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. 1990 Jul;87(14):5307–5311. doi: 10.1073/pnas.87.14.5307

Purification of a DNA supercoiling factor from the posterior silk gland of Bombyx mori.

T Ohta 1, S Hirose 1
PMCID: PMC54312  PMID: 2164676

Abstract

A protein factor with an estimated molecular mass of 50 kDa has been purified to homogeneity from the silk gland of Bombyx mori. In the presence of a molar excess of this factor and eukaryotic DNA topoisomerase II, relaxed circular DNA is converted to the negatively supercoiled form. Eukaryotic DNA topoisomerase I cannot substitute for eukaryotic DNA topoisomerase II in the supercoiling reaction. The reaction is dependent on ATP and is inhibited by VP-16, a specific inhibitor of eukaryotic DNA topoisomerase II. When DNA topoisomerase I is subsequently added to the supercoiling reaction mixture, the supercoiled DNA becomes relaxed. These results suggest that when both the 50-kDa protein and eukaryotic DNA topoisomerase II are present in excess, unconstrained negative supercoils are introduced into DNA.

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Selected References

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

  1. Baldi M. I., Benedetti P., Mattoccia E., Tocchini-Valentini G. P. In vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase. Cell. 1980 Jun;20(2):461–467. doi: 10.1016/0092-8674(80)90632-7. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Briggs M. R., Kadonaga J. T., Bell S. P., Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986 Oct 3;234(4772):47–52. doi: 10.1126/science.3529394. [DOI] [PubMed] [Google Scholar]
  4. Brill S. J., Sternglanz R. Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell. 1988 Jul 29;54(3):403–411. doi: 10.1016/0092-8674(88)90203-6. [DOI] [PubMed] [Google Scholar]
  5. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  6. Giaever G. N., Wang J. C. Supercoiling of intracellular DNA can occur in eukaryotic cells. Cell. 1988 Dec 2;55(5):849–856. doi: 10.1016/0092-8674(88)90140-7. [DOI] [PubMed] [Google Scholar]
  7. Hirose S., Suzuki Y. In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling. Proc Natl Acad Sci U S A. 1988 Feb;85(3):718–722. doi: 10.1073/pnas.85.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hirose S., Tabuchi H., Yoshinaga K. GTP induces knotting, catenation, and relaxation of DNA by stoichiometric amounts of DNA topoisomerase II from Bombyx mori and HeLa cells. J Biol Chem. 1988 Mar 15;263(8):3805–3810. [PubMed] [Google Scholar]
  9. Hirose S., Tsuda M., Suzuki Y. Enhanced transcription of fibroin gene in vitro on covalently closed circular templates. J Biol Chem. 1985 Sep 5;260(19):10557–10562. [PubMed] [Google Scholar]
  10. Hsieh T. Knotting of the circular duplex DNA by type II DNA topoisomerase from Drosophila melanogaster. J Biol Chem. 1983 Jul 10;258(13):8413–8420. [PubMed] [Google Scholar]
  11. Hsieh T. Purification and properties of type II DNA topoisomerase from embryos of Drosophila melanogaster. Methods Enzymol. 1983;100:161–170. doi: 10.1016/0076-6879(83)00052-x. [DOI] [PubMed] [Google Scholar]
  12. Hübscher U., Stalder H. P. Mammalian DNA helicase. Nucleic Acids Res. 1985 Aug 12;13(15):5471–5483. doi: 10.1093/nar/13.15.5471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Laskey R. A., Honda B. M., Mills A. D., Finch J. T. Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature. 1978 Oct 5;275(5679):416–420. doi: 10.1038/275416a0. [DOI] [PubMed] [Google Scholar]
  15. Lee M. P., Sander M., Hsieh T. Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites. J Biol Chem. 1989 Dec 25;264(36):21779–21787. [PubMed] [Google Scholar]
  16. Liu L. F., Wang J. C. DNA-DNA gyrase complex: the wrapping of the DNA duplex outside the enzyme. Cell. 1978 Nov;15(3):979–984. doi: 10.1016/0092-8674(78)90281-7. [DOI] [PubMed] [Google Scholar]
  17. Liu L. F., Wang J. C. Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA. Proc Natl Acad Sci U S A. 1978 May;75(5):2098–2102. doi: 10.1073/pnas.75.5.2098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sinden R. R., Carlson J. O., Pettijohn D. E. Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells. Cell. 1980 Oct;21(3):773–783. doi: 10.1016/0092-8674(80)90440-7. [DOI] [PubMed] [Google Scholar]
  20. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tabuchi H., Hirose S. DNA supercoiling facilitates formation of the transcription initiation complex on the fibroin gene promoter. J Biol Chem. 1988 Oct 25;263(30):15282–15287. [PubMed] [Google Scholar]
  22. Tsuda M., Suzuki Y. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5' flanking sequence far upstream. Cell. 1981 Nov;27(1 Pt 2):175–182. doi: 10.1016/0092-8674(81)90371-8. [DOI] [PubMed] [Google Scholar]
  23. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  24. Weintraub H. Assembly and propagation of repressed and depressed chromosomal states. Cell. 1985 Oct;42(3):705–711. doi: 10.1016/0092-8674(85)90267-3. [DOI] [PubMed] [Google Scholar]

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