Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Mar;85(5):1417–1421. doi: 10.1073/pnas.85.5.1417

Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D.

D K Trask 1, M T Muller 1
PMCID: PMC279782  PMID: 2830618

Abstract

The activity of the endogenous DNA topoisomerase type I (EC 5.99.1.2) can be quantified in situ by determining how efficiently the enzyme is trapped in a covalent complex with DNA upon lysis of nuclei with detergents. In this way, we can measure relative levels of topoisomerase binding to DNA at native sites in chromatin. Since the majority of topoisomerase I is localized in the nucleolus at rRNA genes, we have evaluated how low levels of actinomycin D, which terminate transcription of rRNA genes, affect the activity of topoisomerase I. In vivo, as well as in vitro with purified topoisomerase I, we have found that drug treatment extends the half-life of the covalent topoisomerase-DNA complex. Actinomycin D stabilizes the nicked intermediate in the cleavage and resealing reaction but otherwise does not significantly alter the strand-passing ability of topoisomerase I. Sequence-specific cleavages by topoisomerase I were stimulated by actinomycin D at identical sequences recognized by the enzyme in the absence of drug. The localization of topoisomerase I in the nucleolus, coupled with the observation that transcription in this organelle is highly sensitive to actinomycin D and camptothecin treatment, leads us to propose that topoisomerase I contributes to actinomycin D inhibition of transcription.

Full text

PDF
1417

Images in this article

1418Image
on p.1418
1418Image
on p.1418
1419Image
on p.1419
1420Image
on p.1420
1421Image
on p.1421

Selected References

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

  1. Akiyama Y., Kato S. Two cell lines from lymphomas of Marek's disease. Biken J. 1974 Sep;17(3):105–116. [PubMed] [Google Scholar]
  2. Been M. D., Champoux J. J. DNA breakage and closure by rat liver type 1 topoisomerase: separation of the half-reactions by using a single-stranded DNA substrate. Proc Natl Acad Sci U S A. 1981 May;78(5):2883–2887. doi: 10.1073/pnas.78.5.2883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonven B. J., Gocke E., Westergaard O. A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin. Cell. 1985 Jun;41(2):541–551. doi: 10.1016/s0092-8674(85)80027-1. [DOI] [PubMed] [Google Scholar]
  4. Brill S. J., DiNardo S., Voelkel-Meiman K., Sternglanz R. Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA. 1987 Mar 26-Apr 1Nature. 326(6111):414–416. doi: 10.1038/326414a0. [DOI] [PubMed] [Google Scholar]
  5. Christiansen K., Bonven B. J., Westergaard O. Mapping of sequence-specific chromatin proteins by a novel method: topoisomerase I on Tetrahymena ribosomal chromatin. J Mol Biol. 1987 Feb 5;193(3):517–525. doi: 10.1016/0022-2836(87)90264-6. [DOI] [PubMed] [Google Scholar]
  6. Fleischmann G., Pflugfelder G., Steiner E. K., Javaherian K., Howard G. C., Wang J. C., Elgin S. C. Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6958–6962. doi: 10.1073/pnas.81.22.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garg L. C., DiAngelo S., Jacob S. T. Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene. Proc Natl Acad Sci U S A. 1987 May;84(10):3185–3188. doi: 10.1073/pnas.84.10.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gilmour D. S., Elgin S. C. Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor camptothecin. Mol Cell Biol. 1987 Jan;7(1):141–148. doi: 10.1128/mcb.7.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilmour D. S., Pflugfelder G., Wang J. C., Lis J. T. Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell. 1986 Feb 14;44(3):401–407. doi: 10.1016/0092-8674(86)90461-7. [DOI] [PubMed] [Google Scholar]
  10. Gottesfeld J. M. Novobiocin inhibits RNA polymerase III transcription in vitro by a mechanism distinct from DNA topoisomerase II. Nucleic Acids Res. 1986 Mar 11;14(5):2075–2088. doi: 10.1093/nar/14.5.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halligan B. D., Davis J. L., Edwards K. A., Liu L. F. Intra- and intermolecular strand transfer by HeLa DNA topoisomerase I. J Biol Chem. 1982 Apr 10;257(7):3995–4000. [PubMed] [Google Scholar]
  12. Higashinakagawa T., Wahn H., Reeder R. H. Isolation of ribosomal gene chromatin. Dev Biol. 1977 Feb;55(2):375–386. doi: 10.1016/0012-1606(77)90180-4. [DOI] [PubMed] [Google Scholar]
  13. Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed] [Google Scholar]
  14. Hyman R. W., Davidson N. Kinetics of the in vitro inhibition of transcription by actinomycin. J Mol Biol. 1970 Jun 14;50(2):421–438. doi: 10.1016/0022-2836(70)90202-0. [DOI] [PubMed] [Google Scholar]
  15. Kersten W., Kersten H., Szybalski W. Physicochemical properties of complexes between deoxyribonucleic acid and antibiotics which affect ribonucleic acid synthesis (actinomycin, daunomycin, cinerubin, nogalamycin, chormomycin, mithramycin, and olivomycin). Biochemistry. 1966 Jan;5(1):236–244. doi: 10.1021/bi00865a031. [DOI] [PubMed] [Google Scholar]
  16. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Muller M. T., Pfund W. P., Mehta V. B., Trask D. K. Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J. 1985 May;4(5):1237–1243. doi: 10.1002/j.1460-2075.1985.tb03766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ness P. J., Parish R. W., Koller T. Mapping of endogenous nuclease-sensitive regions and of putative topoisomerase sites of action along the chromatin of Dictyostelium ribosomal RNA genes. J Mol Biol. 1986 Apr 5;188(3):287–300. doi: 10.1016/0022-2836(86)90155-5. [DOI] [PubMed] [Google Scholar]
  19. Pater M. M., Mak S. Actinomycin D-induced breakage of human KB cell DNA. Nature. 1974 Aug 30;250(5469):786–788. doi: 10.1038/250786a0. [DOI] [PubMed] [Google Scholar]
  20. Pays E., Gilmour R. S. Specificity of chromatin transcription in vitro. Asymmetric transcription of the globin gene by Escherichia coli RNA polymerase. Biochim Biophys Acta. 1981 May 29;653(3):356–367. doi: 10.1016/0005-2787(81)90192-1. [DOI] [PubMed] [Google Scholar]
  21. Perry R. P., Kelley D. E. Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J Cell Physiol. 1970 Oct;76(2):127–139. doi: 10.1002/jcp.1040760202. [DOI] [PubMed] [Google Scholar]
  22. Perry R. P., Kelley D. E. Persistent synthesis of 5S RNA when production of 28S and 18S ribosomal RNA is inhibited by low doses of actinomycin D. J Cell Physiol. 1968 Dec;72(3):235–246. doi: 10.1002/jcp.1040720311. [DOI] [PubMed] [Google Scholar]
  23. Phillips D. R., Crothers D. M. Kinetics and sequence specificity of drug-DNA interactions: an in vitro transcription assay. Biochemistry. 1986 Nov 18;25(23):7355–7362. doi: 10.1021/bi00371a017. [DOI] [PubMed] [Google Scholar]
  24. Richardson J. P. The binding of RNA polymerase to DNA. J Mol Biol. 1966 Oct 28;21(1):83–114. doi: 10.1016/0022-2836(66)90081-7. [DOI] [PubMed] [Google Scholar]
  25. Ross W. E., Glaubiger D., Kohn K. W. Qualitative and quantitative aspects of intercalator-induced DNA strand breaks. Biochim Biophys Acta. 1979 Mar 28;562(1):41–50. doi: 10.1016/0005-2787(79)90124-2. [DOI] [PubMed] [Google Scholar]
  26. Rowe T. C., Wang J. C., Liu L. F. In vivo localization of DNA topoisomerase II cleavage sites on Drosophila heat shock chromatin. Mol Cell Biol. 1986 Apr;6(4):985–992. doi: 10.1128/mcb.6.4.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shaw J. L., Blanco J., Mueller G. C. Simple procedure for isolation of DNA, RNA and protein fractions from cultured animal cells. Anal Biochem. 1975 May 12;65(1-2):125–131. doi: 10.1016/0003-2697(75)90498-4. [DOI] [PubMed] [Google Scholar]
  28. Sobell H. M. The stereochemistry of actinomycin binding to DNA and its implications in molecular biology. Prog Nucleic Acid Res Mol Biol. 1973;13:153–190. doi: 10.1016/s0079-6603(08)60103-8. [DOI] [PubMed] [Google Scholar]
  29. Thomsen B., Mollerup S., Bonven B. J., Frank R., Blöcker H., Nielsen O. F., Westergaard O. Sequence specificity of DNA topoisomerase I in the presence and absence of camptothecin. EMBO J. 1987 Jun;6(6):1817–1823. doi: 10.1002/j.1460-2075.1987.tb02436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thrash C., Voelkel K., DiNardo S., Sternglanz R. Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J Biol Chem. 1984 Feb 10;259(3):1375–1377. [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Trask D. K., DiDonato J. A., Muller M. T. Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II. EMBO J. 1984 Mar;3(3):671–676. doi: 10.1002/j.1460-2075.1984.tb01865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Trask D. K., Muller M. T. Biochemical characterization of topoisomerase I purified from avian erythrocytes. Nucleic Acids Res. 1983 May 11;11(9):2779–2800. doi: 10.1093/nar/11.9.2779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Uemura T., Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. doi: 10.1002/j.1460-2075.1984.tb02040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vosberg H. P. DNA topoisomerases: enzymes that control DNA conformation. Curr Top Microbiol Immunol. 1985;114:19–102. doi: 10.1007/978-3-642-70227-3_2. [DOI] [PubMed] [Google Scholar]
  36. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  37. Waring M. Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: evidence for molecular models involving intercalation. J Mol Biol. 1970 Dec 14;54(2):247–279. doi: 10.1016/0022-2836(70)90429-8. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES