Production and characterization of recombinant human Ku antigen (original) (raw)

Abstract

Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.

3918

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allaway G. P., Vivino A. A., Kohn L. D., Notkins A. L., Prabhakar B. S. Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector. Biochem Biophys Res Commun. 1990 Apr 30;168(2):747–755. doi: 10.1016/0006-291x(90)92385-d. [DOI] [PubMed] [Google Scholar]
  2. Chan J. Y., Lerman M. I., Prabhakar B. S., Isozaki O., Santisteban P., Kuppers R. C., Oates E. L., Notkins A. L., Kohn L. D. Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen. J Biol Chem. 1989 Mar 5;264(7):3651–3654. [PubMed] [Google Scholar]
  3. Chou C. H., Wang J., Knuth M. W., Reeves W. H. Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen. J Exp Med. 1992 Jun 1;175(6):1677–1684. doi: 10.1084/jem.175.6.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dvir A., Peterson S. R., Knuth M. W., Lu H., Dynan W. S. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11920–11924. doi: 10.1073/pnas.89.24.11920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Falzon M., Fewell J. W., Kuff E. L. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J Biol Chem. 1993 May 15;268(14):10546–10552. [PubMed] [Google Scholar]
  6. Francoeur A. M., Peebles C. L., Gompper P. T., Tan E. M. Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity. J Immunol. 1986 Mar 1;136(5):1648–1653. [PubMed] [Google Scholar]
  7. Gottlieb T. M., Jackson S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell. 1993 Jan 15;72(1):131–142. doi: 10.1016/0092-8674(93)90057-w. [DOI] [PubMed] [Google Scholar]
  8. Greenfield L., Simpson L., Kaplan D. Conversion of closed circular DNA molecules to single-nicked molecules by digestion with DNAase I in the presence of ethidium bromide. Biochim Biophys Acta. 1975 Oct 15;407(3):365–375. doi: 10.1016/0005-2787(75)90104-5. [DOI] [PubMed] [Google Scholar]
  9. Griffith A. J., Blier P. R., Mimori T., Hardin J. A. Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA. J Biol Chem. 1992 Jan 5;267(1):331–338. [PubMed] [Google Scholar]
  10. Gruskin E. A., Lloyd R. S. The DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity. J Biol Chem. 1986 Jul 25;261(21):9607–9613. [PubMed] [Google Scholar]
  11. Gunderson S. I., Knuth M. W., Burgess R. R. The human U1 snRNA promoter correctly initiates transcription in vitro and is activated by PSE1. Genes Dev. 1990 Dec;4(12A):2048–2060. doi: 10.1101/gad.4.12a.2048. [DOI] [PubMed] [Google Scholar]
  12. Higashiura M., Shimizu Y., Tanimoto M., Morita T., Yagura T. Immunolocalization of Ku-proteins (p80/p70): localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes. Exp Cell Res. 1992 Aug;201(2):444–451. doi: 10.1016/0014-4827(92)90293-h. [DOI] [PubMed] [Google Scholar]
  13. Kaczmarski W., Khan S. A. Lupus autoantigen Ku protein binds HIV-1 TAR RNA in vitro. Biochem Biophys Res Commun. 1993 Oct 29;196(2):935–942. doi: 10.1006/bbrc.1993.2339. [DOI] [PubMed] [Google Scholar]
  14. Knuth M. W., Gunderson S. I., Thompson N. E., Strasheim L. A., Burgess R. R. Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter. J Biol Chem. 1990 Oct 15;265(29):17911–17920. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lai J. S., Herr W. Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6958–6962. doi: 10.1073/pnas.89.15.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Landolfi N. F., Capra J. D., Tucker P. W. Interaction of cell-type-specific nuclear proteins with immunoglobulin VH promoter region sequences. Nature. 1986 Oct 9;323(6088):548–551. doi: 10.1038/323548a0. [DOI] [PubMed] [Google Scholar]
  18. Liu E. S., Lee A. S. Common sets of nuclear factors binding to the conserved promoter sequence motif of two coordinately regulated ER protein genes, GRP78 and GRP94. Nucleic Acids Res. 1991 Oct 11;19(19):5425–5431. doi: 10.1093/nar/19.19.5425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. May G., Sutton C., Gould H. Purification and characterization of Ku-2, an octamer-binding protein related to the autoantigen Ku. J Biol Chem. 1991 Feb 15;266(5):3052–3059. [PubMed] [Google Scholar]
  20. Messier H., Fuller T., Mangal S., Brickner H., Igarashi S., Gaikwad J., Fotedar R., Fotedar A. p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2685–2689. doi: 10.1073/pnas.90.7.2685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mimori T., Akizuki M., Yamagata H., Inada S., Yoshida S., Homma M. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. J Clin Invest. 1981 Sep;68(3):611–620. doi: 10.1172/JCI110295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mimori T., Hardin J. A. Mechanism of interaction between Ku protein and DNA. J Biol Chem. 1986 Aug 5;261(22):10375–10379. [PubMed] [Google Scholar]
  23. Mimori T., Hardin J. A., Steitz J. A. Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem. 1986 Feb 15;261(5):2274–2278. [PubMed] [Google Scholar]
  24. Paillard S., Strauss F. Analysis of the mechanism of interaction of simian Ku protein with DNA. Nucleic Acids Res. 1991 Oct 25;19(20):5619–5624. doi: 10.1093/nar/19.20.5619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Quinn J. P., Farina A. R. Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence. FEBS Lett. 1991 Jul 29;286(1-2):225–228. doi: 10.1016/0014-5793(91)80979-d. [DOI] [PubMed] [Google Scholar]
  26. Quinn J. P., Simpson J., Farina A. R. The Ku complex is modulated in response to viral infection and other cellular changes. Biochim Biophys Acta. 1992 Jun 15;1131(2):181–187. doi: 10.1016/0167-4781(92)90074-a. [DOI] [PubMed] [Google Scholar]
  27. Reeves W. H. Antibodies to the p70/p80 (Ku) antigens in systemic lupus erythematosus. Rheum Dis Clin North Am. 1992 May;18(2):391–414. [PubMed] [Google Scholar]
  28. Reeves W. H., Sthoeger Z. M. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem. 1989 Mar 25;264(9):5047–5052. [PubMed] [Google Scholar]
  29. Reeves W. H. Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera. J Exp Med. 1985 Jan 1;161(1):18–39. doi: 10.1084/jem.161.1.18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sharma S. K., Evans D. B., Vosters A. F., McQuade T. J., Tarpley W. G. Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain. Biotechnol Appl Biochem. 1991 Aug;14(1):69–81. [PubMed] [Google Scholar]
  31. Stuiver M. H., Coenjaerts F. E., van der Vliet P. C. The autoantigen Ku is indistinguishable from NF IV, a protein forming multimeric protein-DNA complexes. J Exp Med. 1990 Oct 1;172(4):1049–1054. doi: 10.1084/jem.172.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang T. S., Korn D. Reactivity of KB cell deoxyribonucleic acid polymerases alpha and beta with nicked and gapped deoxyribonucleic acid. Biochemistry. 1980 Apr 29;19(9):1782–1790. doi: 10.1021/bi00550a009. [DOI] [PubMed] [Google Scholar]
  33. Yaneva M., Arnett F. C. Antibodies against Ku protein in sera from patients with autoimmune diseases. Clin Exp Immunol. 1989 Jun;76(3):366–372. [PMC free article] [PubMed] [Google Scholar]
  34. Yaneva M., Busch H. A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex. Biochemistry. 1986 Sep 9;25(18):5057–5063. doi: 10.1021/bi00366a013. [DOI] [PubMed] [Google Scholar]
  35. Yaneva M., Wen J., Ayala A., Cook R. cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. J Biol Chem. 1989 Aug 15;264(23):13407–13411. [PubMed] [Google Scholar]
  36. Zhang W. W., Yaneva M. On the mechanisms of Ku protein binding to DNA. Biochem Biophys Res Commun. 1992 Jul 15;186(1):574–579. doi: 10.1016/s0006-291x(05)80847-2. [DOI] [PubMed] [Google Scholar]
  37. de Vries E., van Driel W., Bergsma W. G., Arnberg A. C., van der Vliet P. C. HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex. J Mol Biol. 1989 Jul 5;208(1):65–78. doi: 10.1016/0022-2836(89)90088-0. [DOI] [PubMed] [Google Scholar]