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Ku70 protein in hetero-trimeric complex with Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) represents a critical component of the nonhomologous-end-joining (NHEJ), the major machinery of DSBs repair in mammalian cells. It has been previously shown that modulation of Ku70 acetylation by histone deacetylases (HDAC) inhibitors induced sensitization of cancer cells to chemotherapeutic agents. However, the effects of such modulation on the repair of Ionizing Radiation (IR)-induced DSBs and the importance of dynamic equilibrium of acetylation/deacetylation have not been studied in details. To address these questions aceto-blocking and aceto-mimicking mutants were designed by replacing Ku70 lysine residues K317, K331 and K338 with arginine and glutamine respectively via site-directed mutagenesis. Transformed human embryonic lung fibroblasts MRC5VA were transfected to create stables cells lines over-expressing Ku70 mutant proteins. Clonogenic survival and γ-H2AX foci assays were performed to study the impact of these mutants on DNA repair proficiency of MRC5VA cells in response to IR. We report here that both Ku70 aceto-blocking and aceto-mimicking mutants rendered MRC5VA cells more susceptible to IR in terms of clonogenic survival and γH2AX foci. Moreover, modelling the possible interactions and structural impact of these aceto-blocking and aceto-mimicking mutants with DNA substrate showed that mimicking acetylation/deacetylation of K331 and K338 could directly compromise KU-DNA interactions, whereas K317 may have a more subtle role via forming a salt bridge with E330 thus optimising the positioning of the helix containing both K331 and K338 residues on the DNA. Our data indicates that dynamic equilibrium of acetylation/deacetylation of Ku70 lysine residues K317, K331 and K338 is critical for optimal repair of IR-induced DSBs, and may offer a novel therapeutic approach for cancer treatment.

DNA double strand breaks feature in neurological disorders, including many forms of neurodegeneration, stroke and acute neurotrauma. Tuxworth et al. show that inhibiting the DNA damage sensing response is neuroprotective, axon regenerative and improves functional recovery in models of neurodegeneration and optic nerve and spinal cord injury., DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double-strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system., Graphical Abstract Graphical Abstract

DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.

DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro . Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX. However, LX ligation activity is substantially reduced by the incorporation of linker histones. This inhibition is independent of the presence of core histone octamers and cannot be restored by addition of Ku alone but can be partially rescued by DNA-PK. The kinase activity of DNA-PK is essential for the recovery of end-joining. DNA-PK efficiently phosphorylates histone H1. Phosphorylated histone H1 has a reduced affinity for DNA and a decreased capacity to inhibit end-joining. Our findings raise the possibility that DNA-PK may act as a linker histone kinase by phosphorylating linker histones in the vicinity of a DNA break and coupling localized histone H1 release from DNA ends, with the recruitment of LX to carry out double-stranded ligation. Thus, by using histone H1-bound DNA as a template, we have reconstituted the end-joining step of DNA nonhomologous end-joining in vitro with a requirement for DNA-PK.

A DNA ligase IV (LIG4)‐null human pre‐B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error‐prone DNA ligase IV‐independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining. Thus, DNA ligase IV is an important factor influencing the fidelity of end joining in vivo. The LIG4‐defective cell lines also showed impaired end joining in an in vitro assay using cell‐free extracts. Elevated degradation of the terminal nucleotide was observed in a LIG4‐defective line, and addition of the DNA ligase IV–XRCC4 complex restored end protection. End protection by DNA ligase IV was not dependent upon ligation. Finally, using purified proteins, we demonstrate that DNA ligase IV–XRCC4 is able to protect DNA ends from degradation by T7 exonuclease. Thus, the ability of DNA ligase IV–XRCC4 to protect DNA ends may contribute to the ability of DNA ligase IV to promote accurate rejoining in vivo.

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T−B−severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue inSaccharomyces cerevisiaefunctions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis . These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.

Background Ionizing radiation (IR) has unique characteristics as a genotoxic agent in terms of the DNA damage it produces. Its primary lesions, DNA double-strand breaks, are responsible for its biological effects. Ku70 in heterotrimeric complex with Ku80 and DNA-PKcs represents a crucial component of the non-homologous end joining (NHEJ) DNA double-strand break (DSB) repair machinery. It has been shown previously that the modulation of Ku70 acetylation mediates HDAC inhibitors-induced sensitization of cancer cells to chemotherapy. However, the effect of such modulation on the DNA repair efficacy in response to IR has not been studied in details. Here we investigated the effect of deacetylation of Ku70 lysines K317, K331 and K338 on cellular responses to IR. Materials and methods Site-directed mutagenesis was used to replace Ku70K317, K331 and K338 with arginine. MRC5VA cells were transfected using lipofectamine and the expression was checked by Western blot and immunoflourescence. For clonogenic survival, irradiated cells were seeded out into 6-well plates along un-irradiated control and cultured for 14 days. The colonies were stained with crystal violet and counted. The cells were grown on coverslips in 6-well dishes, irradiated and stained for g-H2AX. Slides were analysed with a Carl Zeiss microscope and the foci were counted using Photoshop. The data were statistically analysed using the students’ two-tailed t-test. Results The Clonogenic assay revealed that the survival of the mutant cells is significantly lower than the controls at 8Gy of IR. Moreover, the mutant cells retained statistically significant higher percentages of residual foci 24 hours post irradiation than the controls. Conclusions Our work showed that overexpression of Ku70-K317R, Ku70-K331R and Ku70-K338R mutants sensitizes MRC5VA to IR and impairs their capacity to repair DNA DSBs. This finding strongly implies that the deacetylation of Ku70 plays an important role in the regulation of IR-induced DNA damage responses in human cells and may provide a new route for a therapeutic intervention in cancer treatment.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.

In order to investigate the relationships between the induction and rejoining of DNA double-strand breaks (dsb) and their biological consequences it is necessary to measure these lesions uniquely and accurately, especially at relevant low doses of ionizing radiation. Differences in radiosensitivity between cell lines could be due to variations in dsb induction or to differences in the efficiency and/or accuracy of enzymatic repair of these lesions. We have used field-inversion gel electrophoresis to investigate dsb induction and rejoining in V79B parental and XR-V15B ionizing radiation-sensitive mutant cell lines. No difference has been found in the induction of dsb in XR-V15B cells compared with wild type cells; the assay sensitivity permits measurement of dsb induced by doses as low as 1 Gy (p < or = 0.05). The radiosensitivity of the mutant cells is manifested both in a lower fraction of dsb rejoined in the early, fast repair component and longer persistence of unrejoined dsb during post-irradiation incubation. The fraction of dsb remaining unrejoined after prolonged incubations (up to 17 h) correlates well with the higher radiosensitivity of the mutant (as judged by D10 values).

International audience; AIMS: In cancer therapy, research has focused on the development of nanocarriers that can aid diagnosis, deliver therapeutic agents and monitor treatment progress. This study introduces high-resolution synchrotron x-ray fluorescence microscopy (SR-XFM) to investigate intracellular localization of novel lanthanide-coated nanoparticles in human cells and their genotoxicity screening after internalization. MATERIALS & METHODS: Noble metal nanoparticles coated with cerium and luminescent europium complexes have been developed as platforms for bioimaging and potential biodelivery applications. The intracellular distribution after internalization has been analyzed by ultrasensitive SR-XFM and genotoxicity evaluated using γ-H2AX DNA damage foci phosphorylation assay. RESULTS: We demonstrate the unprecedented capability of SR-XFM for extremely sensitive nanoimaging and intracellular elemental distribution analysis of noble metal nanoparticles in cells. Furthermore, we show that, depending on the charge of the coating complex and the presence of the DNA cargo, the internalization of functionalized nanoparticles by human fibroblasts can cause elevated levels of DNA damage detected by histone H2AX phosphorylation. CONCLUSION: The variable genotoxic impact of newly designed nanovectors emphasizes the need for careful and comprehensive testing of biological responses of all new nanoconstructs intended for future clinical applications. This can be greatly facilitated by SR-XFM nanoimaging of nanoparticles in cells at very low concentrations.

Investigating the methods commonly used to evaluate in vitro cytotoxicity of novel compounds, specifically non-covalent DNA binders, identifies that these methods may not be appropriate. The level of anticancer activity depends not only on the incubation time but also on the absolute amount (number of moles) of drug compound applied, rather than the concentration.

Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility loci with other AIDs. The thyroid stimulating hormone receptor (TSHR) represents the primary autoantigen in GD, in which autoantibodies bind to the receptor and mimic its ligand, thyroid stimulating hormone, causing the characteristic clinical phenotype. Although early studies investigating the TSHR and GD proved inconclusive, more recently we provided convincing evidence for association of the TSHR region with disease. In the current study, we investigated a combined panel of 98 SNPs, including 70 tag SNPs, across an extended 800 kb region of the TSHR to refine association in a cohort of 768 GD subjects and 768 matched controls. In total, 28 SNPs revealed association with GD (P

Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.

AHNAK is a high molecular weight protein that is under-expressed in several radiosensitive neuroblastoma cell lines. Using immunoaffinity purification or purified proteins, we show that AHNAK interacts specifically with the DNA ligase IV-XRCC4 complex, a complex that functions in DNA non-homologous end-joining. Furthermore, AHNAK and the DNA ligase IV-XRCC4 complex co-immunoprecipitate demonstrating an in vivo interaction. This interaction is specific and is not observed with other DNA ligases nor with other components of the DNA non-homologous end-joining machinery. We characterised AHNAK as a protein that stimulates the double-stranded (DS) ligation activity of DNA ligase IV-XRCC4. We show that AHNAK has weak DNA-binding activity and forms a stable complex with the DNA ligase IV-XRCC4 complex on DNA. AHNAK is also able to link two DNA molecules to a similar extent to that previously reported for Ku. Together, these findings demonstrate new activities for AHNAK, and raise the possibility that it may function to modulate DNA non-homologous end-joining. © 2003 Elsevier B.V. All rights reserved.

The nature of the primary biochemical defect in the human radiosensitive and cancer-prone syndrome, ataxia telangiectasia (AT), has remained obscure despite many efforts to elucidate it. In this study, AT complementation group D cells and a nearly isogenic corrected AT-hamster hybrid derivative have been analyzed for induction and repair of initial double-strand breaks (DSBs) after exposure to ionizing radiation, using a sensitive field-inversion electrophoresis technique. Results suggesting that initial levels of damage are the same in these two cell types, but indicating differences in the fast component of DNA repair, have been compared and correlated with those resulting from a study of the radioresistant DNA synthesis defect and its correction in the same cell lines. These measurements show that the radioresistant phenotype of the substantially corrected AT-hamster hybrid correlates with its higher level of fast-component DSB repair and higher level of inhibition of DNA synthesis, but that the initial damage induction does not contribute to the phenotype. We propose that the AT gene product(s) is likely to act early in a signaling pathway which controls both DNA repair and progression of cells through the phases of the cell cycle in response to ionizing radiation.

Among the techniques available for the measurement of the induction and rejoining of DNA double-strand breaks (DSBs), pulsed-field gel electrophoresis appears to have the greatest potential to improve the sensitivity limits to study these lesions in the dose range closest to that used in cell survival experiments. Encapsulating the cells in agarose during the experimental procedure allows the accurate and reproducible measurement of rejoining kinetics with a very minimal time delay immediately after irradiation. The method allows direct comparison of the amount of initial DNA damage sustained with repair kinetics in experiments designed to elucidate the mechanisms underlying differences in radiosensitivity between cell lines, together with analysis of the effect of different radiation qualities. The sensitivity limits of the method are 1 Gy for the double-strand break induction experiments and 10 Gy for rejoining experiments. Under selected conditions, no significant degradation of DNA had been observed in rodent cell lines during repair incubation up to 17 h in either irradiated cells or unirradiated controls (background levels for neutron experiments, 2.2 +/- 0.3% at Time 0 compared to 2.3 +/- 0.5% after 17 h of incubation; background levels for X-ray experiments, 2.3 +/- 0.6% at Time 0 and 3.7 +/- 1.1% after 17 h of incubation). In preliminary experiments with the A549 human oat cell carcinoma cell line, DNA DSB background levels remained constant in unirradiated controls up to 4 h in the range reported for the rodent cell line.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] and [2], XRCC4 and DNA ligase IV [3], [3], [4] and [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] and [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9], [10] and [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme–adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.