Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase

Buisson, Rémi; Boisvert, Jessica L.; Benes, Cyril H.; Zou, Lee

doi:10.1016/j.molcel.2015.07.029

Cited by 271 publications

(340 citation statements)

References 69 publications

(96 reference statements)

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“…2A and B). We next checked the effects of AZD7762 treatment on DNA damage signaling in H1299 cells and found that treatment induced DNA damage signaling through ATM, likely due to aberrant DNA replication during S phase (34). Treatment of irradiated H1299 cells with AZD7762 did not markedly affect ATM signaling (Fig.…”

Section: Resultsmentioning

confidence: 96%

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Kucharski

Sharon

et al. 2016

J Virol

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Chicken anemia virus (CAV) is a single-stranded circular DNA virus that carries 3 genes, the most studied of which is the gene encoding VP3, also known as apoptin. This protein has been demonstrated to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of the protein is important for the difference. In normal cells, apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells, apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ataxia telangiectasia mutated (ATM) pathway induces the translocation of apoptin from the cytoplasm to the nucleus, where it induces apoptosis. We found that apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing apoptin with inhibitors of checkpoint kinase 1 (Chk1) and Chk2 causes apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of apoptin. Finally, treatment of virus-producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication. IMPORTANCEThe chicken anemia virus (CAV) protein apoptin is known to induce tumor cell-specific death when expressed. Therefore, understanding its regulation and mechanism of action could provide new insights into tumor cell biology. We have determined that checkpoint kinase 1 and 2 signaling is important for apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for antiviral therapy. Circoviruses are a diverse group of nonenveloped, icosahedral viruses containing circular, single-stranded DNA genomes (1, 2). These viruses have been shown to infect a wide range of hosts, and they are the causative agents of several serious diseases in animals. In particular, chicken anemia virus (CAV), a member of the genus Gyrovirus, is the etiological agent of chicken infectious anemia. CAV infects several bone marrow-derived cells, resulting in severe anemia and immunosuppression in young chickens and compromised immune response in older birds (3, 4). CAV can lead to considerable economic loss during intensive chicken farming, and control of the virus through vaccination is currently standard practice. Recently, a novel circovirus with partial homology to CAV isolated from a skin swab was designated human gyrovirus (HGyV) (5). The identificati...

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Section: Resultsmentioning

confidence: 96%

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Kucharski

Sharon

et al. 2016

J Virol

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“…Firstly, inhibition of ATR kinase in unperturbed cells induces aberrant origin firing leading to accumulation of chromatin-bound RPA1 and RPA2 ( Figure 1D, lane 3 vs. 15) [22,23]. This RPA accumulation, however, was prevented upon co-treatment with DDKi ( Figure 1D, lane 15 vs. 18) consistent with the essential role of DDK in origin firing.…”

Section: (Supplementarymentioning

confidence: 53%

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

Sasi

Coquel

Lin

et al. 2017

Preprint

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Abstract:CDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here we show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1, and we show that EXO1 is also required for nascent strand degradation following exposure to HU, raising the possibility that DDK regulates EXO1 directly. Thus, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.Keywords: DNA replication checkpoint/ replication fork/ fork resection/ fork restart/ DDK peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/207266 doi: bioRxiv preprint first posted online 3 DDK is a two-subunit kinase that is essential to initiate DNA replication at individual replication origins by phosphorylating and activating the MCM2-7 replicative helicase. The regulatory subunit DBF4 binds to CDC7 and is required for its kinase activity [1]. DDK also has roles in replication checkpoint signaling that are less well understood [1]. In budding yeast, DDK is a target of the checkpoint effector kinase Rad53 that is activated following replication stress [2]. Rad53-mediated phosphorylation of Dbf4 modestly reduces DDK activity [2] and also inhibits late origin firing [3,4], but there is also evidence that DDK is required for the complete activation of Rad53 kinase [5]. In fission yeast, DDK subunits Hsk1 and Dfp1 are phosphorylated upon HU treatment by the checkpoint kinase Cds1, orthologous to budding yeast Rad53 and mammalian CHK2 [6][7][8]. Deletion of either Cds1 or its activating protein Mrc1 partially rescues the temperature sensitivity of hsk1-1312 mutants suggesting that Cds1 (like Rad53) inhibits DDK activity [8,9]. Cds1 activation in response to HU, however, was reduced significantly in hsk1(ts) strains at restrictive temperature and the cell cycle arrest was also aberrant as seen by an increased population of 'cut' cells (indicative of mitosis without complete DNA replication) [8,10]. These paradoxical results in yeast could be explained by a negative feedback loop where DDK first helps to initiate replication checkpoint activation and then the checkpoint pathway subsequently alters DDK activity to inhibit late origin firing.Initial studies in human cells showed that the replication checkpoint inhibits DDK activity, presumably to inhibit origin firing [11,...

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“…This is likely to be part of a mechanism of coping with the dNTP depletion through RRM2 accumulation. The reduction in E2F1 levels in the CHK1i-treated CHK1i-hypersensitive cell lines are likely to result in reduced RRM2 levels as reported previously (Buisson et al, 2015), and reduced dNTP levels, thus mimicking the effect of HU in reducing cellular dNTP levels. The D20, BL, MM96L and MM329 cell lines were also the ones with the greatest reduction in the non-phosphorylated RPA2 band …”

Section: Chk1i Promotes Rpa2 Hyper-phosphorylation In Chk1i-hypersenssupporting

confidence: 63%

“…Moreover, normal cell types are not killed with siRNA -mediated CHK1 depletion at concentrations that inhibited CHK1 functions (Brooks et al, 2013, Cho et al, 2005. Nevertheless, CHK1 inhibition is effective in sensitising cells to the agents which promotes DNA damage and replication stress, and CHK1 inhibition alone caused cell death in cancer cells with defective DNA repair pathways and high level of replication stress (Brooks et al, 2013, Chen et al, 2009, Buisson et al, 2015.…”

Section: Introductionmentioning

confidence: 99%

Mechanisms and markers of CHK1 inhibitors sensitivity in melanoma

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Landscape for the treatment of cancer patients has recently been changed with the arrival of targeted therapies in different malignancies with the drugs targeting specific mutations and genetic alterations such as, EGF-receptor blockers and BRAF inhibitors, and the drugs such as PARP inhibitors which exploit defects in the cancers and cause synthetic lethality. In this thesis, I investigated to understand the mechanism of action of a drug that targets the cell cycle checkpoint regulator, checkpoint kinase 1 (CHK1) and explored its potential therapeutic usage in specific cancer types. Checkpoint kinase 1 inhibitor (CHK1i) as a single-agent treatment is effective in some of the cancer types with high levels of replication stress, including melanoma. However, the mechanism and manner of cell-killings induced by CHK1i single-agent treatment is still poorly understood. To identify the patient population who can benefit from CHK1i single-agent treatment, it is important to understand how single-agent CHK1i induced the cell killing. CHK1i has been investigated in pre-clinical studies and clinical trials, and shown to enhance the efficacy of chemotherapeutic drugs, particularly those that promote replication stress such as gemcitabine.Most of those investigations were based on the previous notion that CHK1i abrogates the G2/M phase checkpoint to cause mitotic catastrophe and results in cancer cell death. However, considering numerous roles of CHK1 playing in cell cycle and DNA damage response pathway, this mechanism alone may not represent the entirety of sensitivity to CHK1i. CHK1 has different roles in S and G2/M phases of cell cycle such as controlling the G2/M checkpoint, stabilising the stalled replication fork, and inhibiting the apoptosis together with other DNA damage repair and cell cycle checkpoint regulators. When one of these other regulators/pathways has defects, inhibiting CHK1 can be synthetically lethal to the cancer cells. Another CHK1 function in S phase is regulating CDC25A; CHK1 triggers the destabilisation of CDC25A upon DNA damage and replication stress.The normal role of CDC25A in S phase progression is activation of CDK2 which is required for progression into and through S phase. CDC25A dysregulation and overexpression has been reported in various cancers and shown to create increased replication stress in the cells. Tumours with high level of replication stress have been suggested to be selectively susceptible to the inhibition of CHK1 and its upstream regulator, ATR.In this thesis, it was firstly shown that S phase cell cycle checkpoint defect is a common feature of CHK1i-hypersensitive melanoma cell lines, and this defect is often associated with failure to degrade CDC25A in response to replication stress. Furthermore, CDC25A over-expression and/or dysregulation contributes to CHK1i sensitivity in hypersensitive melanoma cell lines. Secondly, it was demonstrated that CHK1i induced high level of replication stress via RPA hyperphosphorylation and subsequently RPA depletion occurred in h...

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Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase

Cited by 271 publications

References 69 publications

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

Mechanisms and markers of CHK1 inhibitors sensitivity in melanoma

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