Transcriptome data for HEK293T cells depleted of one subunit of DNA-PK complex: Ku70, Ku80 or DNA-PKcs.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA748741 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15209530.fastq.gz | Fastqsanger.gz | |||
| SRR15209531.fastq.gz | Fastqsanger.gz | |||
| SRR15209532.fastq.gz | Fastqsanger.gz | |||
| SRR15209533.fastq.gz | Fastqsanger.gz | |||
| SRR15209534.fastq.gz | Fastqsanger.gz |
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Project description:DNA-PK is a heterotrimeric complex that consists of Ku70 (XRCC6), Ku80 (XRCC5) and DNA-PKcs (PRKDC) subunits. DNA-PK complex is a major player in DNA double strand break (DSB) repair via non-homologous end joining pathway. This process requires all of DNA-PK subunits. Ku70/Ku80 heterodimers firstly bind to DNA-ends at DSB, that increase affinity of DNA-PKcs to DNA-ends. Recruitment of DNA-PKcs subunit to DSB leads to phosphorylation events near DSB, recruitment of another NHEJ-related genes that restore DNA integrity. However, today a lot of evidence demonstrate participation of DNA-PK components in other cellular process, e.g. cytosolic DNA sensing, apoptosis regulation, cellular movement and adhesion. It is important to note that not all subunits of DNA-PK complex are necessary for these process. This demonstrate the independent functions of DNA-PK subunits. Here we for the first time using NGS-sequencing analyzed the transcriptional changes in HEK293T cells under depletion of Ku70, Ku80 or DNA-PKcs to characterize the independent functions of each subunit.
2021-07-23 | GSE180581 | GEO
Project description:The non-homologous end-joining (NHEJ) pathway is critical for DNA double-strand break repair and is essential for lymphocyte development and maturation. The Ku70/Ku80 heterodimer (KU) binds to DNA ends, initiating NHEJ and recruiting additional factors, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that caps the ends and pushes KU inward. The C-terminus of Ku70 in higher eukaryotes includes a flexible linker and a SAP domain, whose physiological role remains poorly understood. To investigate this, we generated a mouse model with knock-in deletion of the SAP domain (Ku70DSAP/DSAP). Ku70DSAP supports KU stability and its recruitment to DNA damage sites in vivo. In contrast to the growth retardation and immunodeficiency seen in Ku70-/- mice, Ku70DSAP/DSAP mice show no defects in lymphocyte development and maturation. Structural modeling of KU on long dsDNA, but not dsRNA suggests that the SAP domain can bind to an adjacent major groove, where it can limit KU’s rotation and lateral movement along the dsDNA. Consistent with this model, in the absence of DNA-PKcs that caps the ends, Ku70DSAP fails to support stable DNA damage-induced KU foci. In DNA-PKcs-/- mice, Ku70DSAP abrogates the leaky T cell development and reduces both the qualitative and quantitative aspects of residual V(D)J recombination. Moreover, purified Ku70DSAP also has reduced affinity for DNA ends and dissociates more readily. These findings revealed a physiological role of the SAP domain in NHEJ by restricting KU rotation and lateral movement on DNA that is largely masked by DNA-PKcs.
2024-12-31 | GSE276069 | GEO
Project description:The classical non-homologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs-/-) abrogates end-processing (e.g., hairpin-opening), but not end-ligation, while expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD) abrogates end-ligation, suggesting a kinases-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs-/- and DNA-PKcsKD/KD mice due to the requirement for both hairpin-opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056-cluster is the best characterized auto-phosphorylation site on human DNA-PKcs. Here we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053 and generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retain normal kinase activity, and undergo efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053-cluster of mouse DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.
2019-04-17 | GSE129895 | GEO
Project description:We report the Ku80/Ku70 complex associated RNA in HEK293T cells using RNA-seq following PAR-Clip.
2024-04-09 | GSE219220 | GEO
Project description:In non-homologous end joining repair of DNA double strand breaks, DNA dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/80 binds the free DNA ends forming the holoenzyme DNA-PK. DNA-PK synapses across the break to tether the broken ends in the initial long range synaptic complex. We generated an integrative structural model of DNA-PK synapsis at a precision of 13.5Å with crosslinking mass spectrometry (XL-MS) restraints. While our hydrogen deuterium exchange (HX) analysis revealed an allosteric axis in DNA-PK connecting DNA binding (including the plug domain) to the kinase domain. Our model presents a symmetrical synapsis primarily through head to head interactions and protection of the DNA by previously uncharacterized a plug domain. The offset of the DNA in our model allows access to downstream processing enzymes, while the combination of DNA binding and kinase loading creates a tensed state that could have roles in the re-arrangement/dissociation of DNA-PKcs as the repair process progresses.
2021-01-20 | PXD017931 | Pride
Project description:The non-homologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of Kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters – PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But mice carrying alanine substitution at all 5 serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. XLF is a non-essential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, MRI, etc.) or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class switch recombination junctions from DNA-PKcsPQR/PQRXlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.
2023-05-01 | GSE228850 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186086 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186085 | GEO
Project description:The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. Beyond DNA, KU also binds to RNA, with unknown significance in mammals. Using mouse models, we uncovered an unexpected role for DNA-PK in ribosomal RNA (rRNA) biogenesis and hematopoiesis. Expression of kinase-dead (KD) DNA-PKcs (DNA-PKcsKD/KD) abroagates cNHEJ2. But DNA-PKcsKD/KDTp53-/- mice develop myeloid disease rather than pro-B cell lymphoma, like other cNHEJ/Tp53-deficient mice3. DNA-PKcs is its own the best substrate. Blocking DNA-PKcs phosphorylation at the T2609, but not the S2056 cluster leads to KU-dependent 18S rRNA processing defects, compromises global protein synthesis in hematopoietic cells and causes bone marrow failure in mice. KU drives assembly of DNA-PKcs on a broad array of cellular RNAs, including the U3 small nucleolar RNA (snoRNA), which is essential for 18S rRNA processing4. U3 activates purified DNA-PK and triggers T2609 phosphorylation. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit (SSU) processome. Together our data show that DNA-PK has RNA-dependent, but cNHEJ-independent, functions during ribosome biogenesis that require DNA-PKcs’ kinase activity and T2609 cluster’s phosphorylation.
2020-01-05 | GSE109026 | GEO
Project description:It has been established that short inverted repeats (SIRs) trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with this structured DNA is unknown. We have previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded DNA template is transiently blocked at DNA hairpins [1](Schmutz et al., 2007). Here, we report the proteomic analysis of proteins bound to the DNA template providing evidence that proteins of the NHEJ pathway, particularly the DNA-PK complex (composed of DNA-PKcs and the Ku70/Ku80 dimer) recognize structured single-stranded DNA. DNA-PKcs inhibition results in the mobilization on the template DNA of the DNA-PK complex, along with other proteins acting downstream in the NHEJ pathway, especially the XRCC4-DNA Ligase 4 complex and the recently identified cofactor PAXX. The retention of NHEJ factors to the template DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that NHEJ proteins may hinder the progression of the DNA synthesis at these sites. Conversely, hijacking of the DNA-PK complex by double-stranded oligos (dsO) results in a large removal of the pausing sites and an elevated DNA extension efficiency. Overall these results raise the possibility that, upon binding to DNA hairpins formed onto ssDNA during fork progression, the DNA-PK complex may play some role in replication fork dynamics in vivo, role that is however not related to repair of double-strand breaks.
2020-02-04 | PXD011156 | Pride