcriptional and transactivating activity are further increased by expression of wild-type ATM but not of S1981A ATM. TonEBP/OREBP reciprocally coimmunoprecipitates with ATM kinase, demonstrating physical association. Additionally, antibody to ATM kinase supershifts TonEBP/OREBP bound to its cognate ORE/TonE DNA element. In AT cells, wortmannin further decreases high NaCl-induced increase in transcriptional activity, consistent with participation of signaling kinase(s) in addition to ATM. In conclusion, signaling via ATM is necessary for full activation of TonEBP/OREBP by high NaCl, but it is not sufficient (Irarrazabal, 2004).
DNA double-strand breaks (DSBs) induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell-cycle checkpoints. A significant fraction of mature ATM-deficient lymphocytes contain telomere-deleted ends produced by failed end joining during V(D)J recombination. These RAG-1/2 endonuclease-dependent, terminally deleted chromosomes persist in peripheral lymphocytes for at least 2 weeks in vivo and are stable over several generations in vitro. Restoration of ATM kinase activity in mature lymphocytes that have transiently lost ATM function leads to loss of cells with terminally deleted chromosomes. Thus, maintenance of genomic stability in lymphocytes requires faithful end joining as well a checkpoint that prevents the long-term persistence and transmission of DSBs. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently damaged by other means in mature cells (Callén, 2007).
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine tract in ataxin-1 (ATXN1). SCA1 pathogenesis studies support a model in which the expanded glutamine tract causes toxicity by modulating the normal activities of ATXN1. To explore native interactions that modify the toxicity of ATXN1, a targeted duplication of the mouse ataxin-1-like (Atxn1l, also known as Boat) locus, a highly conserved paralog of SCA1, was generated, and the role of this protein in SCA1 pathology was tested. Using a knock-in mouse model of SCA1 that recapitulates the selective neurodegeneration seen in affected individuals, it was found that elevated Atxn1l levels suppress neuropathology by displacing mutant Atxn1 from its native complex with Capicua (CIC). These results provide genetic evidence that the selective neuropathology of SCA1 arises from modulation of a core functional activity of ATXN1, and they underscore the importance of studying the paralogs of genes mutated in neurodegenerative diseases to gain insight into mechanisms of pathogenesis (Bowman, 2007).
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