Coupling end resection with the checkpoint response at DNA double-strand breaks

Villa, Matteo; Cassani, Corinne; Gobbini, Elisa; Bonetti, Diego; Longhese, Maria Pia

doi:10.1007/s00018-016-2262-6

Cited by 32 publications

(36 citation statements)

References 113 publications

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“…Generation of ssDNA at DSBs is promoted by several proteins, which control either initiation (Mre11, Rad50, Xrs2 and Sae2) or extension (Dna2, Sgs1 and Exo1) of resection (3,4). To assess whether Npl3 supports DSB processing by promoting the expression of resection factors, we measured the amount of the above proteins by western blot of protein extracts from wild type and npl3Δ cells expressing fully functional tagged versions of these proteins and treated with galactose to induce the HO cut.…”

Section: Resultsmentioning

confidence: 99%

“…Exo1 action is modulated by both positive and negative regulators, which control Exo1 access to DNA and limit excessive DNA degradation (3,4,6–8). Exo1 expression is also induced during yeast meiosis to promote meiotic DSB processing and crossing over (65).…”

Section: Discussionmentioning

confidence: 99%

“…This cleavage creates a substrate for two partially overlapping pathways, which depend on the nucleases Exo1 and Dna2, respectively, and promote the generation of long ssDNA tails (reviewed in 3,4). While Exo1 is a 5΄-3΄ exonuclease capable of efficiently degrading the 5΄ end on duplex DNA, the endonuclease Dna2 requires the helicase activity of Sgs1 (orthologue of mammalian BLM) to efficiently remove small fragments from DNA ends (3,4). …”

Section: Introductionmentioning

confidence: 99%

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The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1

Colombo

Trovesi

Menin

et al. 2017

Nucleic Acids Research

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Eukaryotic cells preserve genome integrity upon DNA damage by activating a signaling network that promotes DNA repair and controls cell cycle progression. One of the most severe DNA damage is the DNA double-strand break (DSB), whose 5΄ ends can be nucleolitically resected by multiple nucleases to create 3΄-ended single-stranded DNA tails that trigger DSB repair by homologous recombination. Here, we identify the Saccharomyces cerevisiae RNA binding protein Npl3 as a new player in DSB resection. Npl3 is related to both the metazoan serine-arginine-rich and the heterogeneous nuclear ribonucleo-proteins. NPL3 deletion impairs the generation of long ssDNA tails at the DSB ends, whereas it does not exacerbate the resection defect of exo1Δ cells. Furthermore, either the lack of Npl3 or the inactivation of its RNA-binding domains causes decrease of the exonuclease Exo1 protein levels as well as generation of unusual and extended EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, indicate that Npl3 participates in DSB resection by promoting the proper biogenesis of EXO1 mRNA.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1

Colombo

Trovesi

Menin

et al. 2017

Nucleic Acids Research

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“…Homology‐dependent DSB repair mechanisms require extensive resection of broken ends which are then used as a platform for localization of DNA damage signalling complexes as well as DNA repair machineries, in order to trigger DNA damage checkpoint, cell cycle arrest and break repair (Symington et al , 2014; Villa et al , 2016). At the late stages of repair, ssDNA must be restored into a double‐stranded state by re‐synthesis of the resected DNA which will otherwise constantly signal for checkpoint activation leading to a persistent cell cycle arrest and cell death.…”

Section: Discussionmentioning

confidence: 99%

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

et al. 2017

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Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.

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“…Homologous recombination requires that the 5′ ends of a DSB are nucleolytically processed (resected) to generate 3′‐ended single‐stranded DNA (ssDNA) that inhibits NHEJ and channels DSB repair into HR (Chiruvella et al , ; Mehta & Haber, ). Genetic studies in yeast implicate the MRX (MRN in humans) complex and Sae2 in early steps of resection (Symington, ; Villa et al , ). In vitro , Mre11 exhibits both 3′–5′ double‐strand DNA (dsDNA)‐specific exonuclease and ssDNA‐specific endonuclease activities (Paull & Gellert, ; Trujillo et al , ).…”

Section: Introductionmentioning

confidence: 99%

The MRX complex regulates Exo1 resection activity by altering DNA end structure

et al. 2018

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Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.

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Coupling end resection with the checkpoint response at DNA double-strand breaks

Cited by 32 publications

References 113 publications

The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1

The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

The MRX complex regulates Exo1 resection activity by altering DNA end structure

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