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PubMed2004

Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation.

The checkpoint kinase Chk1 undergoes ATR-mediated phosphorylation and activation in response to unreplicated DNA, but the precise mechanism of Chk1 activation is not known. In this study, we have analyzed the domain structure of Xenopus Chk1 and explored the mechanism of its activation by ATR-mediated phosphorylation. We show that the C-terminal region of Xenopus Chk1 contains an autoinhibitory region (AIR), which largely overlaps with a bipartite, unusually long ( approximately 85-amino acid) nuclear localization signal. When coexpressed in oocytes or embryos, the AIR can interact with and inhibit the kinase domain of Chk1, but not full-length Chk1, suggesting an autoinhibitory intramolecular interaction in the Chk1 molecule. If linked with the preceding ATR phosphorylation domain that has either phospho-mimic mutation or genuine phosphorylation, however, the AIR can no longer interact with or inhibit the kinase domain, suggesting a conformational change of the AIR by ATR-mediated phosphorylation. Even in full-length Chk1, such phospho-mimic mutation can interfere with the autoinhibitory intramolecular interaction, but only if this interaction is somewhat weakened by an additional mutation in the AIR. These results provide significant insights into the mechanism of Chk1 activation at the DNA replication checkpoint.

Amino Acid Motifs↗
PubMed1998

Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints.

ATR, a phosphatidylinositol kinase-related protein homologous to ataxia telangiectasia mutated (ATM), is important for the survival of human cells following many forms of DNA damage. Expression of a kinase-inactive allele of ATR (ATRkd) in human fibroblasts causes increased sensitivity to ionizing radiation (IR), cis-platinum and methyl methanesulfonate, but only slight UV radiation sensitivity. ATRkd overexpression abrogates the G2/M arrest after exposure to IR, and overexpression of wild-type ATR complements the radioresistant DNA synthesis phenotype of cells lacking ATM, suggesting a potential functional overlap between these proteins. ATRkd overexpression also causes increased sensitivity to hydroxyurea that is associated with microtubule-mediated nuclear abnormalities. These observations are consistent with uncoupling of certain mitotic events from the completion of S-phase. Thus, ATR is an important component of multiple DNA damage response pathways and may be involved in the DNA replication (S/M) checkpoint.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

Hardening neutron spectrum for advanced actinide transmutation experiments in the ATR.

The most effective method for transmuting long-lived isotopes contained in spent nuclear fuel into shorter-lived fission products is in a fast neutron spectrum reactor. In the absence of a fast test reactor in the United States, initial irradiation testing of candidate fuels can be performed in a thermal test reactor that has been modified to produce a test region with a hardened neutron spectrum. Such a test facility, with a spectrum similar but somewhat softer than that of the liquid-metal fast breeder reactor (LMFBR), has been constructed in the INEEL's Advanced Test Reactor (ATR). The radial fission power distribution of the actinide fuel pin, which is an important parameter in fission gas release modelling, needs to be accurately predicted and the hardened neutron spectrum in the ATR and the LMFBR fast neutron spectrum is compared. The comparison analyses in this study are performed using MCWO, a well-developed tool that couples the Monte Carlo transport code MCNP with the isotope depletion and build-up code ORIGEN-2. MCWO analysis yields time-dependent and neutron-spectrum-dependent minor actinide and Pu concentrations and detailed radial fission power profile calculations for a typical fast reactor (LMFBR) neutron spectrum and the hardened neutron spectrum test region in the ATR. The MCWO-calculated results indicate that the cadmium basket used in the advanced fuel test assembly in the ATR can effectively depress the linear heat generation rate in the experimental fuels and harden the neutron spectrum in the test region.

Actinoid Series Elements↗
PubMed1999

A role for ATR in the DNA damage-induced phosphorylation of p53.

Phosphorylation at Ser-15 may be a critical event in the up-regulation and functional activation of p53 during cellular stress. In this report we provide evidence that the ATM-Rad3-related protein ATR regulates phosphorylation of Ser-15 in DNA-damaged cells. Overexpression of catalytically inactive ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to gamma-irradiation and UV light. In gamma-irradiated cells, ATRki expression selectively interfered with late-phase Ser-15 phosphorylation, whereas ATRki blocked UV-induced Ser-15 phosphorylation in a time-independent manner. ATR phosphorylated p53 at Ser-15 and Ser-37 in vitro, suggesting that p53 is a target for phosphorylation by ATR in DNA-damaged cells.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

DNA damage sensors ATM, ATR, DNA-PKcs, and PARP-1 are dispensable for human immunodeficiency virus type 1 integration.

Integration of a DNA copy of the viral RNA genome is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses. While the virally encoded integrase is key to this process, cellular factors yet to be characterized are suspected to participate in its completion. DNA damage sensors such as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related), DNA-PK (DNA-dependent protein kinase), and PARP-1 [poly(ADP-ribose) polymerase 1] play central roles in responses to various forms of DNA injury and as such could facilitate HIV integration. To test this hypothesis, we examined the susceptibility to infection with wild-type HIV-1 and to transduction with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector of human cells stably expressing small interfering RNAs against ATM, ATR, and PARP-1. We found that integration normally occurred in these knockdown cells. Similarly, the VSV-G-pseudotyped HIV-1-based vector could effectively transduce ATM and PARP-1 knockout mouse cells as well as human cells deficient for DNA-PK. Finally, treatment of target cells with the ATM and ATR inhibitors caffeine and wortmannin was without effect in these infectivity assays. We conclude that the DNA repair enzymes ATM, ATR, DNA-PKcs, and PARP-1 are not essential for HIV-1 integration.

Animals↗
PubMed2006

Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit.

In higher eukaryotic organisms, the checkpoint kinase 1 (Chk1) contributes essential functions to both cell cycle and checkpoint control. Chk1 executes these functions, in part, by targeting the Cdc25A protein phosphatase for ubiquitin-mediated proteolysis. In response to genotoxic stress, Chk1 is phosphorylated on serines 317 (S317) and 345 (S345) by the ataxia-telangiectasia-related (ATR) protein kinase. Phosphorylation of Chk1 on these C-terminal serine residues is used as an indicator of Chk1 activation in vivo. Here, we report that inhibition of Chk1 kinase activity paradoxically leads to the accumulation of S317- and S345-phosphorylated Chk1 in vivo and that ATR catalyzes Chk1 phosphorylation under these conditions. We demonstrate that Chk1 phosphorylation by ATR is antagonized by protein phosphatase 2A (PP2A). Importantly, dephosphorylation of Chk1 by PP2A is regulated, in part, by the kinase activity of Chk1. We propose that the ATR-Chk1-PP2A regulatory circuit functions to keep Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 primed to respond rapidly in the event that cells encounter genotoxic stress.

Carbazoles↗
PubMed2006

An ATR- and BRCA1-mediated Fanconi anemia pathway is required for activating the G2/M checkpoint and DNA damage repair upon rereplication.

The timely assembly of prereplicative complexes at replication origins is tightly controlled to ensure that genomic DNA is replicated once per cell cycle. The loss of geminin, a DNA replication inhibitor, causes rereplication that activates a G2/M checkpoint in human cancer cells. Fanconi anemia (FA) is an autosomal recessive and X-linked disorder associated with cancer susceptibility. Here we show that rereplication activates the FA pathway both for the activation of a G2/M checkpoint and for repair processes, like recruitment of RAD51. Both ATR and BRCA1 are required to activate the FA pathway. The G2/M checkpoint-mediated arrest of the cell cycle is critical for the prevention of both apoptosis and the accumulation of cells with rereplicated DNA, because the loss of ATR, BRCA1, or FANCA promotes apoptosis and suppresses the accumulation. The accumulation of cells with rereplicated DNA is restored by the artificial induction of a G2-phase arrest even when ATR, BRCA1, or FANCA is absent. Therefore, the ATR- and BRCA1-mediated FA pathway is required for the activation of a G2/M checkpoint and for DNA damage repair in response to the endogenous signal of rereplication. In its absence, the cells rapidly lose viability when faced with rereplication.

Apoptosis↗
PubMed2001

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.

Adenoviridae↗
PubMed2002

Hypoxia links ATR and p53 through replication arrest.

Previous studies have demonstrated that phosphorylation of human p53 on serine 15 contributes to protein stabilization after DNA damage and that this is mediated by the ATM family of kinases. However, cellular exposure to hypoxia does not induce any detectable level of DNA lesions compared to ionizing radiation, and the oxygen dependency of p53 protein accumulation differs from that of HIF-1, the hypoxia-inducible transcription factor. Here we show that, under severe hypoxic conditions, p53 protein accumulates only in S phase and this accumulation correlates with replication arrest. Inhibition of ATR kinase activity substantially reduces hypoxia-induced phosphorylation of p53 protein on serine 15 as well as p53 protein accumulation. Thus, hypoxia-induced cell growth arrest is tightly linked to an ATR-signaling pathway that is required for p53 modification and accumulation. These studies indicate that the ATR kinase plays an important role during tumor development in responding to hypoxia-induced replication arrest, and hypoxic conditions could select for the loss of key components of ATR-dependent checkpoint controls.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Xenopus Cds1 is regulated by DNA-dependent protein kinase and ATR during the cell cycle checkpoint response to double-stranded DNA ends.

The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.

Amino Acid Sequence↗
PubMed1996

A point mutation in the XNP gene, associated with an ATR-X phenotype without alpha-thalassemia.

We have previously reported the isolation of a gene from Xq13, coding for a putative regulator of transcription (XNP). It is a member of the helicase family, and has now been shown to be the gene involved in the X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome. ATR-X mutations were only found in the 3'-part of the coding sequence, which includes the helicase domains. However, no ATR-X mutation has yet been found in one of the seven conserved helicase domains. In this paper, we report a mutation in XNP, segregating in a family presenting an "ATR-X' phenotype without alpha-thalassemia, that causes a proline to serine transition in the helicase II domain.

Base Sequence↗
PubMed2006

ATR-Chk1 axis protects BCR/ABL leukemia cells from the lethal effect of DNA double-strand breaks.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C (MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (gamma-H2AX). gamma-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR-Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Antineoplastic Agents↗
PubMed2001

Somatic mutations in the DNA damage-response genes ATR and CHK1 in sporadic stomach tumors with microsatellite instability.

Maintenance of genomic stability depends on the appropriate cellular responses to DNA damage and the integrity of the DNA repair systems. We analyzed stomach tumors with microsatellite instability (MSI) for frameshift mutations in several potential targets of the mutator phenotype involved in DNA damage-response pathways, such as the ataxia telangiectasia mutated protein-related protein (ATR)-CHK1-Cdc25c pathway, and DNA repair. High frequency of mutations was found within ATR [5 (21%) of 23], MED1 [10 (43%) of 23], hMSH3 [13 (56%) of 23], and hMSH6 [10 (43%) of 23] genes. Also, a low frequency of mutations within the CHK1 gene was detected in 9% (2 of 23) of tumors. No mutations of hMLH3, ATM, BRCA1, or NBS1 genes were detected. These results confirm ATR, MED1, and CHK1 as targets of the mutator pathway in stomach tumorigenesis, and also suggest a potential role of MED1 increasing, together with hMSH3 and hMSH6, the genomic instability in the mutator pathway as a secondary mutator. Furthermore, these results suggest that the inhibition of the ATR-CHK1 DNA damage-response pathway might be involved in the tumorigenesis of gastric cancer with microsatellite instability.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1992

X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.

We have examined seven pedigrees that include individuals with a recently described X-linked form of severe mental retardation associated with alpha-thalassemia (ATR-X syndrome). Using hematologic and molecular approaches, we have shown that intellectually normal female carriers of this syndrome may be identified by the presence of rare cells containing HbH inclusions in their peripheral blood and by an extremely skewed pattern of X inactivation seen in cells from a variety of tissues. Linkage analysis has localized the ATR-X locus to an interval of approximately 11 cM between the loci DXS106 and DXYS1X (Xq12-q21.31), with a peak LOD score of 5.4 (recombination fraction of 0) at DXS72. These findings provide the basis for genetic counseling, assessment of carrier risk, and prenatal diagnosis of the ATR-X syndrome. Furthermore, they represent an important step in developing strategies to understand how the mutant ATR-X allele causes mental handicap, dysmorphism, and down-regulation of the alpha-globin genes.

Dosage Compensation, Genetic↗
PubMed2004

[A comparative study of malignant tissue diagnosis using ATR and microscopy FTIR spectroscopy].

A comparative FTIR study was undertaken between ATR (attenuated total reflection) and infrared microscopy on their application to measuring the same tissue for cancer diagnosis. The measurements showed that the coincidence of pathological diagnosis with ATR is higher than that of microscopy FTIR method. The result indicated that the measured area of tumor tissue is critical. The area for microscopy measurement is approximately 250 microm2, it reflects the heterogeneity of the tissue. However, the ATR measurement provides more information of malignant tissue for the measured area is 1.9 x 10(5) times than microscopy. Therefore, ATR exhibit the high accuracy in cancer detection for clinical application.

Fiber Optic Technology↗
PubMed1998

Human oral mucosa studies with varying blood glucose concentration by non-invasive ATR-FT-IR-spectroscopy.

During the last few years infrared spectrometry has been investigated as a non-invasive clinical tool for improved understanding of in-vivo processes. Oral mucosa has been suggested as an especially suited subject for drug delivery and in vivo monitoring of endogenous body metabolites due to histological and physicochemical reasons. The attenuated total reflection (ATR) technique was used to characterize the outmost epidermal layer of human oral mucosa by means of Fourier-transform infrared spectroscopy. The penetration depth of the probing radiation in the mid-infrared fingerprint region, using a ZnSe-crystal for the horizontal ATR accessory, is in the order of a few micrometers so that microlayer information can be obtained by such a technique. Spectra of outer human lip and saliva components are presented for comparison. For several test persons, lip spectra were recorded during oral glucose tolerance tests. The individually varying blood glucose concentration was followed by means of frequent blood testing. Variability of the outmost microlayer has been studied using factor analysis of the ATR inner-lip spectra. There is no clear evidence that blood glucose concentration can be followed by ATR-spectroscopy of oral mucosa. Non-invasive spectroscopic methods exploiting trace signals require special attention paid to the variability due to person-to-person differences and changes in physiological conditions.

Blood Glucose↗
PubMed2000

The SOX8 gene is located within 700 kb of the tip of chromosome 16p and is deleted in a patient with ATR-16 syndrome.

SOX proteins are transcription factors that are characterized by a common DNA-binding motif known as the HMG domain. We describe the 5. 4-kb human SOX8 gene that codes for a 446-amino-acid protein and that is expressed strongly in brain and less abundantly in other tissues. SOX8 shows an overall identity of 47% to SOX9 and SOX10. The latter two possess a C-terminal transactivation domain, whereas in SOX8, this domain is located in the central part of the protein. We have mapped SOX8 within 700 kb of the telomeric repeats of band 16p13.3. Hemizygosity for 1 Mb from this region causes the ATR-16 syndrome characterized by alpha-thalassemia and mental retardation. We show that SOX8 is deleted in an ATR-16 patient, and from its location, we deduce that it should be deleted in all previously described cases. Thus, SOX8 is a good candidate gene contributing to the mental retardation phenotype seen in ATR-16 patients.

Amino Acid Sequence↗
PubMed2003

A comparative study of mid-infrared diffuse reflection (DR) and attenuated total reflection (ATR) spectroscopy for the detection of fungal infection on RWA2-corn.

An investigation into the rapid detection of mycotoxin-producing fungi on corn by two mid-infrared spectroscopic techniques was undertaken. Corn samples from a single genotype (RWA2, blanks, and contaminated with Fusarium graminearum) were ground, sieved and, after appropriate sample preparation, subjected to mid-infrared spectroscopy using two different accessories (diffuse reflection and attenuated total reflection). The measured spectra were evaluated with principal component analysis (PCA) and the blank and contaminated samples were classified by cluster analysis. Reference data for fungal metabolites were obtained with conventional methods. After extraction and clean-up, each sample was analyzed for the toxin deoxynivalenol (DON) by gas chromatography with electron capture detection (GC-ECD) and ergosterol (a parameter for the total fungal biomass) by high-performance liquid chromatography with diode array detection (HPLC-DAD). The concentration ranges for contaminated samples were 880-3600 microg/kg for ergosterol and 300-2600 microg/kg for DON. Classification efficiency was 100% for ATR spectra. DR spectra did not show as obvious a clustering of contaminated and blank samples. Results and trends were also observed in single spectra plots. Quantification using a PLS1 regression algorithm showed good correlation with DON reference data, but a rather high standard error of prediction (SEP) with 600 microg/kg (DR) and 490 microg/kg (ATR), respectively, for ergosterol. Comparing measurement procedures and results showed advantages for the ATR technique, mainly owing to its ease of use and the easier interpretation of results that were better with respect to classification and quantification.

Food Analysis↗