Search tips
Search criteria 


Logo of jexpmedHomeEditorsContactInstructions for Authors
J Exp Med. 1992 June 1; 175(6): 1677–1684.
PMCID: PMC2119251

Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen


The Ku antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA. DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear. High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding. In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays. Appropriate folding of the p70 protein was crucial for efficient DNA binding. The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609). Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition. Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding. Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding. The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.

Full Text

The Full Text of this article is available as a PDF (981K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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. [PMC free article] [PubMed]
  • Yaneva M, Ochs R, McRorie DK, Zweig S, Busch H. Purification of an 86-70 kDa nuclear DNA-associated protein complex. Biochim Biophys Acta. 1985 Jul 26;841(1):22–29. [PubMed]
  • Mimori T, Hardin JA. Mechanism of interaction between Ku protein and DNA. J Biol Chem. 1986 Aug 5;261(22):10375–10379. [PubMed]
  • Stuiver MH, Coenjaerts FE, van der Vliet PC. 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. [PMC free article] [PubMed]
  • Amabis JM, Amabis DC, Kaburaki J, Stollar BD. The presence of an antigen reactive with a human autoantibody in Trichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome. Chromosoma. 1990 Apr;99(2):102–110. [PubMed]
  • Knuth MW, Gunderson SI, Thompson NE, Strasheim LA, Burgess RR. 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]
  • 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]
  • Reeves WH, Pierani A, Chou CH, Ng T, Nicastri C, Roeder RG, Sthoeger ZM. Epitopes of the p70 and p80 (Ku) lupus autoantigens. J Immunol. 1991 Apr 15;146(8):2678–2686. [PubMed]
  • Porges AJ, Ng T, Reeves WH. Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species. J Immunol. 1990 Dec 15;145(12):4222–4228. [PubMed]
  • Chan EK, Tan EM. Human autoantibody-reactive epitopes of SS-B/La are highly conserved in comparison with epitopes recognized by murine monoclonal antibodies. J Exp Med. 1987 Dec 1;166(6):1627–1640. [PMC free article] [PubMed]
  • Reeves WH. Antinuclear antibodies as probes to explore the structural organization of the genome. J Rheumatol Suppl. 1987 Jun;14 (Suppl 13):97–105. [PubMed]
  • Dieckmann CL, Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed]
  • Landt O, Grunert HP, Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. [PubMed]
  • Allaway GP, Vivino AA, Kohn LD, Notkins AL, Prabhakar BS. 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. [PubMed]
  • Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. [PubMed]
  • McKay RD. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. [PubMed]
  • Johnson AD, Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. [PubMed]
  • Roberts MR, Miskimins WK, Ruddle FH. Nuclear proteins TREF1 and TREF2 bind to the transcriptional control element of the transferrin receptor gene and appear to be associated as a heterodimer. Cell Regul. 1989 Nov;1(1):151–164. [PMC free article] [PubMed]
  • Reeves WH, Sthoeger ZM. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem. 1989 Mar 25;264(9):5047–5052. [PubMed]
  • Chan JY, Lerman MI, Prabhakar BS, Isozaki O, Santisteban P, Kuppers RC, Oates EL, Notkins AL, Kohn LD. 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]
  • Pabo CO, Sauer RT. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. [PubMed]
  • Harrison SC, Aggarwal AK. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. [PubMed]
  • Chou PY, Fasman GD. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. [PubMed]
  • Sturm RA, Herr W. The POU domain is a bipartite DNA-binding structure. Nature. 1988 Dec 8;336(6199):601–604. [PubMed]
  • Stuiver MH, Celis JE, van der Vliet PC. Identification of nuclear factor IV/Ku autoantigen in a human 2D-gel protein database. Modification of the large subunit depends on cellular proliferation. FEBS Lett. 1991 Apr 22;282(1):189–192. [PubMed]
  • Griffith AJ, Blier PR, Mimori T, Hardin JA. 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]
  • Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. [PubMed]
  • Treacy MN, He X, Rosenfeld MG. I-POU: a POU-domain protein that inhibits neuron-specific gene activation. Nature. 1991 Apr 18;350(6319):577–584. [PubMed]
  • Clerc RG, Corcoran LM, LeBowitz JH, Baltimore D, Sharp PA. The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains. Genes Dev. 1988 Dec;2(12A):1570–1581. [PubMed]
  • Herr W, Sturm RA, Clerc RG, Corcoran LM, Baltimore D, Sharp PA, Ingraham HA, Rosenfeld MG, Finney M, Ruvkun G, et al. The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products. Genes Dev. 1988 Dec;2(12A):1513–1516. [PubMed]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press