Abstract
Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- Gaillard C., Strauss F. Sequence-specific single-strand-binding protein for the simian virus 40 early promoter stimulates transcription in vitro. J Mol Biol. 1990 Sep 20;215(2):245–255. doi: 10.1016/S0022-2836(05)80343-2. [DOI] [PubMed] [Google Scholar]
- 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]
- Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Mimori T., Ohosone Y., Hama N., Suwa A., Akizuki M., Homma M., Griffith A. J., Hardin J. A. Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1777–1781. doi: 10.1073/pnas.87.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Roberts M. R., Miskimins W. K., Ruddle F. H. 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. doi: 10.1091/mbc.1.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg H., Singer M., Rosenberg M. Highly reiterated sequences of SIMIANSIMIANSIMIANSIMIANSIMIAN. Science. 1978 Apr 28;200(4340):394–402. doi: 10.1126/science.205944. [DOI] [PubMed] [Google Scholar]
- Strauss F., Varshavsky A. A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell. 1984 Jul;37(3):889–901. doi: 10.1016/0092-8674(84)90424-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Tan E. M. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989;44:93–151. doi: 10.1016/s0065-2776(08)60641-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Yaneva M., Ochs R., McRorie D. K., 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. doi: 10.1016/0304-4165(85)90270-3. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]