Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates

Neelsen, Kai J.; Zanini, Isabella M.Y.; Herrador, Raquel; Lopes, Massimo

doi:10.1083/jcb.201212058

Cited by 171 publications

(179 citation statements)

References 40 publications

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“…We show that EME2 depletion and PLK1 inhibition efficiently restore the bulk of DNA replication in cells treated with WEE1 inhibitors, arguing that a significant fraction of replication intermediates targeted for cleavage is capable of supporting DNA synthesis effectively. Therefore, even though most naturally-occurring MUS81 substrates are likely to be stalled RFs (Beck et al, 2012;Naim et al, 2013;Neelsen et al, 2013;Ying et al, 2013), we envision that RF remodeling may not be a prerequisite for substrate recognition and cleavage. In agreement with this model, WEE1 inhibition triggers massive DNA breakage and chromosome pulverization without significantly altering the recruitment of RPA to replicating chromosomes ( Figure 3H and I).…”

Section: Wee1 Prevents Unscheduled Processing Of Vital Replication Inmentioning

confidence: 99%

“…Interestingly, besides licensing premature entry into mitosis, down-regulation of WEE1 or CHK1 causes CDK-dependent breakage of replicating chromosomes (Beck et al, 2010;Beck et al, 2012;Dominguez-Kelly et al, 2011;Forment et al, 2011). In turn, CDC25A overexpression leads to replication fork (RF) reversal, which is also followed by CDK1-dependent DNA cleavage (Neelsen et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…Yet, several studies established that it involves MUS81, a structure-selective endonuclease with important roles in the metabolism of replication intermediates (Beck et al, 2012;Dominguez-Kelly et al, 2011;Forment et al, 2011;Kim et al, 2013;Neelsen et al, 2013;Techer et al, 2016). In mammals, MUS81 forms heterodimeric complexes with the non-catalytic subunits EME1 or EME2 (Pepe and West, 2014a).…”

Section: Introductionmentioning

confidence: 99%

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A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis

et al. 2016

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While DNA replication and mitosis occur in a sequential manner, precisely how cells maintain their temporal separation and order remains elusive. Here, we unveil a double-negative feedback loop between replication intermediates and an M-phase-specific structure-selective endonuclease, MUS81-SLX4, which renders DNA replication and mitosis mutually exclusive. MUS81 nuclease is constitutively active throughout the cell cycle but requires association with SLX4 for efficient substrate targeting. To preclude toxic processing of replicating chromosomes, WEE1 kinase restrains CDK1 and PLK1-mediated MUS81-SLX4 assembly during S phase. Accordingly, WEE1 inhibition triggers widespread nucleolytic breakage of replication intermediates, halting DNA replication and leading to chromosome pulverization. Unexpectedly, premature entry into mitosis-licensed by unrestrained CDK1 activity during S phaserequires MUS81-SLX4, which inhibits DNA replication. This suggests that ongoing replication assists WEE1 in delaying entry into M phase and, indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates, which safeguards chromosome segregation. Unexpectedly, premature entry into mitosis -licensed by unrestrained CDK1 activity during S-phase-requires MUS81-SLX4, which inhibits DNA replication. This suggests that ongoing replication assists WEE1 in delaying entry into M-phase and, indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates, which safeguards chromosome segregation.3

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Section: Wee1 Prevents Unscheduled Processing Of Vital Replication Inmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis

et al. 2016

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“…We also show that accumulation of these unusual replication intermediates is accompanied by DDR activation and chromatin recruitment of DSB repair factors, suggesting the conceptually attractive hypothesis that the formation of DNA ends (regressed arms) by fork reversal could contribute to checkpoint signaling. It should be noted, however, that other experimental conditions recently reported to induce similar levels of reversed replication forks have not been associated with DDR activation (11,(32)(33)(34), excluding the possibility that fork reversal per se is sufficient to induce checkpoint activation and chromatin recruitment of DSB repair factors. Checkpoint activation upon PAR accumulation may directly reflect the recently reported physical association of PAR with checkpoint factors, such as CHK1 (35).…”

Section: Discussionmentioning

confidence: 94%

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Chaudhuri

Ahuja

Herrador

et al. 2015

Molecular and Cellular Biology

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Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribosyl) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibition per se slows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and single-stranded DNA (ssDNA) gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG-defective cells displayed both ataxia-telangiectasia-mutated (ATM) and ataxia-Rad3-related (ATR) activation, as well as chromatin recruitment of standard double-strand-break-repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation to be essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restarting are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy.C ellular responses are crucial for the adaptability and survival of a cell exposed to different types of endogenous and exogenous stress. The DNA damage response (DDR) consists of one such defense mechanism in response to different types of insults to the DNA. Poly(ADP)ribosylation of proteins is one of the quickest cellular responses to DNA damage and is brought about by proteins of the poly(ADP) ribose polymerase family (PARP), mostly PARP1 (1). Upon being recruited to sites of the DNA damage, NAD ϩ is used as a substrate by PARP to synthesize negatively charged poly(ADP-ribosyl)ation (PAR) polymers onto itself and also its target proteins (1). Through this posttranslational modification, PARP targets a variety of nuclear proteins to facilitate the recruitment of DNA repair factors to sites of damage (2, 3). Accordingly, PARP-1 or PARP-2-deficient mice and mouse embryonic fibroblasts show chromosomal aberrations and various DNA repair defects (4-6).Inhibition of PARP has become a promising therapeutic approach for the treatment of certain types of cancer (7). It was shown that PARP inhibitors could selectively kill homologous recombination (HR)-deficient cancer cells (8, 9). The reason behind the sensitivity of HR-deficient cells to PARP inhibition is thought to be the accumulation of single-stranded DNA (ssDNA) breaks in the absence of PAR synthesis, leading to replication fork collapse and double-stranded breaks (DSBs), which would then require HR factors for repair (8,10). Recently, PARP activity has also been reported to play a ro...

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“…S6B,C). The latter two features closely resemble the effects of oncogene activation (Neelsen et al 2013) and thus most likely reflect the licensing defects common to these genetic conditions (Hook et al 2007;Blow and Gillespie 2008) and their consequences in terms of nucleotide depletion (Bester et al 2011) and/or interference with transcription (Jones et al 2013). Although ssDNA sensors (e.g., RPA chromatin loading and PCNA ubiquitylation) ( Fig.…”

Section: Deregulation Of Origin Licensing Induces Ssdna Gaps On Replimentioning

confidence: 99%

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

et al. 2013

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Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.

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Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates

Abstract: Processing of unusual replication intermediates such as reversed forks by MUS81 contributes to oncogene-induced double-strand breaks and depends on mitotic entry.

Cited by 171 publications

References 40 publications

A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis

A Mechanism for Controlled Breakage of Under-replicated Chromosomes during Mitosis

Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

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