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

Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.
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PMID:Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage. 1103 Mar 44

Abundant CDK2/cyclin A activity is present in human cancer cells, suggesting that rapid S phase CDK2 inhibition would be an effective anti-cancer approach. The dynamic change of chromatin-loading and -dissociation of MCM proteins requires S phase CDK2 activity. CDK2 inhibition during replication leads to increased MCM complex association with DNA and triggers rereplication. Overreplication-induced DSB and RPA-ssDNA intermediates activate ATM and ATR, resulting in a p53 response which selectively deletes cells with unresolved rereplication.
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PMID:A model for CDK2 in maintaining genomic stability. 1549 12

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.
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PMID:Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases. 1644 60

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.
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PMID:ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks. 1660 1

The functionally related ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases are critical regulators of DNA damage responses in mammalian cells. ATM and ATR share highly overlapping substrate specificities and show a strong preference for the phosphorylation of Ser or Thr residues followed by Gln. In this report we used a polyreactive phosphospecific antibody (alpha-pDSQ) that recognizes a subset of phosphorylated Asp-Ser-Gln sequences to purify candidate ATM/ATR substrates. This led to the identification of phosphorylation sites in the carboxyl terminus of the minichromosome maintenance protein 3 (MCM3), a component of the hexameric MCM DNA helicase. We show that the alpha-DSQ antibody recognizes tandem DSQ phosphorylation sites (Ser-725 and Ser-732) in the carboxyl terminus of murine MCM3 (mMCM3) and that ATM phosphorylates both sites in vitro. ATM phosphorylated the carboxyl termini of mMCM3 and human MCM3 in vivo and the phosphorylated form of MCM3 retained association with the canonical MCM complex. Although DNA damage did not affect steady-state levels of chromatin-bound MCM3, the ATM-phosphorylated form of MCM3 was preferentially localized to the soluble, nucleoplasmic fraction. This finding suggests that the carboxyl terminus of chromatin-loaded MCM3 may be sequestered from ATM-dependent checkpoint signals. Finally, we show that ATM and ATR jointly contribute to UV light-induced MCM3 phosphorylation, but that ATM is the predominant UV-activated MCM3 kinase in vivo. The carboxyl-terminal ATM phosphorylation sites are conserved in vertebrate MCM3 orthologs suggesting that this motif may serve important regulatory functions in response to DNA damage. Our findings also suggest that DSQ motifs are common phosphoacceptor motifs for ATM family kinases.
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PMID:Identification of carboxyl-terminal MCM3 phosphorylation sites using polyreactive phosphospecific antibodies. 1724 5

DNA replication is tightly regulated, but paradoxically there is reported to be an excess of MCM DNA replication proteins over the number of replication origins. Here, we show that MCM levels in primary human T cells are induced during the G(0)-->G(1) transition and are not in excess in proliferating cells. The level of induction is critical as we show that a 50% reduction leads to increased centromere separation, premature chromatid separation (PCS) and gross chromosomal abnormalities typical of genomic instability syndromes. We investigated the mechanisms involved and show that a reduction in MCM levels causes dose-dependent DNA damage involving activation of ATR & ATM and Chk1 & Chk2. There is increased DNA mis-repair by non-homologous end joining (NHEJ) and both NHEJ and homologous recombination are necessary for Mcm7-depleted cells to progress to metaphase. Therefore, a simple reduction in MCM loading onto DNA, which occurs in cancers as a result of aberrant cell cycle control, is sufficient to cause PCS and gross genomic instability within one cell cycle.
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PMID:Reducing MCM levels in human primary T cells during the G(0)-->G(1) transition causes genomic instability during the first cell cycle. 2044 Feb 61

DNA replication is an essential process for cell division and as such it is a process that is directly targeted by several anticancer drugs. CDC7 plays an essential role in the activation of replication origins and has recently been proposed as a novel target for drug discovery. The MCM DNA helicase complex (MCM2-7) is a key target of the CDC7 kinase, and MCM phosphorylation status at specific sites is a reliable biomarker of CDC7 cellular activity. In this work we describe a cell-based assay that utilizes the "In Cell Western Technique" (ICW) to identify compounds that affect cellular CDC7 activity. By screening a library of approved drugs and kinase inhibitors we found several compounds that can affect CDC7-dependent phosphorylation of MCM2 in HeLa cells. Among these, Mitoxantrone, a topoisomerase inhibitor, and Ryuvidine, previously described as a CDK4 inhibitor, cause a reduction in phosphorylated MCM2 levels and a sudden blockade of DNA synthesis that is accompanied by an ATM-dependent checkpoint response. This study sheds light on the previously observed cytotoxity of Ryuvidine, strongly suggesting that it is related to its effect of causing DNA damage.
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PMID:A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response. 2490 48

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4(DDB2). Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation.
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PMID:UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation. 2954 85

MYC-induced nuclear antigen (MINA53) is a JmjC (jumonji C domain)-containing protein, which is highly expressed in many cancers including glioblastoma. We have revealed in our previous report that MINA53 is a poor prognostic indicator for glioblastoma patients, and knockdown of MINA53 could reduce glioblastoma malignancy. In this study, we found that MINA53 knockdown could decrease the DNA replication initiation in glioblastoma cells. Through further investigations, we revealed that MINA53 could regulate the expression of the CDC45-MCM-GINS (CMG) complex genes, which are vital for DNA replication initiation. Knockdown of MINA53 reduced the CMG genes expression and thus induced DNA replication stress and DNA damage. Furthermore, MINA53 knockdown diminished DNA damage response (DDR) by reducing the ATM/ATR-H2AX pathway activity and finally led glioblastoma cells to apoptosis and death. We further applied a genotoxic drug Doxorubicin and found that MINA53 deficiency sensitized glioblastoma cells to Doxorubicin. Our study reveals that MINA53 is involved in DNA replication initiation and DNA damage response, and provides support for MINA53 as a novel and potential therapeutic target for glioblastoma treatment.
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PMID:MINA53 deficiency leads to glioblastoma cell apoptosis via inducing DNA replication stress and diminishing DNA damage response. 3033 81