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Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair

ABSTRACT: RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNAse H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-stranded break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous recombination (HR)-mediated DSB repair process and RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment, and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair, and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE84882 | GEO | 2016/10/28

SECONDARY ACCESSION(S): PRJNA335432

REPOSITORIES: GEO

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Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair

Project description:RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNAse H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-stranded break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous recombination (HR)-mediated DSB repair process and RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment, and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair, and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

2016-10-28 | GSE80398 | GEO

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis

Project description:Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. We have previously shown that a class of DSB-induced small RNAs (diRNAs) facilitates homologous recombination (HR)-mediated DSB repair in Arabidopsis thaliana. Here we show that INVOLVED IN DE NOVO 2 (IDN2), a double-stranded RNA (dsRNA) binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA-binding ARGONAUTE 2 (AGO2) leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from ssDNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

2017-02-28 | GSE94305 | GEO

Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation

Project description:DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD was able to bind chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced the ssDNA fraction upon camptothecin treatment and AsiSI-induced DSB resection and reduced RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage . Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

2019-02-26 | PXD012045 | Pride

RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

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

RNA processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

2014-11-20 | GSE63444 | GEO

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart

Project description:R-loops represent a major source of replication stress but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest and restart in S. cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate normally their chromosomes under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Indeed, these cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative step in resuming replication. Similar impairments in nascent DNA resection at HU-arrested forks were observed in human cells lacking RNase H2. However, the addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation, fully restored fork resection. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

2023-10-14 | GSE215896 | GEO

Monitoring Homology Search during DNA Double-Strand Break Repair in vivo

Project description:Homologous recombination (HR) is crucial for genetic exchange, accurate repair of DNA double-strand breaks and pivotal for genome integrity. HR uses homologous sequences for repair, but how homology search, the exploration of the genome for homologous DNA sequences, is conducted in the nucleus remains poorly understood. Here, we use time-resolved chromatin immunoprecipitations of repair proteins to monitor homology search in vivo. We found that homology search proceeds by a probing mechanism, which commences around the break and samples preferentially on the broken chromosome. However, elements thought to instruct chromosome loops mediate homology search shortcuts, and centromeres, which cluster within the nucleus, may facilitate homology search on other chromosomes. Our study thus revealed crucial parameters for homology search in vivo and emphasizes the importance of linear distance, chromosome architecture and proximity for recombination efficiency. 2 new custom ChIP-chip platforms used; both Nimblegen; differ in oligo density: (platform 1: 2006-07-18_Scerevisiae_ChIP_Stefan Jentsch MPI Biochemistry S.cerevisiae 385K Tiling Array Version 1) ( platform 2: 100304_Scer2_MS_Chip_Stefan Jentsch MPI Biochemistry S.cerevisiae 135K Tiling Array Version 2) ChIP-chip profiling of DSB repair factors (Rad51, Rad52, RPA, gamma-H2A) upon single inducible DSBs in S.cerevisiae

2013-03-21 | E-GEOD-44844 | biostudies-arrayexpress

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

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

Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA damage repair

Project description:The microRNA biogenesis enzyme Drosha was found to be important for DNA repair and this function appears to be distinct to its role in miRNA-mediated repression. Novel small RNAs were reported previously to be produced from the sequences around a DNA break. Utilising an endonuclease system (AsiSI) we were unable to detect such small RNA around 100 cuts within the endogenous genome. Sequencing of R-loops (DNA:RNA hybrids) was performed and an increase in R-loop formation was observed around many DNA break sites. Loss of Drosha appears to perturb this damage dependent formation of R-loops. RNase H1 over-expression appears to reduce repair at these break sites. Drosha appears to be important for facilitating R-loop formation at DNA break sites to aid in the repair process.

2018-02-18 | GSE97648 | GEO

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