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: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: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:Class Switch Recombination (CSR) is a B cell specific genomic alteration induced by activation induced cytidine deaminase (AID)-dependent DNA break, followed by repair and recombination at the immunoglobulin heavy-chain locus. The involvement of several chromatin-associated factors in promoting AID-induced DNA break formation has been reported. However, the involvement of chromatin adaptors at the repair phase of CSR remains unknown. Here, we provide evidence that acetylated histone reader Brd4 is critical to the repair and recombination step of CSR. Brd4 was recruited to the AID-induced DNA break region, and depletion of Brd4 from the S region chromatin by knockdown or a chemical inhibitor JQ1 causes CSR impairment without affecting AID-induced DNA break generation. Such inhibition of Brd4 suppressed the accumulation of 53BP1 and UNG at the cleaved S regions, perturbed switch donor-switch acceptor microhomology length and reduced Igh/c-myc translocation. We conclude that Brd4 serves as a histone-reader platform required for the recruitment of CSR repair components. Brd4 were depleted from the chromatin by either siRNA treatment or JQ1 (40nM) addition in CH12F3-2A cells in the presence of CIT stimulation. RNA from each samples were extracted and relative difference in transcript level were compared with control RNAi- and DMSO-treated, CIT-stimulated samples.
2014-06-20 | E-GEOD-52239 | biostudies-arrayexpress
Project description:Class Switch Recombination (CSR) is a B cell specific genomic alteration induced by activation induced cytidine deaminase (AID)-dependent DNA break, followed by repair and recombination at the immunoglobulin heavy-chain locus. The involvement of several chromatin-associated factors in promoting AID-induced DNA break formation has been reported. However, the involvement of chromatin adaptors at the repair phase of CSR remains unknown. Here, we provide evidence that acetylated histone reader Brd4 is critical to the repair and recombination step of CSR. Brd4 was recruited to the AID-induced DNA break region, and depletion of Brd4 from the S region chromatin by knockdown or a chemical inhibitor JQ1 causes CSR impairment without affecting AID-induced DNA break generation. Such inhibition of Brd4 suppressed the accumulation of 53BP1 and UNG at the cleaved S regions, perturbed switch donor-switch acceptor microhomology length and reduced Igh/c-myc translocation. We conclude that Brd4 serves as a histone-reader platform required for the recruitment of CSR repair components.
2014-06-20 | GSE52239 | GEO
Project description:Here we found that ILF3 prefers to bind telomere R-loops and protects telomere from aberrant homologous recombination. ILF3 knockout induces TERRA aggregation onto telomere and activates telomere DNA damage response (DDR). Furthermore, ILF3 deficiency disrupts telomere homeostasis and induces abnormal ALT-mediated telomere lengthening
2022-03-04 | PXD032093 |
Project description:Meiotic recombination ensures proper chromosome segregation and genetic diversity during gametogenesis, and its disruption leads to infertility. It is known that the dual histone methylation writer–reader system, in which PRDM9 deposits H3K4me3 and H3K36me3 marks at nucleosomes to specify recombination hotspots and ZCWPW1 functions as a reader recognizing these marks, is essential for meiotic recombination. However, the regulatory mechanism of this system remains unclear. Here, Deficiency of ZCWPW2 causes recombination defects, with impairment in homologous synapsis and DNA double-strand break repair. CUT&Tag and mass spectrometry analyses revealed that ZCWPW2 recognizes PRDM9-mediated dual histone methylation and interacts with ZCWPW1 to form a complex that recruits recombination-associated proteins to hotspots. Additionally, ZCWPW2 independently binds to promoter regions to regulate meiotic transcription. Lactylome and proteome analyses demonstrated that the ZCWPW1–ZCWPW2 complex interacts with and maintains the activity of lactate dehydrogenase LDHA, and together with ZCWPW2-driven transcription of lactylation writer EP300, to promote lactylation of recombination-associated proteins and stabilize their expression at hotspots. Collectively, we identified ZCWPW2 as a previously unrecognized yet indispensable factor supporting the function of PRDM9/ZCWPW1 during meiotic recombination, elucidated the molecular mechanism of the PRDM9/ZCWPW1/ZCWPW2 system in regulating recombination, and uncovered a critical role for lactylation in meiosis.
2026-01-07 | PXD070427 | Pride
Project description:Meiotic recombination ensures proper chromosome segregation and genetic diversity during gametogenesis, and its disruption leads to infertility. It is known that the dual histone methylation writer–reader system, in which PRDM9 deposits H3K4me3 and H3K36me3 marks at nucleosomes to specify recombination hotspots and ZCWPW1 functions as a reader recognizing these marks, is essential for meiotic recombination. However, the regulatory mechanism of this system remains unclear. Here, Deficiency of ZCWPW2 causes recombination defects, with impairment in homologous synapsis and DNA double-strand break repair. CUT&Tag and mass spectrometry analyses revealed that ZCWPW2 recognizes PRDM9-mediated dual histone methylation and interacts with ZCWPW1 to form a complex that recruits recombination-associated proteins to hotspots. Additionally, ZCWPW2 independently binds to promoter regions to regulate meiotic transcription. Lactylome and proteome analyses demonstrated that the ZCWPW1–ZCWPW2 complex interacts with and maintains the activity of lactate dehydrogenase LDHA, and together with ZCWPW2-driven transcription of lactylation writer EP300, to promote lactylation of recombination-associated proteins and stabilize their expression at hotspots. Collectively, we identified ZCWPW2 as a previously unrecognized yet indispensable factor supporting the function of PRDM9/ZCWPW1 during meiotic recombination, elucidated the molecular mechanism of the PRDM9/ZCWPW1/ZCWPW2 system in regulating recombination, and uncovered a critical role for lactylation in meiosis.
2026-01-07 | PXD068838 | Pride
Project description:DNA double-strand break (DSB) repair is required to maintain genome integrity and organismal fitness. This process occurs within chromatin and although accurate repair requires extensive changes in chromatin structure and composition, chromatin effectors facilitating DSB repair remain largely unknown in plants. Using reverse genetics, we identified two chromatin readers, YAF9A and YAF9B, as new factors required for DSB repair in Arabidopsis. While only YAF9B is induced by DNA damage, both YAF9A and YAF9B are required for DSB repair, including via homologous recombination (HR)-like pathways. Mechanistically, we found YAF9A and YAF9B denote distinct remodeling complexes: While YAF9A associates with both NuA4 and SWR1, YAF9B only associates with NuA4, defining a DNA-damage-specific version of this remodeling complex and revealing the first link between NuA4 and DSB repair in plants. We also uncovered direct roles for YAF9A and YAF9B in DNA repair, rather than indirect roles in gene regulation, as yaf9 mutants showed normal transcriptional responses to DNA damage. Finally, we demonstrate that the YAF9B reader domain is required for DSB repair. These results link YAF9A, YAF9B, and their respective remodeling complexes, to DSB repair in a histone reader-dependent manner, expanding our understanding of how chromatin effectors regulate DSB repair in plants.
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Project description:DNA double-strand break (DSB) repair via homologous recombination (HR) is critical for maintaining genomic integrity and requires proper DNA end resection to generate ssDNA overhangs. Yet, how this critical step is regulated in cells remains to be understood. Here, we report that GNL3, a nucleolar GTP-binding protein, plays a critical role in HR repair via regulating DNA end resection dependently on its SUMOylation. Ectopic expression of wild-type, but not the SUMO-defective K196R mutant, GNL3 completely abolished DNA damage response induced by knockdown of endogenous GNL3. GNL3 interacts with the BLM-DNA2 helicase-nuclease complex and is critical for DNA end resection and subsequent RPA and RAD51 loading. The GNL3-BLM interaction requires SUMOylation and SUMO-interacting motifs (SIMs) of both proteins. We further showed that USP36, a nucleolar deubiquitinating enzyme, acts as a novel SUMO ligase for GNL3, whereas SUMOylated GNL3 can be deSUMOylated by the SUMO protease SENP3. Interestingly, several breast cancer-derived variants of GNL3 that interfere with its SUMOylation of SIM failed to interact with the BLM-DNA2 complex. Knockdown of GNL3 sensitizes breast cancer cells to the treatment with etoposide or Olaparib. Together, our results reveal that GNL3 SUMOylation is critical for HR repair and targeting GNL3 SUMOylation may induce HR deficiency and sensitize breast cancers to DNA damage-inducing agents.
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Project description:The Saccharomyces cerevisae RAD3 gene is homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Mutant alleles of RAD3 have been identified (rad3-101 and rad3-102) that have partial defects in DNA repair associated with a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. We have further characterized these events using a system in which the reciprocal products of mitotic recombination between homologs are recovered as red and white sectored colonies. Both rad3-101 and rad3-102 elevate the frequency of sectored colonies about 100-fold. Subsequent mapping of these events shows that three-quarters of crossovers between homologs induced in hyper-Rec rad3 mutants reflect DSBs formed in at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. In addition to examining crossovers on chromosome V, we mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.
2015-12-30 | GSE76395 | GEO