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. 1988 Apr;85(8):2543–2547. doi: 10.1073/pnas.85.8.2543

cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment.

P D'Arpa 1, P S Machlin 1, H Ratrie 3rd 1, N F Rothfield 1, D W Cleveland 1, W C Earnshaw 1
PMCID: PMC280033  PMID: 2833744

Abstract

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.

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Selected References

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

  1. Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Champoux J. J. Proteins that affect DNA conformation. Annu Rev Biochem. 1978;47:449–479. doi: 10.1146/annurev.bi.47.070178.002313. [DOI] [PubMed] [Google Scholar]
  5. Cleveland D. W. Peptide mapping in one dimension by limited proteolysis of sodium dodecyl sulfate-solubilized proteins. Methods Enzymol. 1983;96:222–229. doi: 10.1016/s0076-6879(83)96020-2. [DOI] [PubMed] [Google Scholar]
  6. Cleveland D. W., Sullivan K. F. Molecular biology and genetics of tubulin. Annu Rev Biochem. 1985;54:331–365. doi: 10.1146/annurev.bi.54.070185.001555. [DOI] [PubMed] [Google Scholar]
  7. Dean F. B., Stasiak A., Koller T., Cozzarelli N. R. Duplex DNA knots produced by Escherichia coli topoisomerase I. Structure and requirements for formation. J Biol Chem. 1985 Apr 25;260(8):4975–4983. [PubMed] [Google Scholar]
  8. Earnshaw W. C., Sullivan K. F., Machlin P. S., Cooke C. A., Kaiser D. A., Pollard T. D., Rothfield N. F., Cleveland D. W. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J Cell Biol. 1987 Apr;104(4):817–829. doi: 10.1083/jcb.104.4.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Goto T., Wang J. C. Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7178–7182. doi: 10.1073/pnas.82.21.7178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  15. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  16. Liu L. F., Miller K. G. Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3487–3491. doi: 10.1073/pnas.78.6.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McCoubrey W. K., Jr, Champoux J. J. The role of single-strand breaks in the catenation reaction catalyzed by the rat type I topoisomerase. J Biol Chem. 1986 Apr 15;261(11):5130–5137. [PubMed] [Google Scholar]
  18. Minden J. S., Marians K. J. Replication of pBR322 DNA in vitro with purified proteins. Requirement for topoisomerase I in the maintenance of template specificity. J Biol Chem. 1985 Aug 5;260(16):9316–9325. [PubMed] [Google Scholar]
  19. 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]
  20. Nagata K., Guggenheimer R. A., Hurwitz J. Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4266–4270. doi: 10.1073/pnas.80.14.4266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shero J. H., Bordwell B., Rothfield N. F., Earnshaw W. C. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 1986 Feb 14;231(4739):737–740. doi: 10.1126/science.3003910. [DOI] [PubMed] [Google Scholar]
  23. Sternberger N. H., Sternberger L. A., Ulrich J. Aberrant neurofilament phosphorylation in Alzheimer disease. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4274–4276. doi: 10.1073/pnas.82.12.4274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tse-Dinh Y. C., Wang J. C. Complete nucleotide sequence of the topA gene encoding Escherichia coli DNA topoisomerase I. J Mol Biol. 1986 Oct 5;191(3):321–331. doi: 10.1016/0022-2836(86)90129-4. [DOI] [PubMed] [Google Scholar]
  25. Uemura T., Tanagida M. Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis. EMBO J. 1986 May;5(5):1003–1010. doi: 10.1002/j.1460-2075.1986.tb04315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  28. Yang L., Wold M. S., Li J. J., Kelly T. J., Liu L. F. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1987 Feb;84(4):950–954. doi: 10.1073/pnas.84.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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