DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA701448 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13690474.fastq.gz | Fastqsanger.gz | |||
| SRR13690475.fastq.gz | Fastqsanger.gz | |||
| SRR13690476.fastq.gz | Fastqsanger.gz | |||
| SRR13690477.fastq.gz | Fastqsanger.gz | |||
| SRR13690478.fastq.gz | Fastqsanger.gz |
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Project description:The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.
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Project description:SETMAR functions in illegitimate DNA recombination and non-homologous end joining repair
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Project description:The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.
| S-EPMC8015226 | biostudies-literature
Project description:Purpose: The recombinant endonucleases introduce double-strand breaks (DSBs) in genomic DNA at targeted sites and non-homologous end-joining (NHEJ) pathways repair the breaks introducing indels into the genome. To investigate whether there is another NHEJ pathway or if A-NHEJ and C-NHEJ are redundant, we examined NHEJ edits in HeLa cells. Methods: To test among these possibilities, we engineered a NHEJ reporter system using targeted TALEN induced double strand DNA breaks. C-NHEJ was blocked in XRCC4(-/-) cells and A-NHEJ was blocked pharmacologically with mirin. To assess how NHEJ was maintained, differentially expressed genes were determined by RNA-Seq. Results: When both C-NHEJ and A-NHEJ are blocked, edits were still observed. No changes in expression of DNA repair genes was observed. However, when both NHEJ pathways were blocked, genes from several pathways including DNA damage response, hypoxic response, chromatin packaging, p53 response and two genes that stimulate homologous recombination were differentially expressed. Conclusions: NHEJ is maintained when both NHEJ pathways are blocked through a transcriptional response that must compensate for the inhibited steps in both pathways.
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Project description:Mutations in the ATM tumor suppressor gene confer cellular hypersensitivity to various DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms towards such drugs, we performed genome-wide CRISPR-Cas9 loss-of-function screens in cells treated with the DNA topoisomerase I poison topotecan. Our ensuing characterizations of hits established that loss of terminal components of the non-homologous end joining (NHEJ) machinery or components of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. Our findings indicate that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib is due to delayed engagement of homologous recombination repair (HRR) at a subset of DNA-replication-fork associated single ended double-strand breaks (seDSBs), which allows non-homologous end joining (NHEJ) mediated repair, resulting in toxic chromosome fusions. Thus, restoration of legitimate repair in ATM-deficient cells – either by preventing the DNA ligation step of NHEJ or by enhancing HRR engagement by deregulating the BRCA1-A complex – markedly suppresses this toxicity. We conclude that the crucial role for ATM at seDSBs is to prevent toxic LIG4-mediated NHEJ at damaged replication forks. Furthermore, our observation that suppressor mutations in ATM-mutant backgrounds are fundamentally different to those that operate in BRCA1-mutant scenarios suggests new opportunities for patient stratification in the clinic, as well as additional therapeutic vulnerabilities that might be exploited in drug-resistant cancers.
2019-11-12 | PXD011108 | Pride
Project description:The critical role of the alternative end joining (AEJ) repair mechanism in V(D)J recombination for B and T lymphocyte development, particularly in the absence of the key non-homologous end joining (NHEJ) component Ku70, is not well understood. AEJ involves functionally redundant factors such as Parp1 vs Polθ and Lig3 vs Lig1, making it unclear which factors are responsible for repairing V(D)J recombination. Additionally, the regulatory mechanism of AEJ remains enigmatic. In this study, we utilized the murine Igκ antigen locus as a model and employed gene editing and HTGTS-Seq pipelines to systematically evaluate potential factors involved in V(D)J recombination. We assessed various aspects of recombination, including efficiency, V gene usage, end resection, joining fidelity, repair patterns, and inter-chromosome translocation. Our findings highlight the Parp1-XRCC1-Lig3 axis as a key mediator of V(D)J recombination, regulated by 53BP1 and ATM. This study advances our understanding of AEJ in DNA damage repair and contributes to our knowledge of lymphocyte development in immunology.
2023-12-31 | GSE246239 | GEO
Project description:RIF1 acts downstream of 53BP1 to coordinate DNA double strand break repair pathway choice between non-homologous end joining (NHEJ) and homologous recombination (HR). Here we identified ASF1 as an endogenous RIF1-associated protein. We showed that ASF1 forms complex with RIF1 and regulates RIF1-dependent functions in DNA damage response.
2022-06-09 | PXD027727 | Pride
Project description:Transcriptome changes of Hela cells with blocking classical and alternative non-homologous end-joining (NHEJ) pathways
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Project description:To generate antibodies with different effector functions, B cells undergo Immunoglobulin class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical non-homologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ~25% CSR can be achieved by the alternative end-joining (A-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ mediated repair of endonuclease generated breaks requires DNA end-resection, we show that CtIP-mediated DNA end-resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppress resection without altering the MH-pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ mediated CSR by suppressing inter-chromosomal translocations independent of end-resection. Finally, temperol analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favoriate substrates for MH-mediated end-joining contributing to the robustness and resection-indepndence of A-EJ-mediated CSR.
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Project description:Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.
2021-01-20 | E-MTAB-8382 | biostudies-arrayexpress