Structure-Specific DNA Endonuclease Mus81/Eme1 Generates DNA Damage Caused by Chk1 Inactivation

Forment, Josep V.; Blasius, Melanie; Guerini, Ilaria; Jackson, Stephen

doi:10.1371/journal.pone.0023517

Cited by 97 publications

(123 citation statements)

References 29 publications

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“…Although it is not clearly essential for fork collapse in all cell types, the Mus81-Eme1 complex in Schizosaccharomyces pombe plays a crucial role in DSB generation at stalled replication forks (Froget et al 2008), and a similar relationship has been observed in mammalian cells when CHK1 is inhibited (Forment et al 2011). These findings in aggregate suggest that PLK1 could play an active facilitative role in replication fork collapse at the level of both the replisome and cleaving aberrant fork structures.…”

mentioning

confidence: 71%

“…The SLX4-endonuclease complex has previously been implicated as a mediator of replication fork collapse, particularly in cooperation with the MUS81-EME1 complex (Froget et al 2008;Forment et al 2011;Matos et al 2011;Gallo-Fernandez et al 2012;Munoz-Galvan et al 2012;Schwartz et al 2012;Szakal and Branzei 2013). To examine the involvement of this complex in the DSBs generated upon fork stalling in ATR-deficient cells, Crelox conditional SLX4 cells were used to delete Slx4 and determine the effect of its absence on DSB generation following ATR inhibition and fork stalling.…”

Section: Rnf4 and Plk1 Are Required For Slx4-dependent Dsb Formation mentioning

confidence: 99%

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RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

et al. 2013

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The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atrdeleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atrdeleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.

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confidence: 71%

Section: Rnf4 and Plk1 Are Required For Slx4-dependent Dsb Formation mentioning

confidence: 99%

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

et al. 2013

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“…4B). The MUS81 structurespecific endonuclease generates DSBs during persistent exposure to replication stress agents or in response to silencing or inhibition of replication fork repair proteins such as WRN, SMARCAL1, and CHK1 (Hanada et al 2007;Franchitto et al 2008;Forment et al 2011;Betous et al 2012). Therefore, we tested whether siRNA depletion of MUS81 affects DSB formation in HU/ATRi-treated cells.…”

Section: Atr Inhibition Causes Nascent-strand Ssdna Formationmentioning

confidence: 99%

ATR phosphorylates SMARCAL1 to prevent replication fork collapse

et al. 2013

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The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess singlestranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.

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“…The massive amount of DNA breakage is likely mediated by DNA endonuclease activity, and recent studies suggest that this is mediated by the endonuclease MUS81 (12,14,15). Notably, the mechanisms by which oncogenes or inhibition of checkpoint kinases can lead to endonuclease-mediated DNA breakage are poorly understood.…”

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confidence: 99%

Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

Beck

Nähse-Kumpf

Larsen

et al. 2012

Molecular and Cellular Biology

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Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage. DNA replication is tightly monitored to ensure that the genome is replicated precisely once per cell cycle and that DNA replication is complete before mitosis begins. Conditions for DNA synthesis are rarely ideal, and a number of obstacles must often be dealt with, such as a damaged DNA template and shortage of deoxynucleoside triphosphates (dNTPs), to allow replication fork progression. Stalling replication forks pose serious threats to genome integrity because they can collapse through disassembly of the replication complex and break (6,11,24). Such damaged forks may subsequently undergo incorrect repair, leading to genetic changes like chromosomal rearrangements (6,24). Recent data have also revealed that activated oncogenes can induce DNA replication stress, defined here as replication-associated DNA damage (2, 3, 10). Oncogene-induced replication stress can lead to additional tumor-promoting genetic changes, but it may also serve as a tumor barrier by activation of cell cycle arrest, apoptosis, and/or senescence during early tumor development (32).WEE1 and CHK1 kinases have major roles in suppressing DNA replication stress (4,23,27,42), and attenuation of their function can contribute to carcinogenesis and cause cell death (40). The massive amount of DNA breakage is likely mediated by DNA endonuclease activity, and recent studies suggest that this is mediated by the endonuclease MUS81 (12,14,15). Notably, the mechanisms by which oncogenes or inhibition of checkpoint kinases can lead to endonuclease-mediated DNA breakage are poorly understood. It is also not fully understood if these breaks also play a role in inducing fork stalling or if they are temporally delayed events secondary to the fork stalling.As both oncogenes and checkpoint kinases are regulators of cyclin-dependent kinase (CDK) activity, we previously proposed that most of the DNA replication str...

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Structure-Specific DNA Endonuclease Mus81/Eme1 Generates DNA Damage Caused by Chk1 Inactivation

Cited by 97 publications

References 29 publications

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

ATR phosphorylates SMARCAL1 to prevent replication fork collapse

Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

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