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Autor: Falck, J

22 záznamů v Medvik Filtry

Článek

CDK targeting of NBS1 promotes DNA-end resection, replication restart and homologous recombination

Embo reports. 2012 ; 13 (6) : 561-568.

EMBO Rep
ISSN 1469-221X
Medvik
Zdroj

The conserved MRE11-RAD50-NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.

Článek

HCLK2 is required for activity of the DNA damage response kinase ATR

The Journal of biological chemistry. 2009 ; 284 (7) : 4140-4147.

J Biol Chem
ISSN 0021-9258
Medvik
Zdroj

ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

Článek

Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

The Journal of cell biology. 2009 ; 186 (5) : 655-663.

J Cell Biol
ISSN 0021-9525
Medvik
Zdroj

Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA-mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.

Článek

Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage-modified chromatin

The Journal of cell biology. 2008 ; 181 (2) : 213-226.

J Cell Biol
ISSN 0021-9525
Medvik
Zdroj

DNA double-strand breaks (DSBs) trigger accumulation of the MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN-MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.

Článek

ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks

Nature cell biology. 2006 ; 8 (1) : 37-45.

Nat Cell Biol
ISSN 1465-7392
Medvik
Zdroj

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.

Článek

Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway

Experimental cell research. 2005 ; 302 (2) : 162-169.

Exp Cell Res
ISSN 0014-4827
Medvik
Zdroj

When exposed to DNA-damaging insults such as ionizing radiation (IR) or ultraviolet light (UV), mammalian cells activate checkpoint pathways to halt cell cycle progression or induce cell death. Here we examined the ability of five commonly used anticancer drugs with different mechanisms of action to activate the Chk1/Chk2-Cdc25A-CDK2/cyclin E cell cycle checkpoint pathway, previously shown to be induced by IR or UV. Whereas exposure of human cells to topoisomerase inhibitors camptothecin, etoposide, or adriamycin resulted in rapid (within 1 h) activation of the pathway including degradation of the Cdc25A phosphatase and inhibition of cyclin E/CDK2 kinase activity, taxol failed to activate this checkpoint even after a prolonged treatment. Unexpectedly, although the alkylating agent cisplatin also induced degradation of Cdc25A (albeit delayed, after 8-12 h), cyclin E/CDK2 activity was elevated and DNA synthesis continued, a phenomena that correlated with increased E2F1 protein levels and consequently enhanced expression of cyclin E. These results reveal a differential impact of various classes of anticancer chemotherapeutics on the Cdc25A-degradation pathway, and indicate that the kinetics of checkpoint induction, and the relative balance of key components within the DNA damage response network may dictate whether the treated cells arrest their cell cycle progression.

Článek

Alternative splicing and mutation status of CHEK2 in stage III breast cancer

Oncogene. 2004 ; 23 (52) : 8535-8544.

ISSN 0950-9232
Medvik
Zdroj

The DNA damage checkpoint kinase, CHK2, promotes growth arrest or apoptosis through phosphorylating targets such as Cdc25A, Cdc25C, BRCA1, and p53. Both germline and somatic loss-of-function CHEK2 mutations occur in human tumours, the former linked to the Li-Fraumeni syndrome, and the latter found in diverse types of sporadic malignancies. Here we examined the status of CHK2 by genetic and immunohistochemical analyses in 53 breast carcinomas previously characterized for TP53 status. We identified two CHEK2 mutants, 470T>C (Ile157Thr), and a novel mutation, 1368insA leading to a premature stop codon in exon 13. The truncated protein encoded by CHEK2 carrying the 1368insA was stable yet mislocalized to the cytoplasm in tumour sections and when ectopically expressed in cultured cells. Unexpectedly, we found CHEK2 to be subject to extensive alternative splicing, with some 90 splice variants detected in our tumour series. While all cancers expressed normal-length CHEK2 mRNA together with the spliced transcripts, we demonstrate and/or predict some of these splice variants to lack CHK2 function and/or localize aberrantly. We conclude that cytoplasmic sequestration may represent a novel mechanism to disable CHK2, and propose to further explore the significance of the complex splicing patterns of this tumour suppressor gene in oncogenesis.

Článek

Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation

Oncogene. 2004 ; 23 (17) : 3122-3127.

ISSN 0950-9232
Medvik
Zdroj

The ATM kinase is a tumour suppressor and a key activator of genome integrity checkpoints in mammalian cells exposed to ionizing radiation (IR) and other insults that elicit DNA double-strand breaks (DSBs). In response to IR, autophosphorylation on serine 1981 causes dissociation of ATM dimers and initiates cellular ATM kinase activity. Here, we show that the kinetics and magnitude of ATM Ser1981 phosphorylation after exposure of human fibroblasts to low doses (2 Gy) of IR are altered in cells deficient in Nbs1, a substrate of ATM and a component of the MRN (Mre11-Rad50-Nbs1) complex involved in processing/repair of DSBs and ATM-dependent cell cycle checkpoints. Timely phosphorylation of both ATM Ser1981 and the ATM substrate Smc1 after IR were rescued via retrovirally mediated reconstitution of Nbs1-deficient cells by wild-type Nbs1 or mutants of Nbs1 defective in the FHA domain or nonphosphorylatable by ATM, but not by Nbs1 lacking the Mre11-interaction domain. Our data indicate that apart from its role downstream of ATM in the DNA damage checkpoint network, the MRN complex serves also as a modulator/amplifier of ATM activity. Although not absolutely required for ATM activation, the MRN nuclease complex may help reach the threshold activity of ATM necessary for optimal genome maintenance and prevention of cancer.

Článek

CHEK2 variant I157T may be associated with increased breast cancer risk

International journal of cancer. 2004 ; 111 (4) : 543-547.

Int J Cancer
ISSN 0020-7136
Medvik
Zdroj

Cell cycle checkpoint kinase 2 (CHEK2) is a transducer of cellular responses to DNA damage. The CHEK2 1100delC has previously been shown to be a low-penetrance breast cancer susceptibility allele. We have evaluated the role of another CHEK2 variant, I157T in the FHA domain of the gene, for association with breast cancer. I157T was found at a significantly higher frequency in the population-based series of breast cancer patients (77/1035, 7.4%, odds ratio [OR] = 1.43, 95% confidence interval [CI] = 1.06-1.95, p = 0.021) than among population controls (100/1885, 5.3%). The frequency in the familial breast cancer patients was not elevated (28/507, 5.5%, OR = 1.04, 95% CI = 0.68-1.61). The I157T protein, that undermines cellular responses to ionizing radiation and shows deficiency in substrate recognition in vivo, was expressed at normal level in tumor tissues as well as in cultured cells. The I157T protein was stable and it dimerized with the wild-type CHEK2 co-expressed in human cells. These functional properties of the I157T protein suggest that this variant may have negative effect on the pool of normal CHEK2 protein in heterozygous carrier cells by formation of heterodimers with wild-type CHEK2. The I157T variant may be associated with breast cancer risk, but the risk is lower than for 1100delC. Copyright 2004 Wiley-Liss, Inc.

MeSH
checkpoint kinasa 2 MeSH
genetická predispozice k nemoci * MeSH
imunohistochemie MeSH
lidé MeSH
nádory prsu * etiologie genetika MeSH
odds ratio MeSH
poškození DNA MeSH
protein-serin-threoninkinasy * genetika MeSH
replikace DNA MeSH
studie případů a kontrol MeSH
tolerance záření MeSH
tumor supresorové geny MeSH
zárodečné mutace * MeSH
Check Tag
lidé MeSH
ženské pohlaví MeSH

Článek

Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention

EMBO journal. 2004 ; 23 (13) : 2674-2683.

EMBO J
ISSN 0261-4189
Medvik
Zdroj

Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11-Rad50-Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double-strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1-independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1's FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB-flanking chromatin and caused aberrant pan-nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB-induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA-mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB-flanking chromatin. Our data suggest that Mdc1 functions as an H2AX-dependent interaction platform enabling a switch from transient, Mdc1-independent recruitment of Nbs1 to DSBs towards sustained, Mdc1-dependent interactions with the surrounding chromosomal microenvironment.

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