Exo1 Processes Stalled Replication Forks and Counteracts Fork Reversal in Checkpoint-Defective Cells

Cotta‐Ramusino, Cecilia; Fachinetti, Daniele; Lucca, Chiara; Doksani, Ylli; Lopes, Massimo; Sogo, José M.; Foiani, Marco

doi:10.1016/j.molcel.2004.11.032

Cited by 246 publications

(266 citation statements)

References 40 publications

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“…7). These restart events may entail nucleolytic degradation rather than branch migration of the reversed forks (12), in agreement with the nucleolytic processing of reversed forks previously reported in yeast (38,39). Regardless of their source, postreplicative ssDNA gaps can certainly contribute to explain the observed accumulation of RAD51 in PARG-depleted cells.…”

Section: Discussionsupporting

confidence: 85%

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: Discussionsupporting

confidence: 85%

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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“…However, based on the exo1Δ mutational spectra and genetic interaction with REV3, we previously hypothesized that EXO1 participates in at least one MMR-independent mutation avoidance pathway [12]. In agreement with our supposition, recent studies have identified a potential role for EXO1 in the maintenance and repair of stalled replication forks [20][21][22].…”

Section: Introductionsupporting

confidence: 79%

“…In addition to the role of EXO1 in PRR proposed here, EXO1 has been implicated to function in cell cycle checkpoint, fork maintenance and fork repair pathways [20][21][22]51,52]. EXO1 has been shown to play subtle roles in end-processing for mitotic recombination and function redundantly with at least one other unidentified nuclease.…”

Section: Discussionmentioning

confidence: 78%

“…The MRX complex and Exo1p were shown to collaborate in producing long ssDNA tails at DSB ends and promote Mec1p association with the DSBs. The long ssDNA tails produced by Exo1p can also revoke fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed replication forks in a rad53-deficient background [22]. Consistent with a role in replication fork maintenance, EXO1 has been demonstrated to have a synthetic interaction with SGS1 mutations [20].…”

Section: Discussionmentioning

confidence: 99%

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A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

et al. 2007

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Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved postreplication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.

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“…Human exonuclease 1 (hExo1) is a 5Ј33Ј exonuclease (2, 3) that exists in Ia and Ib isoforms (2) and shares 27% identity with yeast Exo1 (2). Discovered in yeast (4), Exo1 participates in recombination (4,5), telomere maintenance (6, 7), mismatch repair (2,3,8), and processing of stalled replication forks (9). Consistent with its recombination function, mice devoid of Exo1 demonstrate a reduction in ssDNA formation at DSBs (7).…”

mentioning

confidence: 99%

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

Nimonkar

Özsoy

Genschel³

et al. 2008

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

269

236

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The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5 33 double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.Bloom syndrome ͉ Rad51 ͉ recombination ͉ RecQ ͉ DNA pairing H omologous recombination contributes toward the maintenance of genomic integrity by accurately repairing doublestranded DNA (dsDNA) breaks. The recombinational repair of dsDNA breaks (DSBs) proceeds by successive reactions that start with processing of the broken DNA ends to reveal single-stranded DNA (ssDNA) (1). Processing requires the action of a helicase and/or nuclease. The resultant 3Ј-terminated ssDNA is used by a DNA strand exchange protein as the substrate for assembly of the nucleoprotein filament that is the active species in the search for homologous DNA and subsequent DNA pairing. It is unclear which enzymes in eukaryotes resect the DNA break to initiate recombination; however, much is known about the process in bacteria. In Escherichia coli, recombinational DNA repair occurs by either the RecBCD or the RecF pathway (1). RecBCD is a helicase/nuclease that processes dsDNA to produce 3Ј-tailed ssDNA onto which RecA is loaded. In the RecF pathway, a separate helicase and nuclease are used for resection of DSBs and ssDNA-gaps: RecQ, a 3Ј35Ј helicase, and RecJ, a 5Ј33Ј exonuclease (1).

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Exo1 Processes Stalled Replication Forks and Counteracts Fork Reversal in Checkpoint-Defective Cells

Cited by 246 publications

References 40 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

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

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