Critical residues in the Ku70 von Willebrand A domain mediate Ku interaction with the LigIV-XRCC4 complex in non-homologous end-joining
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Laila Bayat
LAB HEAD: Caroline Schild
PROVIDER: PXD047914 | Pride | 2024-08-16
REPOSITORIES: pride
Dataset's files
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| pp_2022_013_1b.pepXML | Pepxml | |||
| pp_2022_013_1b.raw | Raw | |||
| pp_2022_013_2.pepXML | Pepxml | |||
| pp_2022_013_2.raw | Raw | |||
| pp_2022_013_3.pepXML | Pepxml |
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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:DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damages as they can lead to mutations and chromosomal rearrangements, which underlie cancer development and progression. Non-homologous end-joining (NHEJ) is the dominant pathway for the repair of DSBs in human cells, which involves the DNA binding proteins Ku70/Ku80. Other DNA binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair. However, their role in NHEJ has remained elusive. Here we show that ZNF384, which is a member of the C2H2 family of ZnF proteins that binds DNA in vitro, is recruited to DSBs in vivo. ZNF384 recruitment requires the PARP/PAR-dependent expansion of damaged chromatin followed by binding of ZNF384 to the exposed DNA via its unique C2H2 motifs. Mass-spectrometry-based experiments revealed that ZNF384 interacts with the Ku70/Ku80 NHEJ complex via its N-terminus. This interaction promotes the assembly of Ku70/Ku80 at DBSs as revealed by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. By regulating Ku70/80 dynamics, ZNF384 facilitates the assembly of several downstream NHEJ factors (e.g. APLF and XRCC4) and repair by cNHEJ at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds to DSBs and Ku70/80 to facilitate their binding and the subsequent build-up of a functional cNHEJ repairosome at these lesions, high-lighting a role for ZNF384 in DSB repair and genome maintenance.
2021-11-15 | PXD020417 | Pride
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:We conducted proximity-dependent biotin identification (BioID) using wild-type Ku70 in HEK293 cells. Ku70 is part of a protein complex known as Ku, which is involved in a variety of functions throughout the cell. By identifying candidate protein interactors, we can infer new cellular processes that may involve the Ku complex.
2019-01-03 | PXD010931 | Pride
Project description:The Ku heterodimer (Ku70/Ku80) is a main component of the nonhomologous end-joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs). Ku binds the broken DNA end and recruits other proteins to facilitate the processing and ligation of the broken end. While Ku interacts with many proteins involved in DNA damage/repair-related functions, few interactions have been mapped to the N-terminal von Willebrand A (vWA) domain, a predicted protein interaction domain. The mutagenesis of Ku70 vWA domain S155/D156 unexpectedly increased cell survival following ionizing radiation (IR) treatment. DNA repair appeared unaffected, but defects in the activation of apoptosis and alterations in the DNA damage signaling response were identified. In particular, Ku70 S155A/D156A affected the IR-induced transcriptional response of several activating transcription factor 2 (ATF2)-regulated genes involved in apoptosis regulation. ATF2 phosphorylation and recruitment to DNA damage-induced foci was increased in Ku70-deficient cells, suggesting that Ku represses ATF2 activation. Ku70 S155A/D156A substitutions further enhanced this repression. S155A substitution alone was sufficient to confer enhanced survival, whereas alteration to a phosphomimetic residue (S155D) reversed this effect, suggesting that S155 is a phosphorylation site. Thus, these findings infer that Ku links signals from the DNA repair machinery to DNA damage signaling regulators that control apoptotic pathways.
| S-EPMC3255697 | biostudies-literature
Project description:Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(?/?) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of ?H2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.
| S-EPMC3711201 | biostudies-other
Project description:Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1-deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.
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Project description:In this study we characterize and investigate the effect of the loss of Ku70 protein in a conditional Ku70 knockout TREx-293 cell system. Loss of Ku70 protein levels was directly correlated with a loss of cell viability, supporting the observation that Ku performs an essential role in human cells. Interestingly, critical loss of average telomere length was not observed in Ku70 knockouts generated. Global quantitative proteomic analysis of whole cell extracts from Ku70 knockouts following depletion of Ku70 may indicate that Ku’s essential role in humans is not at telomeres, but a non-canonical role which is not yet fully understood.
2023-07-27 | PXD036297 | Pride
Project description:RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.
| S-EPMC4216052 | biostudies-literature
Project description:We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.
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