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PubMed1996

Sequence-specific DNA binding by Ku autoantigen and its effects on transcription.

DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription.

Animals↗
PubMed2002

A monoclonal antibody specific for BK virus large T-antigen (clone BK.T-1) also binds the human Ku autoantigen.

A monoclonal antibody directed against the BK virus large T-antigen (clone BK.T-1) has previously been used to evaluate BKV-T antigen (BKV-TAg) expression. However, our experience showed a consistent reactivity to uninfected human, but not rodent cells and tissues. Using immunoprecipitation, Western analysis, amino acid sequencing and end-point dilution analysis, we analysed the BK.T-1 antibody reactivity and identified the bound cellular protein. The results clearly show that the antibody recognizes the large subunit (Ku86) of the Ku autoantigen, the regulatory component of the DNA-PKcs. We also demonstrated that the antibody retained its original reactivity in BKV-TAg transformed hamster kidney cells. The cross-reaction of the BK.T-1 antibody suggests a possible similarity between BKV-TAg and Ku86, but makes the antibody unsuitable for studies of BKV large T-antigen in the human cells.

Animals↗
PubMed1994

Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat.

We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2- to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (U5RE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: 110-, 80- and 70-kDa proteins. The 110-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70- and 80-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV-I basal transcriptional repression.

Amino Acid Sequence↗
PubMed1990

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

Amino Acid Sequence↗
PubMed2000

Ku autoantigen: a multifunctional DNA-binding protein.

Ku is a heterodimeric protein composed of approximately 70- and approximately 80-kDa subunits (Ku70 and Ku80) originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. Ku has high binding affinity for DNA ends and that is why originally it was known as a DNA end binding protein, but now it is known to also bind the DNA structure at nicks, gaps, hairpins, as well as the ends of telomeres. It has been reported also to bind with sequence specificity to DNA and with weak affinity to RNA. Ku is an abundant nuclear protein and is present in vertebrates, insects, yeast, and worms. Ku contains ssDNA-dependent ATPase and ATP-dependent DNA helicase activities. It is the regulatory subunit of the DNA-dependent protein kinase that phosphorylates many proteins, including SV-40 large T antigen, p53, RNA-polymerase II, RP-A, topoisomerases, hsp90, and many transcription factors such as c-Jun, c-Fos, oct-1, sp-1, c-Myc, TFIID, and many more. It seems to be a multifunctional protein that has been implicated to be involved directly or indirectly in many important cellular metabolic processes such as DNA double-strand break repair, V(D)J recombination of immunoglobulins and T-cell receptor genes, immunoglobulin isotype switching, DNA replication, transcription regulation, regulation of heat shock-induced responses, regulation of the precise structure of telomeric termini, and it also plays a novel role in G2 and M phases of the cell cycle. The mechanism underlying the regulation of all the diverse functions of Ku is still obscure.

Adenosine Triphosphatases↗
PubMed1998

Autoantigen Ku in the brain. Developmentally regulated expression and subcellular localization.

A double-stranded DNA end-binding factor with high levels of expression in brain and testis of adult mice was identified as the Ku protein, earlier described as an autoantigen in connective tissue diseases and found to be essential for recombination of the immunoglobulin genes and DNA repair. High Ku levels were found in the cerebellum and pituitary gland, lower levels in the hippocampus, hypothalamus and white matter structures. Ku levels were much higher in embryonic rat brain than in the adult brain, suggesting a role of the Ku protein in brain development. In embryonic rat brain, Ku was associated with cell nuclei, but was predominantly located in the cytosol in the adult rat cerebellum and hippocampus. The abundant expression of Ku in the brain suggests the involvement of Ku autoantibodies in the pathogenesis of neuropsychiatric complications in connective tissue diseases.

Animals↗
PubMed1994

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.

Adenosine Triphosphatases↗
PubMed1993

Somatostatin specifically binds p86 subunit of the autoantigen Ku.

The heterodimer Ku, first described as a nuclear autoantigen, is a regulatory factor of DNA replication and transcription. We have expressed the p86 subunit of Ku in Escherichia coli as a fusion protein with glutathione-S-transferase, using the vector pGEX-2T. After splitting up by thrombin, p86 was isolated by Sephacryl S200 gel filtration. The recombinant protein was found to have the same electrophoretic migration and to react with the same monoclonal antibody as the somatostatin-binding protein we recently isolated from the human gastric tumor cell HGT1 [7]. Furthermore, using the analog [125I]Tyr-11 somatostatin-14 as a tracer, we found that, like the HGT1 cell-purified protein, recombinant p86 specifically bound somatostatin with high affinity (KD = 2.3 +/- 0.3 nM) and large capacity (10,300 +/- 1,700 pmol/mg protein). These findings suggest that p86 subunit of Ku stands for the protein we previously isolated from the HGT1 cell. It could represent a new somatostatin receptor subtype perhaps involved in the antimitogenic effect of this peptide.

Acute Disease↗
PubMed1990

Mapping of epitopes on the 86 kDa subunit of the Ku autoantigen.

Patients with rheumatic disorders develop autoantibodies to a nuclear protein antigen termed Ku. The Ku antigen has been identified as a non-histone DNA-binding protein complex composed of two polypeptides: 86 kDa and 70 kDa. Initial competition experiments with four monoclonal antibodies specific for the 86 kDa subunit of Ku protein suggested there was more than one epitope on this polypeptide. To determine the region for these epitopes, cDNA deletions were made from both 5' and 3' ends. The immunoreactivity of the expressed proteins was assayed by immunoblotting. A 40 amino acids region located near the C-terminus of the polypeptide between amino acids 667-708 was essential for binding the monoclonal and autoimmune antibodies. Within this region, we differentiated three epitopes for the monoclonal antibodies. The sequence critical for binding of the autoimmune antibodies was in the same region, between amino acids 689-708. None of the four monoclonal antibodies blocked the binding of the autoantibodies. The implications of these results for the etiology of the anti-Ku autoantibody response are discussed.

Amino Acid Sequence↗
PubMed1998

Inhibition of Ku autoantigen binding activity to the E2F motif after ultraviolet B irradiation of melanocytic cells.

In human melanocytes and a human melanoma cell line (MM96L), the level of Ku sequence-specific binding to a 37-mer oligonucleotide containing a single E2F-1 binding site of the c-myc promoter (E2cM) significantly decreased 12 24h after cytostatic exposure to 300 J/m2 ultraviolet B radiation (UVB). No UVB-induced loss was found in fibroblasts, while HeLa cells showed an earlier (4 h) but less significant decrease than melanocytic cells. Equitoxic doses of gamma radiation, cisplatin or UVC had little effect on E2cM-specific binding. The loss of Ku binding in MM96L cells was not the result of translocation of Ku or a decrease in Ku protein or DNA-dependent protein kinase activity. The level of E2cM-specific binding in MM96L cells was increased by tunicamycin (2 microg/ml), an inhibitor of N-linked glycosylation, and decreased by the glucosidase inhibitor castanospermine (50 microg/ml). These results, which parallel the reported loss in melanocytes of the cell cycle regulator pRB after UVB, suggest that the DNA binding activity of Ku is affected by post-translational modification and may play a role in regulating the cell cycle response to UVB.

Antibodies, Monoclonal↗
PubMed1998

Sequence-specific binding of Ku autoantigen to single-stranded DNA.

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3-4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t1/2 on = 2.0 min) and was exceptionally stable, with an off rate of t1/2= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.

Antigens, Nuclear↗
PubMed1996

The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination.

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G1 and G2 or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.

Antigens, Nuclear↗
PubMed2004

Interplay between Drosophila Bloom's syndrome helicase and Ku autoantigen during nonhomologous end joining repair of P element-induced DNA breaks.

P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. The broken DNA ends generated during transposition can be repaired via the homology-directed synthesis-dependent strand annealing or by nonhomologous end joining (NHEJ). Genetic studies have demonstrated an interaction between the gene (mus309, for mutagen-sensitive) encoding the Drosophila Bloom's syndrome helicase homolog (DmBLM) and the Ku70 gene, which is involved in NHEJ. We have used RNA interference (RNAi) to knock down expression of DmBLM and one or both of the Drosophila Ku subunits, DmKu70 or DmKu80. Our results show that upon reduction of DmKu, an increase in small deletions (1-49 bp) and large deletions (>/=50 bp) flanking the site of P element-induced breaks is observed, and a reduction in large deletions at these sites is found upon reduction of DmBLM. Moreover, double RNAi of DmKu and DmBLM results in an increase in small deletions characteristic of the DmKu RNAi and also partially suppresses the reduction in repair efficiency observed with DmKu RNAi. These results suggest that there are DNA double-strand break recognition and/or processing events involving DmKu and DmBLM that, when eliminated by RNAi, lead to deletions. Finally, these results raise the possibility that, unlike the situation in mammals, where BLM appears to function exclusively in the homologous repair pathway, in Drosophila, DmBLM may be directly involved in, or at least influence the double-strand break recognition that leads to the NHEJ repair pathway.

Adenosine Triphosphatases↗
PubMed1994

DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen.

A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.

Adenosine Triphosphatases↗
PubMed1992

Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies.

Sera from certain patients with SLE, scleroderma and other autoimmune diseases react with the two subunits of the Ku protein: 86 and 70 kDa. Previous experiments indicated that a region of 40 amino acids near the C-terminus of the 86 kDa subunit between amino acids 667 and 708 was critical for binding of monoclonal and some autoimmune antibodies. In the present study, a series of additional 5' deletions and site-specific mutations in the critical region were produced and the immunoreactivities of the recombinant proteins were examined. ELISA and immunoblot analyses showed that three non-competing monoclonal antibodies specific for the 86 kDa subunit require stretches of amino acids significantly longer than 40 amino acids for reactivity, suggesting that the antigen is recognized in a folded state with perhaps more than one contact point. The reactivities of 12 of 24 anti-Ku positive autoimmune sera screened depended on the same amino acid sequences required for binding of the monoclonal antibodies, site-specific mutations reduced the reactivities of monoclonal and autoantibodies in a similar way. Preincubation of native Ku protein with the monoclonal antibodies shifted the electrophoretic mobility of Ku protein-DNA complex, suggesting that these monoclonal antibodies bind to epitopes on the surface of the native Ku protein. Taken together, the results from the deletion and site-directed mutagenesis demonstrate that both monoclonal and autoantibodies recognize non-linear epitopes of the 86 kDa polypeptide. These findings indicate that in a large portion of patients the anti-Ku autoimmune response is similar to the normal immune response to the Ku antigen in mice.

Antibodies, Monoclonal↗
PubMed1996

Thermotolerance and heat shock proteins: possible involvement of Ku autoantigen in regulating Hsp70 expression.

Here we characterize and compare the phenomenon of thermotolerance and permanent heat resistance in mammalian cells. The biochemical and molecular mechanisms underlying the induction of thermotolerance, and the role that heat shock proteins play in its development and decay are discussed. Finally, we describe a novel constitutive HSE-binding factor (CHBF/Ku) that appears to be involved in the regulation of the heat shock response.

Antigens, Nuclear↗