ATR-dependent pathways control hEXO1 stability in response to stalled forks

El-Shemerly, Mahmoud; Heß, Daniel; Pyakurel, Aswin; Moselhy, Said S; Ferrari, Stefano

doi:10.1093/nar/gkm1052

Cited by 73 publications

(81 citation statements)

References 40 publications

(50 reference statements)

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“…DDK is therefore required for the initiation of replicationcheckpoint activation, and we also show for the recovery of stalled forks. An active replicationcheckpoint would then attenuate nucleolytic activity at stalled forks to prevent excessive degradation of DNA by described mechanisms [41,[47][48][49]. Budding yeast cells lacking the checkpoint kinase Rad53, which inhibits DDK, exhibited extremely long tracks of ssDNA in presence of replication stress, which were abrogated upon deletion of Exo1 [33] again consistent with our model.…”

Section: Low Dose Ddki Causes Aberrant Mitotic Structuressupporting

confidence: 85%

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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Section: Low Dose Ddki Causes Aberrant Mitotic Structuressupporting

confidence: 85%

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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“…hEXO1 is expressed at low levels in human nonproliferating tissues (23)(24)(25)(26), and the endogenous protein is barely detectable in total crude extracts from cell lines because of its low abundance and limited affinity to the available Abs (23). However, hEXO1 can be recovered and analyzed by Western blot analysis after immunoprecipitation.…”

Section: Resultsmentioning

confidence: 99%

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

Sertic

Pizzi

Cloney

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells.local UV damage | UV lesion processing | ssDNA | ubiquitin

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“…Studies in both human cells and budding yeast have indicated that phosphorylation of Exo1 negatively regulates its activity. Exo1 phosphorylation appears to be regulated by the Mec1/ATR pathways in budding yeast and human cells (11,30). Extrapolating to fission yeast, it is plausible that eliminating Rad3 enhances Exo1 activity and thereby allows Exo1 to substitute for Ctp1.…”

Section: Discussionmentioning

confidence: 99%

Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3^ATR and Tel1^ATM Checkpoint Kinases at Double-Strand Breaks

Limbo

Porter-Goff

Rhind

et al. 2011

Molecular and Cellular Biology

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Rad3, the Schizosaccharomyces pombe ortholog of human ATR and Saccharomyces cerevisiae Mec1, activates the checkpoint kinase Chk1 in response to DNA double-strand breaks (DSBs). Rad3 ATR/Mec1 associates with replication protein A (RPA), which binds single-stranded DNA overhangs formed by DSB resection. In humans and both yeasts, DSBs are initially detected and processed by the Mre11-Rad50-Nbs1Xrs2 (MRN) nucleolytic protein complex in association with the Tel1 ATM checkpoint kinase and the Ctp1 CtIP/Sae2 DNA-end processing factor; however, in budding yeast, neither Mre11 nuclease activity or Sae2 are required for Mec1 signaling at irreparable DSBs. Here, we investigate the relationship between DNA end processing and the DSB checkpoint response in fission yeast, and we report that Mre11 nuclease activity and Ctp1 are critical for efficient Rad3-to-Chk1 signaling. Moreover, deleting Ctp1 reveals a Tel1-to-Chk1 signaling pathway that bypasses Rad3. This pathway requires Mre11 nuclease activity, the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp complex, and Crb2 checkpoint mediator. Ctp1 negatively regulates this pathway by controlling MRN residency at DSBs. A Tel1-to-Chk1 checkpoint pathway acting at unresected DSBs provides a mechanism for coupling Chk1 activation to the initial detection of DSBs and suggests that ATM may activate Chk1 by both direct and indirect mechanisms in mammalian cells.DNA double-strand breaks (DSBs), formed by clastogens or from endogenous damage, trigger multiple cellular responses that are critical for maintaining genome integrity. Of particular importance is the cell cycle checkpoint that restrains the onset of mitosis while DSB repair is under way. Chk1 is the critical effector of this checkpoint in the fission yeast Schizosaccharomyces pombe and mammalian cells, whereas the budding yeast Saccharomyces cerevisiae uses both Chk1 and Rad53 (orthologous to human Chk2 and fission yeast Cds1) to delay anaphase entry and mitotic exit. These kinases are regulated by ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) checkpoint kinases (5). Curiously, the regulatory connections between ATM/ATR and Chk1/Chk2 orthologs are not strictly conserved between species (Fig. 1A). In mammals, ATM activates Chk2 while ATR activates Chk1. In S. cerevisiae and S. pombe, ATR orthologs (Mec1 and Rad3, respectively) activate Chk2 orthologs and Chk1, while Tel1 (ATM ortholog) is primarily involved in telomere maintenance (14,38,40).The functions of ATM and ATR orthologs are intimately tied to the detection and nucleolytic processing of DSBs. ATM Tel1 localizes at DSBs by interacting with Mre11-Rad50-Nbs1Xrs2 (MRN) protein complex, which directly binds DNA ends (12,20,24,50,52). The MRN complex is essential for ATM Tel1 function in all species. The Mre11 subunit of MRN complex has DNase activities that are critical for radioresistance in S. pombe and mice but not in budding yeast (3,19,22,50). In fission yeast, MRN complex also recruits Ctp1 DNA end-processing factor to DSBs (25,49). Ctp1 is structurall...

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ATR-dependent pathways control hEXO1 stability in response to stalled forks

Cited by 73 publications

References 40 publications

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

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

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3^ATR and Tel1^ATM Checkpoint Kinases at Double-Strand Breaks

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ATR-dependent pathways control hEXO1 stability in response to stalled forks

Cited by 73 publications

References 40 publications

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

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

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks

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Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3^ATR and Tel1^ATM Checkpoint Kinases at Double-Strand Breaks