186,375 research outputs found

    Cellular and clinical impact of Haploinsufficiency for genes involved in ATR signaling

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    Ataxia telangiectasia and Rad3-related (ATR) protein, a kinase that regulates a DNA damage-response pathway, is mutated in ATR-Seckel syndrome (ATR-SS), a disorder characterized by severe microcephaly and growth delay. Impaired ATR signaling is also observed in cell lines from additional disorders characterized by microcephaly and growth delay, including non-ATR-SS, Nijmegen breakage syndrome, and MCPH1 (microcephaly, primary autosomal recessive, 1)-dependent primary microcephaly. Here, we examined ATR-pathway function in cell lines from three haploinsufficient contiguous gene-deletion disorders--a subset of blepharophimosis-ptosis-epicanthus inversus syndrome, Miller-Dieker lissencephaly syndrome, and Williams-Beuren syndrome--in which the deleted region encompasses ATR, RPA1, and RFC2, respectively. These three genes function in ATR signaling. Cell lines from these disorders displayed an impaired ATR-dependent DNA damage response. Thus, we describe ATR signaling as a pathway unusually sensitive to haploinsufficiency and identify three further human disorders displaying a defective ATR-dependent DNA damage response. The striking correlation of ATR-pathway dysfunction with the presence of microcephaly and growth delay strongly suggests a causal relationship

    Claspin and the Activated Form of ATR-ATRIP Collaborate in the Activation of Chk1

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    Claspin is necessary for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. ATR possesses a regulatory partner called ATRIP. We have studied the respective roles of ATR-ATRIP and Claspin in the activation of Chk1. ATR-ATRIP bound well to various DNA templates in Xenopus egg extracts. ATR-ATRIP bound to a single-stranded DNA template was weakly active. By contrast, the ATR-ATRIP complex on a DNA template containing both single- and double-stranded regions displayed a large increase in kinase activity. This observation suggests that ATR-ATRIP normally undergoes activation upon association with specific nucleic acid structures at DNA replication forks. Without Claspin, activated ATR-ATRIP phosphorylated Chk1 weakly in a cell-free reaction. The addition of Claspin to this reaction strongly stimulated the phosphorylation of Chk1 by ATR-ATRIP. Claspin also induced significant autophosphorylation of Chk1 in the absence of ATR-ATRIP. Taken together, these results indicate that the checkpoint-dependent phosphorylation of Chk1 is a multistep process involving activation of the ATR-ATRIP complex at replication forks and presentation of Chk1 to this complex by Claspin

    Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target validation in breast cancer

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    Background: ATR-Chk1 signalling network is critical for genomic stability. ATR-Chk1 may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. Patients and methods: We investigated ATR and phosphorylated CHK1Ser345 protein (pChk1) expression in 1712 breast cancers (Nottingham Tenovus series). ATR and Chk1 mRNA were evaluated in 1950 breast cancers (METABRIC cohort). Pre-clinically, biological consequences of ATR gene knockdown or ATR inhibition by small molecule inhibitor (VE-821) were investigated in MCF-7 and MDA-MB-231 breast cancer cell lines and in non-tumorigenic breast epithelial cells (MCF10A). Results: High ATR and high cytoplasmic pChk1 expression was significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analysis, high ATR and high cytoplasmic pChk1 protein expression was associated with shorter breast cancer specific survival (BCSS). In multivariate analysis, high ATR remains an independent predictor of adverse outcome. At the mRNA level, high Chk1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her-2 overexpression, triple-negative phenotype and molecular classes associated with aggressive behaviour and shorter survival.. Pre-clinically, Chk1 phosphorylation at serine 345 following replication stress (induced by gemcitabine or hydroxyurea treatment) was impaired in ATR knockdown and in VE-821 treated breast cancer cells. Doxycycline inducible knockdown of ATR suppressed growth, which was restored when ATR was re-expressed. Similarly, VE-821 treatment resulted in a dose dependent suppression of cancer cell growth and survival (MCF7 and MDA-MB-231) but had no effect on non-tumorigenic breast epithelial cells (MCF10A). Conclusions: We provides evidence that ATR and Chk1 are promising biomarkers and rational drug target for personalized therapy in breast cancer

    Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

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    A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be define

    Ataxia-telangiectasia Mutated (ATM)-dependent Activation of ATR Occurs through Phosphorylation of TopBP1 by ATM

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    ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not to DNA replication stress. TopBP1 has been identified as a direct activator of ATR. We show that ATM regulates Xenopus TopBP1 by phosphorylating Ser-1131 and thereby strongly enhancing association of TopBP1 with ATR. Xenopus egg extracts containing a mutant of TopBP1 that cannot be phosphorylated on Ser-1131 are defective in the ATR-dependent phosphorylation of Chk1 in response to DSBs but not to DNA replication stress. Thus, TopBP1 is critical for the ATM-dependent activation of ATR following production of DSBs in the genome

    Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP

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    ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA

    The Rad9-Hus1-Rad1 Checkpoint Clamp Regulates Interaction of TopBP1 with ATR

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    TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR

    Evidence for Respiratory Neuromodulator Interdependence after Cholinergic Disruption in the Ventral Respiratory Column

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    Reverse dialysis of the muscarinic receptor antagonist, atropine (ATR, 50 mM), into the pre-Bötzinger Complex region of the ventral respiratory column (VRC) of awake and sleeping goats increases breathing frequency and serotonin (5-HT), substance P (SP), glycine, and GABA concentrations in the effluent dialysate. Herein, we report data from goats in which we reverse dialyzed 5 mM ATR or specific antagonists of M2 or M3 muscarinic receptors into the VRC. The effects on frequency of all three antagonists were not significantly different from time control studies. 5 mM ATR and the M3 antagonist increased SP sevenfold less than 50 mM ATR. The antagonists had no effect on 5-HT, glycine, and/or GABA, suggesting that the increases in glycine and GABA with 50 mM ATR were secondary to the larger increases in 5-HT and/or SP. These data are suggestive of neuromodulator interdependence, whereby attenuation of one neuromodulator is compensated for by local changes in other neuromodulators to stabilize breathing
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