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A cell-free system to study the damage response at unprotected DNA ends in Drosophila

ABSTRACT: We discovered that the cell-free chromatin assembly system senses free DNA ends and mounts a DNA double-strand break response. DNA ends are first recognized by the Ku complex and later resected. The phosphorylation of H2A.V (homologous to gH2A.X) initiates at DNA breaks and spreads over ten thousands of base pairs of DNA within a few minutes. The phosphorylation of gH2A.V remains tightly associated with the damaged DNA in cis and does not transfer to intact DNA circles in the same reaction. Our descriptions of the damage-related proteome and phospho-proteome provide a rich resource for in-depth mechanistic analyses of the DNA chromosome break response in this model system.

OTHER RELATED OMICS DATASETS IN: PXD013130PXD012990

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE126415 | GEO | 2019/07/22

REPOSITORIES: GEO

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Sensing and signalling of DNA breaks in extracts of preblastoderm Drosophila embryos

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

The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA

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

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-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

Exploration and clinical validation of molecular typing of early colorectal cancer

Project description:Interventions: Case series:None Primary outcome(s): exon genes;transcriptional expression;proteome;protein phosphorylation group Study Design: Sequential

| 2739647 | ecrin-mdr-crc

Integrative Structural Model of DNA-PK Synapsis

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2021-01-20 | PXD017931 | Pride

Ku70 and Ligase IV deficiencies reveal distinct alternative end-joining outcomes in G1-arrested progenitor B cells

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.

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RecBCD Coordinates Repair of Two Ends at a DNA Double-Strand Break, Preventing Aberrant Chromosome Amplification [ChIP-seq]

Project description:In the bacterium Escherichia coli, RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification

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HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer’s disease pathology

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

PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF

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

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