The m6A reader IGF2BP2 promotes homologous recombination-mediated DNA repair by eliminating DNA-RNA hybrids
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Yang Li
PROVIDER: PXD067817 | iProX | Thu Aug 28 00:00:00 BST 2025
REPOSITORIES: iProX
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Project description:Diploid Saccharomyces cerevisiae cells undergo meiosis when they are starved of nitrogen in the presence of a non-fermentable carbon source. Nutrient starvation triggers expression of Ime1, a master regulatory protein required to activate transcription of meiotic “early genes” that mediate premeiotic S phase and Prophase I processes, including recombination and chromosome synapsis. During prophase, the highly conserved, topisomerase-like protein, Spo11, generates ~200 double strand breaks that are used to identify homologous chromosomes and generate crossovers between them. DNA-RNA hybrids are formed when an RNA molecule anneals to a complementary strand of DNA and are present at the ends of double strand breaks during Prophase I of meiosis in a variety of organisms. One way of removing DNA/RNA hybrids is degradation of the RNA by RNase H. Phenotypic characterization of organisms lacking RNase H activity has demonstrated that regulation of the level of DNA-RNA hybrids is important for meiotic double strand break repair. Sen1 is an essential 5’-3’ DNA-RNA helicase that can remove DNA-RNA hybrids by unwinding the RNA. Sen1 is orthologous to the mammalian Senataxin (SETX) helicase. Mouse mutants lacking either Senataxin or RNase H activity exhibit male infertility and defects in double strand break repair. SETX is also required for meiotic sex chromosome inactivation, making it unclear whether SETX’s role in meiotic recombination is direct or an indirect consequence due to defects in SETX functions that affect transcription. Using a variety of orthogonal approaches, this work demonstrates that SEN1 has multiple, temporally distinct functions that promote yeast meiosis. First, it enables the timely expression of IME1-regulated early genes. Second, it helps prevent/remove DNA-RNA hybrids that form during premeiotic S phase. Third, it facilitates repair of Spo11 double strand breaks generated during Prophase I, as well as chromosome synapsis.
2025-08-01 | GSE292703 | GEO
Project description:RNA:DNA hybrids accumulate at the vicinity of DNA double-strand breaks (DSBs) and were shown to regulate homologous recombination repair. The mechanism responsible for the formation of these non-canonical RNA:DNA structures remains unclear although they were proposed to arise consequently to RNA Polymerase II or III loading followed by DSB-induced de novo transcription at the break site. Here, we found no evidence of RNA polymerases recruitment at DSBs. Rather, strand-specific R-loop mapping revealed that RNA:DNA hybrids are mainly generated at DSBs occurring in transcribing loci, from the hybridization of pre-existing RNA to the 3’ overhang left by DNA end resection. We further identified the H3K4me3 reader Spindlin 1 and the transcriptional regulator PAF1 as promoting RNA:DNA hybrid accumulation at DSBs, through their role in mediating transcriptional repression in cis to DSBs. Altogether, we provide evidence that RNA:DNA hybrids accumulate at DSBs occurring in transcribing loci as a result of DSB-induced transcriptional shut-down.
2025-03-30 | E-MTAB-13197 | biostudies-arrayexpress
Project description:The BRCA2 tumor suppressor is a DNA double-strand break repair factor essential to maintain genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2 interacting partner. We show that DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid helicase activity of DDX5. Impaired BRCA2-DDX5 interaction, as observed in cells bearing the breast cancer variant BRCA2-T207A, reduces DDX5 association with DSBs, decreases the number of RPA foci upon irradiation and impairs RAD51 repair foci formation. Our findings are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.
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Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
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Project description:Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.
2020-06-15 | PXD019156 | Pride
Project description:DNA–RNA hybrids triggered by double-strand breaks (DSBs) are crucial intermediates during DSB repair, and their timely resolution requires numbers of RNA helicases, including DEAD box 1 (DDX1). However, how these helicases are recruited to DSB-induced hybrids in time remains largely unclear. Here, we revealed that squamous cell carcinoma antigen recognized by T cells 3 (SART3) promotes DDX1 binding to DNA–RNA hybrids at DSBs for optimal homologous recombination (HR) repair. SART3 itself can associate with DNA–RNA hybrids and PAR chains, and is recruited to DSBs in both PARylation- and hybrid-dependent fashion. SART3 also associates with DDX1 and is necessary for DDX1 enrichment at DSBs. The defective SART3-DDX1 association observed in cells expressing the cancer-associated variant SART3-R836W not only abrogates the accumulation of DDX1 at DSBs, but also impairs hybrid removal and HR efficiency, leading to hypersensitivity of tumor cells to drug treatments. Interestingly, beyond impairing hybrid removal through DDX1, SART3 loss also inhibits DNA end resection, causing a more profound DSB repair defect and chemosensitivity. The stimulatory role of SART3 in end resection is mediated by its function to enhance USP15-BARD1 association and BRCA1-BARD1 retention. Together, our study reveals a previously unknown role of SART3 in DSB repair, rendering SART3 a promising target for cancer therapy.
2025-01-30 | PXD053595 | Pride
Project description:Transcriptionally active loci are particularly prone to breakage and mounting evidence suggest that DNA Double-Strand Breaks arising in active genes are handled by a dedicated repair pathway, Transcription-Coupled DSB Repair (TC-DSBR), that entails R-loop accumulation and dissolution. Here, we uncovered a function for the Bloom RecQ DNA helicase (BLM) in TC-DSBR in human cells. BLM is recruited in a transcription dependent-manner at DSBs where it fosters resection, RAD51 binding and accurate Homologous Recombination repair. However, in an R-loop dissolution-deficient background, we found that BLM promotes cell death. We report that upon excessive R-loop accumulation, DNA synthesis is enhanced at DSBs, in a anner that depends on BLM and POLD3. Altogether our work unveils a role for BLM at DSBs in active chromatin, and highlights the toxic potential of RNA:DNA hybrids that accumulate at transcription-associated DSBs.
2022-04-13 | E-MTAB-11592 | biostudies-arrayexpress
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 | GSE84882 | GEO
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.
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Project description:DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is only replicated once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both initiation of DNA replication and elongation of DNA replication. Although only two DNA helicase activities are required to establish a bidirectional replication fork from each origin of replication, a large excess of MCM complexes is loaded and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most of them are displaced from the DNA during S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of the reduction of the MCM complex. The alteration of MCM proteins induced a change in the activation of important factors of the DDR in response to Etoposide treatment. The phosphorylation of γ-H2AX, CHK1 and CHK2 is affected following DNA damage induced by treatment with Etoposide without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we have identified a decrease of HR, but not NHEJ, associated with a decrease of the MCM complex demonstrating a role for the MCM complex in homologous recombination.
2019-11-12 | PXD009539 | Pride