Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Phosphorylation at the S2056 1 cluster of DNA-PKcs is dispensable for lymphocyte development and class switch recombination in mice.

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE129895 | GEO | 2019/04/17

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF-deficient mice

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

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

Project description:The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end-resection), and preferential usage of micro-homology – all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

2020-07-18 | GSE154210 | GEO

DNA-PKcs has KU-dependent function in rRNA processing and hematopoiesis

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

DNA damage-induced phosphorylation of CtIP at a conserved ATM/ATR site promotes lymphomagenesis in NHEJ/p53-double deficient mice

Project description:Human lymphoid malignancies are often characterized by oncogenic translocations involving the antigen receptor gene loci. CtIP is a DNA end-resection factor that has been widely implicated in alternative end-joining (A-EJ) mediated chromosomal translocations in reporters. The ATM and ATR kinases phosphorylate CtIP at T859 (T855 in mouse) and other sites to promote DNA end-resection. Using two classical non-homologous end-joining (cNHEJ) and Tp53-double deficient mouse models, we identified a role of CtIP T855 phosphorylation in the neonatal development of Xrcc4-/-Tp53-/- mice and the IgH-Myc translocation driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, we found that CtIP T855 phosphorylation is important for the progression of DNA end-resection, while dispensable for hairpin opening and inter-sister DNA break ligation. Moreover, we found that CtIP-T855 phosphorylation supports the proliferation of Myc-driven lymphoma cells by promoting the transition from ATM-mediated to ATR-mediated G2/M cell cycle checkpoint. Correspondingly, the CtIP-T855A mutation delays splenomegaly in l-Myc mice. Collectively, our findings suggest that DNA damage-induced CtIP phosphorylation has a checkpoint function during lymphomagenesis independent of its role in A-EJ-mediated chromosomal translocation

2021-12-31 | GSE174496 | GEO

Transcriptome data for HEK293T cells depleted of one subunit of DNA-PK complex: Ku70, Ku80 or DNA-PKcs.

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

Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

Project description:The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs-/- B cells. Meanwhile, the residual joints from DNA-PKcsKD/KDcells and the efficient Sμ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sμ-Sγ1 joints are more resistant to inversions and extensive resection than Sμ-Se and Sμ-Sμ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

2018-07-26 | GSE117628 | GEO

Integrative Structural Model of DNA-PK Synapsis

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

CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining

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.

2020-09-18 | GSE156392 | GEO

The impact of DNA-PKcs in V(D)J recombination of mouse IgK antigen locus

Project description:DNA-PKcs is a critical component of the non-homologous end joining (NHEJ) pathway, playing a role in the re-ligation of DNA double-strand breaks (DSBs) generated by RAG1/2 during V(D)J recombination in antigen loci. When NHEJ is deficient, DSBs can be repaired through alternative end joining (A-EJ). In this study, we investigated the consequences of DNA-PKcs deletion on V-J recombination at the IgK antigen locus. Our results reveal that DNA-PKcs deletion leads to inefficient V-J recombination, exhibiting phenotypes such as reduced efficiency, increased end resection, and decreased micro-insertions in the repair pattern. These findings are consistent with previously reported functions of Ku70, another key player in NHEJ. However, unlike Ku70, additional deletion of DNA-PKcs does not restore the efficiency of repair in the absence of Lig4. Instead, it reduces both end resection and microhomology-mediated repair. These observations highlight the context-dependent role of DNA-PKcs in end processing. The presence or absence of Lig4 appears to influence the function of DNA-PKcs, further emphasizing the intricate interplay between repair factors in the DNA damage response. Overall, our findings provide insights into the distinct functions of DNA-PKcs and Ku70 in V(D)J recombination and underscore the complex regulatory mechanisms underlying repair efficiency in different genetic backgrounds.

2023-09-01 | GSE232924 | GEO

Role of Polycomb group proteins in the DNA damage response – a reassessment

Project description:We have investigated whether components of Polycomb repressive complex 1 (PRC1) are recruited to double-strand breaks (DSBs) generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DNA repair proteins were detected at a subset of AsiSI sites that are predominantly at the 5’ ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression. Single chromatin preparations from treated and untreated cells (+/- OHT), immunoprecipitated with two antibodies (pDNA-PKcs and MEL18) were sequenced as technical replicates, along with the corresponding input DNAs

2014-07-06 | E-GEOD-55605 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data