Dna2 removes toxic RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis
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ABSTRACT:
PROVIDER: PRJNA899853 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22246726_1.fastq.gz | Fastqsanger.gz | |||
| SRR22246726_2.fastq.gz | Fastqsanger.gz | |||
| SRR22246727_1.fastq.gz | Fastqsanger.gz | |||
| SRR22246727_2.fastq.gz | Fastqsanger.gz | |||
| SRR22246728_1.fastq.gz | Fastqsanger.gz |
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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
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
Project description:During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many programmed DSBs and thus many ssDNAs occur during meiosis. However, it is unclear how these ssDNAs are removed for the complete repair of meiotic DSBs. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) results in an abundant accumulation of RPA and an expansion of RPA from DSBs to broader regions in Saccharomyces cerevisiae. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, Dna2 induction 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 decreases and increases RPA accumulation in dna2-md, respectively. In addition, blocking DNA synthesis during meiotic recombination dramatically decreases RPA accumulation in dna2-md. Together, our findings show that meiotic DSB repair requires Dna2 to remove ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.
| S-EPMC10450173 | biostudies-literature
Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. Strand-specific transcriptome analysis of biological replicates of WT cells (JKM139 strain) at T0, or 60 and 240 minutes after HO induction, and of xrn1∆, rrp6∆ and trf4∆ cells at T0.
2014-11-20 | E-GEOD-63444 | biostudies-arrayexpress
Project description:RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.
2023-03-15 | PXD036935 | Pride
Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.
2014-11-20 | GSE63444 | GEO
Project description:Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is loaded to replication forks remains unexplored. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) binds RPA and is required for the association of RPA with replication forks. Cells lacking Rtt105 exhibit dramatic genome instability and severe defects in DNA replication. Intriguingly, both the RPA nuclear import and its ability to bind DNA replication forks were greatly compromised in rtt105 mutant cells, however, targeting RPA to the nucleus cannot rescue the defect of RPA binding to replication forks. Importantly, Rtt105 promotes the binding of RPA to ssDNA but does not associate with the final RPA-ssDNA complex in vitro. Moreover, single-molecule studies revealed that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to nucleus and assemble RPA onto ssDNA at replication forks.
2018-07-27 | GSE101536 | GEO
Project description:Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is guided to DNA replication forks remains unclear. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) is an RPA-binding protein and is required for the binding of RPA at replication forks. Cells lacking Rtt105 exhibit extensive genome instability and severe defects in DNA replication. Both the nuclear import of RPA and the extent of genome-wide binding of RPA at replication forks were greatly compromised in rtt105 mutant cells. Mechanistically, Rtt105 is required for the interaction of RPA with Kap95, an importin protein known to be essential for RPA’s nuclear import. Remarkably, Rtt105 strongly enhances RPA binding with ssDNA substrates in vitro. We therefore propose a model in which Rtt105 functions as a RPA chaperone that both escorts RPA during nuclear important and delivers RPA to replication forks.
2018-07-27 | GSE87356 | GEO
Project description:Chromatin replication requires tight coordination of nucleosome assembly machinery with DNA replication machinery. While significant progress has been made in characterizing histone chaperones in this process, the mechanism of whereby nucleosome assembly couples with DNA replication remains largely unknown. Here we show that replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein that is essential for DNA replication provides a binding platform for H3-H4 deposition by histone chaperons and is required for nucleosome formation on nascent chromatin. RPA binds free histone H3-H4 but not nucleosomal histones, and a RPA coated ssDNA stimulates assembly of H3-H4 onto double strand DNA in vitro. RPA mutant with reduced H3-H4 binding exhibits synthetic genetic interaction with mutations at key factors involved in replication-coupled (RC) nucleosome assembly, and are defective in assembly of replicating DNA into nucleosomes in cells. These results reveal a novel function for RPA in nucleosome assembly and a mechanism whereby nucleosome assembly is coordinated with DNA replication.
2017-01-27 | GSE83648 | GEO
Project description:AID promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in G1. RPA is a ssDNA-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR) such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in S-G2/M is extensive, ATM-independent, and associated with Rad51 accumulation. RPA in S-G2/M increases in NHEJ-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during CSR represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. Chip-Seq of RPA from mouse activated B cells (n = 40), mouse thymocytes (n = 6), and MEFs (n = 1). Different genotypes and/or inhibitors were used.
2013-01-02 | E-GEOD-42451 | biostudies-arrayexpress