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USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

ABSTRACT: USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

PROVIDER: PRJNA1122641 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29362466.fastq.gz	Fastqsanger.gz
	SRR29362467.fastq.gz	Fastqsanger.gz
	SRR29362468.fastq.gz	Fastqsanger.gz
	SRR29362469.fastq.gz	Fastqsanger.gz

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USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

Project description:Mammalian DNA replication relies on various DNA helicases and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN: FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks..

2024-08-21 | GSE269605 | GEO

A whole genome-screening strategy to identify DNA2 binding sites and genomic regions that require DNA2 for replication

Project description:We report that DNA2 predominantly bound to the centromeric α-satellite regions. The centromeric regions contained 58% of the DNA2-associated DNA, representing a 33.5-fold enrichment over genomic input. To define the under-replicated DNA regions in DNA2-null cells, we conducted whole-genome DNA sequencing of the under-replicated BrdU negative DNA. After normalization to genomic input DNA from the same cells, 12.9% of the peaks from the under-replicated DNA aligned with the centromeric DNA regions, representing an 8.5-fold enrichment. In addition, among the peaks that overlapped between the DNA2 pull-down and under-replicated regions, two thirds fell into the centromeric regions, representing a 48-fold enrichment.

2018-04-10 | GSE108619 | GEO

Dna2 removes toxic RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis

Project description:During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strand can displace the parental strand to generate single-strand DNA (ssDNA) flaps. Many programmed DSBs and thus many ssDNA flaps occur during meiosis. However, how these flaps are removed remains enigmatic. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) in Saccharomyces cerevisiae causes an abundant accumulation of RPA-ssDNA flaps and an expansion of RPA from DSBs to broader regions. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, inducing Dna2 expression at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreased and increased RPA accumulation in dna2-md, respectively. Together, our findings show that meiotic DSB repair requires Dna2 to remove RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.

2023-08-30 | GSE217628 | GEO

Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

Project description:Targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.

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RPA promotes processive ssDNA digestion by Dna2 via their bimodal interaction in Saccharomyces cerevisiae

Project description:Single-stranded DNA (ssDNA) widely exists as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors out-compete RPA and access to ssDNA. Using the budding yeast, Saccharomyces cerevisiae, as a model system, we discovered that Dna2, a key nuclease in DNA replication and repair, employs a bimodal interface to act with RPA both in cis and in trans. The cis-action renders RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA actively delivers its bound ssDNA to Dna2. The trans-action mediated by an acidic patch from Dna2, on the other hand, enables Dna2 to operatie with a sub-optimal amount of RPA or to overcome DNA secondary structures. Genetically, this trans-action mode is not required for cell viability, but indispensable for successful DSB repair.

2022-02-22 | PXD028637 | Pride

Dynamic Control of RNA-DNA Hybrid Formation Orchestrates DNA2 Activation at Stalled Forks by RNAPII and DDX39A [ChIP-seq]

Project description:The accurate processing of stalled forks by the DNA2 nuclease is pivotal for replication fork restart, as excessive degradation poses a threat to genomic stability. However, the regulation of DNA2 activity at stalled forks remains elusive. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. Furthermore, we provide evidence that DDX39A, functioning as an RNA-DNA resolver, unwinds these hybrids, allowing DNA2 access to stalled forks. This orchestrated process facilitates controlled DNA2-dependent stalled fork processing and restart. Nevertheless, premature removal of RNA-DNA hybrids at stalled forks leads to DNA2-dependent excessive degradation of nascent DNA. Finally, we reveal that loss of DDX39A enhances the protection of stalled forks in BRCA1/2-deficient cells, consequently conferring chemoresistance within this specific cellular context. Our results suggest that the dynamic regulation of RNA-DNA hybrid formation at stalled forks by RNAPII and DDX39A precisely governs the timing of DNA2 activation, contributing to stalled fork processing and restart, ultimately promoting genome stability.

2024-12-19 | GSE252172 | GEO

Dynamic Control of RNA-DNA Hybrid Formation Orchestrates DNA2 Activation at Stalled Forks by RNAPII and DDX39A [RNA-seq]

2024-12-19 | GSE252171 | GEO

A whole genome-screening strategy to identify DNA2 binding sites and genomic regions that require DNA2 for replication

Project description:A whole genome-screening strategy to identify DNA2 binding sites and genomic regions that require DNA2 for replication

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The RNA helicase Ded1p suppresses translation initiation from near-cognate start codons

Project description:The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian ortholog DDX3 are critical for translation initiation. Mutations in DDX3 are linked to tumorigenesis and intellectual disability, and the enzyme is targeted by diverse viruses. How Ded1p and its orthologs engage RNAs to impact translation initiation has been a longstanding, unresolved question. Here we show that Ded1p associates with the pre-initiation complex at the mRNA entry channel of the small ribosomal subunit and that the helicase unwinds mRNA structure ahead of the scanning pre-initiation complex. Defective Ded1p causes pervasive translation in 5’UTRs, starting from near-cognate initiation codons located 5' of mRNA structures and concomitant decrease of protein synthesis from of the main ORFs. The data indicate that Ded1p functions to suppress translation initiation on near-cognate codons proximal to mRNA structure and show how the helicase is targeted to specific RNA sites without common sequence signatures. Our results reveal a straightforward mechanism for the activation of upstream open reading frames and suggest that mRNA structure and proximal near-cognate initiation codons encode a widespread regulatory program for translation initiation that is sensitive to RNA helicase function.

2017-01-24 | GSE93959 | GEO

Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

Project description:Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

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