Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE166631 | GEO | 2021/03/15

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Tyrosine kinase c-Abl couples RNA polymerase II transcription to an RNA-dependent DNA damage response

Project description:Various modes of DNA repair counteract genotoxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilises damage-induced small RNA for efficient repair of DSBs. However, production and processing of RNA is poorly understood. Here we show that localised induction of DSBs triggers phosphorylation of RNA polymerase II (RNAPII) on carboxy-terminal domain (CTD) residue tyrosine-1 in an Mre11-Rad50-Nbs1 (MRN) complex-dependent manner. CTD Tyr1-phosphorylated RNAPII synthetises, strand-specific, damage-responsive transcripts (DARTs). DART synthesis occurs via formation of transient RNA-DNA hybrid (R-loop) intermediates. Impaired R-loop formation attenuates DART synthesis, impairs recruitment of repair factors and delays the DDR. Collectively, we provide mechanistic insight in RNA-dependent DSB repair.

2019-02-04 | GSE96825 | GEO

A truncated MRE11 form induced by SPRTN is deficient in DNA repair and radiosensitises cancer cells

Project description:he human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses 3’-5’ exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified the approximate cleavage site of TR-MRE11 at 559-580 amino acids, its DNA damage repair function and the factors regulating TR-MRE11 accumulation. The nuclease enzymatic activity of TR-MRE11 was dramatically reduced, associated with a lack of DNA binding efficiency, whilst TR-MRE11 still interacted efficiently with RAD50 and NBS1. Lack of the MRE11 C-terminal resulted in deficient HR repair and increased cellular radiosensitivity. Knockdown of SprT-like N-terminal domain (SPRTN), an essential metalloprotease for DNA-protein crosslink repair, resulted in failure of MRE11 cleavage, with TR-MRE11 protein levels being positively correlated with SPRTN protein expression. The presence of this DNA repair-defective C-terminal truncation could explain the finding of high MRE11 expression, by immunohistochemistry using an antibody against MRE11 prior to the C-terminal, being associated with survival following radical radiotherapy in cancer patients. Ultimately, understanding the functional differences between intact and repair-defective MRE11 may lead to improvements in patient outcomes through a more informed choice of treatment.

2021-02-23 | PXD017964 | Pride

RNA-seq analysis of PRMT5-regulated genes in irradiated/non-irradiated LNCaP cells

Project description:DNA Double-strand break (DSB) repair is critical for cell survival and genome integrity. Upon recognition of DSBs, repair proteins are transiently upregulated to facilitate repair through homologous recombination (HR) or non-homologous end joining (NHEJ). We present evidence that PRMT5 cooperates with pICln to function as a master epigenetic activator of DNA damage response (DDR) genes involved in HR, NHEJ, and G2 arrest (including RAD51, BRCA1, and BRCA2) to upregulate gene expression upon DNA damage. Contrary to the predominant role of PRMT5 as an epigenetic repressor, our results demonstrate that PRMT5 and pICln can activate gene expression, potentially independent of PRMT5’s obligate cofactor MEP50. Targeting PRMT5 or pICln hinders repair of DSBs in multiple cancer cell lines, and both PRMT5 and pICln expression positively correlates with DDR genes across 32 clinical cancer data sets. Thus, targeting PRMT5 or pICln may be explored in combination with radiation or chemotherapy for cancer treatment.

2019-12-05 | GSE111620 | GEO

UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168

Project description:Maintenance of genome integrity requires tight control of DNA damage signalling and repair activities at DNA lesions, as well as their restriction at telomeres. Key components of the mechanisms underlying appropriate responses to DNA damage include phosphorylation events by DNA damage response (DDR) kinases, as well as regulatory and proteolytic ubiquitination events by the ubiquitin machinery. How these events are coordinated and controlled in a concerted manner to achieve productive DNA repair is not well understood. Here we identify the ubiquitin-conjugating enzyme UBE2D3 (or UBCH5C) as a critical multi-level regulator of ATM kinase-induced DDR signalling that controls the activity of the ubiquitin-ligase RNF168 to promote non-homologous end-joining (NHEJ) mediated DNA repair at telomeres. We find that UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both of which are mediated by RNF168 upon DNA damage-induced ATM activation. In addition, we find that UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated KAP1-S824 phosphorylation, important for heterochromatic DNA repair. Mechanistically, we show that defective KAP1-S824 phosphorylation upon UBE2D3-deficiency is linked to RNF168 hyperaccumulation and caused by aberrant PP2A phosphatase activity, the counteraction of which restores both KAP1-S824 phosphorylation and telomeric NHEJ in UBE2D3-deficient cells. Our results identify UBE2D3 as a novel multi-level regulator of NHEJ that orchestrates the activities of RNF168 in DNA repair. Moreover, we reveal the existence of a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and PP2A-mediated restriction of ATM-dependent KAP1 phosphorylation.

2024-04-30 | PXD044410 | Pride

Human U2OS DNApk GLASS-ChIP LC-MS/MS

Project description:The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA- PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA- PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA- PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

2023-10-24 | PXD045033 | Pride

Altered levels of MOF and decreased levels of H4K16ac correlate with a defective DNA damage response (DDR).

Project description:Full title: Altered levels of MOF (member of MYST family histone acetyl transferase) and decreased levels of H4K16ac correlate with a defective DNA damage response (DDR). The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that altered levels of H4K16ac correlate with a defective DNA damage response (DDR) to ionizing radiation (IR). The defect however is not due to altered expression of proteins involved in DDR. Specific inhibition of H4K16ac deacetylation in MOF-depleted cells rescued IR-induced abrogation of DDR. MOF was found associated with DNA-dependent protein kinase catalytic subunit (DNAPKcs), a protein involved in non-homologous end joining (NHEJ) repair, whose ATMdependent IR-induced phosphorylation was abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased the repair of DNA double-strand breaks (DSBs) by NHEJ and homologous recombination (HR). In addition, the MOF protein activity associates with chromatin upon DNA damage and colocalizes with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac, plays a critical role in the cellular DNA damage response. Keywords: Cell type comparison HEK293 cells were transfected with plasmids encoding hMOF for over-expression of the histone acetyl transferase that leads to elevated levels of acetylation of Lysine 16 of histone H4. siRNA mediated knock-down of hMOF was performed to deplete the H4K16ac levels. Total RNA samples for expression profiling was obtained from wild type (293 cells without any treatment), hMOF over-expressed and hMOF knock-down 293 cell lines. Each sample was analyzed in triplicates using EGPF dsRNA treated samples as control.

2010-03-23 | E-GEOD-20193 | biostudies-arrayexpress

Altered levels of MOF and decreased levels of H4K16ac correlate with a defective DNA damage response (DDR).

2010-03-15 | GSE20193 | GEO

Transcriptome changes of Hela cells with blocking classical and alternative non-homologous end-joining (NHEJ) pathways

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.

2020-12-31 | GSE135274 | GEO

Choice of Template Delivery Mitigates the Genotoxic Risk and Adverse Impact of Editing in Human Hematopoietic Stem Cells (Targeted deep sequencing)

Project description:Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long- term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

2022-10-06 | GSE197386 | GEO

Choice of Template Delivery Mitigates the Genotoxic Risk and Adverse Impact of Editing in Human Hematopoietic Stem Cells (RAAVIoli sequencing)

2022-10-06 | GSE197388 | GEO