Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

Zellweger, Ralph; Dalcher, Damian; Mutreja, Karun; Berti, Matteo; Schmid, Jonas; Herrador, Raquel; Vindigni, Alessandro; Lopes, Massimo

doi:10.1083/jcb.201406099

Cited by 600 publications

(856 citation statements)

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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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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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“…-MBD cells to 10 nM CPT, a dose that typically causes mild replicative stress without inducing substantial DNA breaks (34) (Fig. 4A), triggered a significant increase in the number of chromosomal aberrations (Fig.…”

Section: Resultsmentioning

confidence: 99%

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress

Bleuyard

Fournier

Nakato

et al. 2017

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

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The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.nherited mutations in the partner and localiser of BRCA2 (PALB2) gene predispose to breast and pancreatic cancer (1-3) and also congenital malformations, growth retardation, and early childhood cancer in a rare subgroup of Fanconi anemia (FA-N) patients (4, 5). The best characterized function of PALB2 is to physically link the two main breast cancer susceptibility gene products, BRCA1 and BRCA2, at sites of DNA damage (6-8), thus playing a pivotal role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA1 facilitates DNA-end resection to produce single-stranded (ss) DNA (9) and concomitantly attracts PALB2, together with BRCA2 and RAD51, to DSB sites (6-8). In turn, BRCA2 promotes the loading of the RAD51 recombinase onto ssDNA (10-12), which is critical for the strand invasion and exchange phase of HR (13). This mechanism of repair factor recruitment is important for the timely activation of HR at sites of DNA damage, and its perturbation in individuals with impaired PALB2 function is presumed to cause human pathologies. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, we and others have provided evidence that PALB2 also associates with chromatin in the absence of DNA damage (14-17). A recent genome-wide analysis of PALB2 chromatin occupancy revealed enrichment at highly active genes, with PALB2 occupying the entire body of these genes (18). PALB2 was shown to support the transcription of a subset of NF-κB-and retinoic acid-responsive genes. However, PALB2 does...

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“…3 C and Fig. S3 A), which does not lead to extensive DNA breakage (Zellweger et al, 2015). Using native BrdU staining to visualize ssDNA formation in S phase cells, we found that the increased amount of ssDNA generated in response to CPT-induced replication stress was suppressed by TRA IP knockdown (Fig.…”

Section: Tra Ip Facilitates Ssdna Formation and Checkpoint Signalingmentioning

confidence: 95%

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

Hoffmann¹,

Smedegaard²,

Nakamura³

et al. 2016

J Exp Med

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Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

Abstract: Genotoxic treatments in human cells consistently induce uncoupling of replication forks and their remodeling into four-way junctions by the RAD51 recombinase.

Cited by 600 publications

References 61 publications

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

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

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

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