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At least 865 records · Page 48Linked to original sources
PubMed1995

DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cell line CHO-K1, has been demonstrated to be defective in DNA double-strand break repair and also in its proficiency to undergo V(D)J recombination. Recent work has provided both genetic and biochemical evidence that the M(r) 80,000 subunit of the Ku protein is able to complement the radiosensitivity and the V(D)J recombination defect in the xrs-6 mutant. We demonstrate here that complementation of the radiosensitive phenotype in xrs-6 cells by the introduction of Ku80 cDNA is accompanied by the concomitant restoration of DNA double-strand break rejoining proficiency to almost that of the parental CHO-K1 cells, as measured both by neutral single-cell microgel electrophoresis (Comet) technique and by pulsed-field gel electrophoresis. These results provide further biochemical evidence for the involvement of the Ku protein in the repair of DNA double-strand breaks.

Animals↗
PubMed1994

Systemic lupus erythematosus. Antibodies to DNA, DNA-binding proteins, and histones.

Pathogenic autoantibodies to DNA are frequently associated with autoantibodies to chromatin-associated proteins such as histones or the Ku (p70/p80) antigens. In view of the increasing evidence that autoantibody production is antigen-driven, and because DNA is packaged with proteins in the cell, we suggest that anti-DNA antibodies may arise in response to DNA-protein complexes rather than naked DNA. Recent studies of the specificities of autoantibodies directed against the components of nucleosomes and transcriptional complexes are consistent with this hypothesis. The possible clinical significance of immune recognition of various types of chromatin complexes in autoimmune disease is discussed.

Antibodies, Antinuclear↗
PubMed1994

Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant.

Double-strand DNA break repair is important in maintaining the genetic integrity of the genome. Using a mobility shift assay, we find that a protein, or complex of proteins, that is present in mammalian and yeast cells binds to the ends of double-strand DNA and renders the ends resistant to exonuclease digestion. Additionally, a mammalian DNA double-strand repair-deficient mutant, xrs, has no observable DNA end binding activity, while a revertant cell has wild-type activity. In addition, mobility supershift assays using monoclonal antibodies to the human Ku antigen (M(r) 70,000 subunit) reveal that one of the proteins of this end binding activity may be the Ku antigen or a protein with similar antigenic determinants. These observations suggest that this DNA end-binding protein may function in DNA repair.

Animals↗
PubMed1993

Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.

Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner. Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein. Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature. Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA. Nick binding was also observed directly using radiolabeled singly nicked circular DNA. The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent. Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate. A similar inhibition was not observed at nicks. These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo.

Animals↗
PubMed1996

Induction and repair of chromosome aberrations in scid cells measured by premature chromosome condensation.

Severe combined immunodeficient (scid) murine cells, which are defective in both repair of DNA double-strand breaks and V(D)J recombination, are deficient in DNA-dependent protein kinase (DNA-PK), a protein which forms an activated complex with the DNA end-binding Ku proteins (p80 and p70) upon association with damaged DNA. Xrs 5 cells are deficient in the Ku p80 protein and also fail to form an active DNA-PK repair complex. Since both scid and xrs cells are defective in the same protein complex, we compared the kinetics of chromosome repair in scid cells to results published previously for xrs 5 cells. C.B-17 cells, scid cells and scid cells complemented with a single human chromosome 8 were irradiated with 6 Gy and allowed to repair from 0-24 h before fusion to HeLa cells for chromosome condensation. Breaks and dicentrics were visualized by fluorescence in situ hybridization. All cells had the same initial amount of chromosome damage, but scid cells had a slower rate of rejoining, more unrejoined breaks and more dicentrics than C.B-17 and scid cells with human chromosome 8. The scid cells appear to respond differently than xrs 5 cells, despite both cells lacking an essential component of the same DNA repair complex.

Algorithms↗
PubMed1996

Ku protein complex is involved in nucleotide excision repair of DNA.

The repair of UV-C (254 nm) DNA lesions by nucleotide excision repair (NER) has been studied in the rodent cell line xrs6 belonging to complementation group 5 of ionising radiation sensitive (IRs) mutants. xrs6 cell line shows a defect in the DNA-end binding protein complex Ku which is involved in the repair of double-strand breaks (DSB) due to IR. In agreement with IR sensitivity, a bleomycin sensitive phenotype of xrs6 cell line was found as compared to the parental CHO-K1 line (factor > 8 fold). xrs6 exhibited also a slight (factor 2) but reproducible sensitivity to UV-C-light, while a revertant cell line for Ku DNA-end binding activity, xrs6rev, showed a restoration of both IR and UV-C sensitivities to the parental level. The NER activity of these cell lines was measured in vitro in nuclear protein extracts in the presence of plasmid DNA repair substrate damaged with UV-C lesions repaired by NER: xrs6 cell extracts exhibited only 55% of NER activity as compared to the control CHO-K1 and xrs6rev cell extracts. These results indicate that the Ku DSB repair protein is involved also in the NER process.

Animals↗
PubMed1996

The role of Ataxia telangiectasia and the DNA-dependent protein kinase in the p53-mediated cellular response to ionising radiation.

The DNA-dependent protein kinase (DNA-PK), whose catalytic subunit shows structural similarities to the Ataxia telangiectasia (AT) gene product (ATM), has also been implicated in the p53-mediated signal transduction pathway that activates the cellular response to DNA damage produced by ionizing radiation. DNA-PK activity however was not found to be related to the transcriptional induction of WAFl/CIP1(p2l) in AT lymphoblastoid cell lines, following treatment with ionizing radiation. Normal protein and transcription levels of Ku70 and Ku80, as well as DNA-PK activity, were found in six different AT cell lines, 1-4 h following exposure to ionizing radiation, timepoints where reduced and delayed transcriptional induction of WAF1/CIP1 (p21) was observed. WAF1/CIP1 (p21) was found to be transcriptionally induced by p53 in normal cell lines over this same time period following exposure to ionizing radiation. These results suggest that despite the findings that in vitro DNA-PK may phosphorylate p53, in vivo it would not appear to play a central role in the activation of p53 as a transcription factor nor can it substitute for the ATM gene product in the cellular response following exposure to ionizing radiation.

Animals↗
PubMed1996

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways.

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.

Antigens, Nuclear↗
PubMed1996

Scleroderma-polymyositis overlap syndrome associated with anti-Ku antibody and rimmed vacuole formation.

Scleroderma-polymyositis overlap syndrome is strongly associated with certain autoantibodies, such as anti-PM-Scl antibody and anti-Ku antibody. We describe a case of scleroderma-polymyositis overlap syndrome associated with anti-Ku antibody, which revealed rimmed vacuole formation in addition to intranuclear and intracytoplasmic filamentous inclusions in muscle biopsy specimens.

Adult↗
PubMed1997

Ku proteins join DNA fragments as shown by atomic force microscopy.

The binding of the Ku protein to DNA was investigated using the atomic force microscope. Ku was found to bind predominantly to the ends of double-stranded DNA. Experiments with plasmid DNA revealed that Ku does not bind to circular plasmids but does bind to plasmids that have been linearized by treatment with ionizing radiation. The binding of Ku to poly(dG-dC) x poly(dG-dC) polynucleotides and to a 400-bp DNA EcoRI fragment resulted in a shift in the fragment size distribution to include longer fragments, with internally binding Ku. Furthermore, we observed images consistent with fragments joined together by Ku, showing an interaction with two ends of DNA. These observations suggest that Ku may play a role in physically orienting DNA for ligation by binding the ends of adjacent DNA molecules.

Antigens, Nuclear↗
PubMed1997

Long-term morphine treatment increases Ku protein DNA end-binding activity.

Human neuroblastoma SH-SY5Y and small-cell lung carcinoma U1690 cells of neuroendocrine origin were exposed to morphine for 1 h, 3 h or 5 days. These treatments did not alter activities of AP-1, NF-kappa B and YY1 transcription factors in SH-SY5Y cells or NF-kappa B and YY1 in U1690 cells. Five-day morphine treatment, however, caused a twofold increase in the activity of a sequence-non-specific, spermidine-activated DNA-binding factor in U1690 cells. The morphine effect was prevented by the antagonist naloxone. The DNA-binding factor bound preferentially to double-stranded DNA ends. This fact and data on subunit composition, molecular masses of subunits, and supershift/inhibition by specific antibodies in a band shift assay, show the spermidine-activated factor to be identical with the Ku protein, the DNA-binding subunit of DNA-dependent protein kinase. The effect observed may be one of the mechanisms through which opioids influence gene regulation.

Antigens, Nuclear↗
PubMed1997

Regulation of Ku expression in normal murine B cells by stimuli that promote switch recombination.

DNA-dependent protein kinase, the catalytic subunit associated with the Ku heterodimer (Ku70/Ku86), has been implicated in switch recombination. Therefore, we tested whether certain stimuli known to promote switch recombination may act in part by inducing Ku expression. We find that resting B cells contain relatively low levels of nuclear Ku, but that Ku expression can be up-regulated by culturing the cells with two switch stimuli. First, IL-4 and CD40 engagement in combination, but neither of these stimuli acting alone, strongly induce Ku expression; Ku levels rise within 24 h, about 2 days before switch recombination is detected. Second, dextran-conjugated anti-IgD Abs strongly induce Ku expression, which is variably enhanced by IL-5, but not by IL-4. Our data suggest that switch recombination may be regulated, at least in part, through changes in the nuclear expression of Ku.

Animals↗
PubMed1998

Analysis of DNA binding proteins associated with hemin-induced transcriptional inhibition. The hemin response element binding protein is a heterogeneous complex that includes the Ku protein.

Hemin inhibits transcription of the tartrate resistant acid phosphatase (TRAP) gene. Using deletion mutagenesis of the mouse TRAP 5'-flanking region, we previously identified a 27-bp DNA segment containing a central GAGGC tandem repeat sequence (the hemin response element [HRE]), which bound nuclear proteins (hemin response element binding proteins [HREBPs]) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin. We now have used affinity binding to HRE-derivatized beads to identify four HREBP components with apparent molecular masses of 133-, 90-, 80-, and 37-kD, respectively. The 80- and 90-kD components correspond to the p70 and p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity. Based on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1. The 133-kD component appeared to be a unique protein. KuAg participation in HREBP complexes was specific as it was present in HREBPs bound to HRE microcircles. Results of depletion/reconstitution experiments suggested that KuAg does not bind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins. We conclude that HREBP is a heterogeneous complex composed of KuAg, ref1, and a unique 133-kD protein. We speculate that the role of heme may be to promote interactions among these components, thereby facilitating HRE binding and downregulation of hemin responsive genes.

Acid Phosphatase↗
PubMed1998

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells.

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

Androstadienes↗
PubMed2001

Monoclonal antibodies derived from BALB/c mice immunized with apoptotic Jurkat T cells recognize known autoantigens.

It has been postulated that post-translational modifications and relocalization of proteins during apoptosis may lead to presentation of these molecules to the immune system in such a way that normal mechanisms of tolerance are bypassed. In the present study, Jurkat cells were induced to undergo apoptosis by treatment with the chemotherapeutic agent Ara-C. BALB/c mice were then immunized with the apoptotic cells and hybridomas were generated. Using an indirect immunofluorescence assay, the monoclonal antibodies produced were screened by flow cytometry for those monoclonal antibodies demonstrating reactivity with permeabilized apoptotic Jurkat cells but not with non-permeabilized normal or apoptotic Jurkat cells. Of 281 monoclonal antibodies, 20 monoclonal antibodies with these properties were selected for further analysis. Using 32P- or 35S-metabolically labelled Jurkat cells, these selected monoclonal antibodies were screened for their ability to recognize autoantigens by immunoprecipitation and Western blotting. Well characterized autoimmune sera were then used to confirm the identity of autoantigens by immunoblotting. We demonstrate that immunization of normal mice with apoptotic Jurkat cells results in the formation of antibodies targeting multiple autoantigens or autoantigen complexes, including Ku, rRNPs, snRNPs and vimentin. These findings are consistent with the hypothesis that apoptosis can contribute to the development of autoimmunity.

Animals↗
PubMed2003

Molecular cloning and characterization of rKAB1, which interacts with KARP-1, localizes in the nucleus and protects cells against oxidative death.

The Ku autoantigen/KARP-1 (Ku86 autoantigen related protein-1) plays an important role in the double-strand break repair of mammalian DNA as a DNA-binding component of DNA-dependent protein kinase (DNA-PK) complex. KARP-1 is differently transcribed from the human Ku86 autoantigen gene locus and it is implicated in the control of DNA-dependent protein kinase activity. We cloned rKAB1, a rat homolog of KAB1 (KARP-1 binding protein 1 of human) from a rat hippocampal cDNA library. rKAB1 mRNA was specifically expressed in the brain and the thymus. EGFP-tagged rKAB1 protein localized in cell nucleus and in the condensed chromosome during the mitotic cell division. We found that rKAB1 works as a protective protein against cell damage by oxidative stress.

Amino Acid Sequence↗
PubMed1994

DNA-dependent protein kinase (Ku protein-p350 complex) assembles on double-stranded DNA.

The Ku protein is an autoantigen that consists of 70- and 80-kDa polypeptides. It associates with double-stranded DNA at free ends. In the present study, we examined the ability of anti-Ku antibodies to immunoprecipitate various structures from extracts of HeLa cells prepared at different salt concentrations. Under physiological conditions, these antibodies identified a complex containing the Ku protein and the 350-kDa component (p350) of DNA-dependent protein kinase (DNA-PK), which appeared to be closely associated on the DNA strand. In reconstitution experiments with cell extracts and biochemically purified components, the Ku protein-p350 complex formed only in the presence of double-stranded DNA. The reconstituted complex was catalytically active. Together with previous studies, these results indicate that the Ku protein interacts with DNA to create a binding site for p350 as the DNA-PK holoenzyme assembles.

Antigens, Nuclear↗
PubMed2001

Secondary necrosis is a source of proteolytically modified forms of specific intracellular autoantigens: implications for systemic autoimmunity.

OBJECTIVE: Specific autoantigens targeted in systemic autoimmunity undergo posttranslational modifications, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunologic consequences of defective clearance of apoptotic cells, we sought to determine whether autoantigens cleaved during apoptosis undergo an additional wave of proteolysis as apoptosis progresses to secondary necrosis in the absence of phagocytosis. METHODS: Apoptosis was induced in Jurkat cells with etoposide, anti-Fas antibody, or staurosporine (STS), and in HeLa cells with STS. Progression to secondary necrosis was assessed morphologically and quantified by trypan blue uptake. Autoantigen proteolysis during cell death was examined by immunoblotting of cell lysates using highly specific human autoantibodies as detecting probes. RESULTS: Cells treated with the different apoptosis inducers underwent a rapid apoptosis that gradually progressed to secondary necrosis. During the initial apoptotic stages, several autoantigens, including poly(ADP-ribose) polymerase, topoisomerase I (or Scl-70), SSB/La, and U1-70 kd, were cleaved into their signature apoptotic fragments. Progression of apoptosis to secondary necrosis was associated with additional proteolysis of these and other autoantigens in a caspase-independent manner. Some autoantigens (e.g., ribosomal RNP, Ku, and SSA/Ro) appeared to be resistant to proteolysis during cell death. CONCLUSION: In the absence of phagocytosis, apoptotic cells may undergo secondary necrosis, a process associated with additional proteolytic degradation of specific autoantigens. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells and serve as a source of modified forms of specific autoantigens that might stimulate autoantibody responses under proinflammatory conditions.

Amino Acid Chloromethyl Ketones↗