A cell-free system to study the damage response at unprotected DNA ends in Drosophila
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE126415 | GEO | 2019/07/22
REPOSITORIES: GEO
Similar Datasets
Project description:Preblastoderm Drosophila embryos replicate their genomes at maximal speed. The assembly of chromatin on the newly synthesized chromosomes relies entirely on high concentrations of maternal nucleosome assembly and remodelling factors. Extracts from preblastoderm embryos can be turned into a system for highly efficient assembly of complex chromatin. We serendipitously discovered that this chromatin assembly system contains numerous activities that recognise unprotected DNA ends and initiate DNA damage signalling cascades. Within minutes the phosphorylation of H2A.V (homologous to H2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The phosphorylation of H2A.V remains tightly associated with the damaged DNA in cis and does not spread to intact DNA circles in the same reaction. The ultrafast cycles of syncytial nuclei divisions of cleavage-stage embryos do not allow sufficient time to repair DNA breaks. Rather, fatally damaged nuclei are eliminated by a programmed ‘mitotic catastrophe’. Our descriptions of the damage-related proteome and DNA break-dependent phospho-proteome suggest that certain aspects of the DNA damage checkpoint that leads to the elimination of nuclei can be studied in this cell-free system. Our data provide a rich resource for in-depth mechanistic analyses of the DNA break response in this model system.
2019-10-14 | PXD013130 | Pride
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: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
Project description:Interventions: Case series:None
Primary outcome(s): exon genes;transcriptional expression;proteome;protein phosphorylation group
Study Design: Sequential
| 2739647 | ecrin-mdr-crc
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:Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but is thought to predominate in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG1/2-initiated DSBs during V(D)J recombination in G1-phase lymphocyte progenitors, cycling cells deficient for core C-NHEJ factors join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination to C-NHEJ has been attributed to RAG-mediated exclusion of A-EJ; however, it remains unclear whether A-EJ is similarly excluded from more general DSBs in G1. Here, we report that Ku actively and robustly suppresses A-EJ of RAG1/2 and two classes of engineered endonuclease-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while targeted DSBs remain as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Lig4-deficient cells restores DSB rejoining and translocation to levels observed in Ku70-deficient counterparts. Correspondingly, while V(D)J recombination is abrogated in Ligase4-deficient lines, V(D)J-like joining occurs in Ku70-deficient and Ku70/Lig4 double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Lig4-deficient progenitor B cells are functionally end-joining deficient due to a near complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase IV deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including Ku-deficiency rescuing Lig4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase.
2021-05-08 | GSE162453 | GEO
Project description:In the bacterium Escherichia coli, RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification
2018-07-18 | GSE107972 | GEO
Project description:DNA damage is increased in Alzheimer’s disease (AD), while the underlying mechanisms are unknown. Here, we employ comprehensive phosphoproteome analysis, and identify abnormal phosphorylation of 70 kDa subunit of Ku antigen (Ku70) at Ser77/78, which prevents Ku70-DNA interaction, in human AD postmortem brains. The abnormal phosphorylation inhibits accumulation of Ku70 to the foci of DNA double strand break (DSB), impairs DNA damage repair and eventually causes transcriptional repression-induced atypical cell death (TRIAD). Cells under TRIAD necrosis reveal senescence phenotypes. Extracellular high mobility group box 1 (HMGB1) protein, which is released from necrotic or hyper-activated neurons in AD, binds to toll-like receptor 4 (TLR4) of neighboring neurons, and activates protein kinase C alpha (PKCα) that executes Ku70 phosphorylation at Ser77/78. Administration of human anti-HMGB1 antibody to post-symptomatic AD model mice decreases neuronal DSBs, suppresses secondary TRIAD necrosis of neurons, prevents escalation of neurodegeneration, and ameliorates cognitive symptoms. TRIAD shares multiple features with senescence. These results newly unravel the HMGB1-Ku70 axis that accounts for the increase of neuronal DNA damage and secondary enhancement of TRIAD, the cell death phenotype of senescence, in AD.
2022-02-15 | PXD028089 | Pride
Project description:In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a new c-NHEJ core component. We report here, that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift towards higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapping with XLF’s function.
2016-10-06 | PXD004659 | Pride
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