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Proc Natl Acad Sci U S A. Dec 1987; 84(24): 8971–8975.
PMCID: PMC299673

Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli.


We show that, despite differences in primary structure, substrate preference, and mechanism of catalysis, yeast DNA topoisomerase I can functionally substitute for Escherichia coli DNA topoisomerase I. A family of plasmids expressing the yeast TOP1 gene or 5'-deletion mutations of it were used to complement the temperature-sensitive phenotype of an E. coli topA mutant. These plasmids were then isolated from the cells by a rapid lysis procedure and examined for their degrees of supercoiling. Functional complementation of a conditional-lethal mutation in topA, which encodes E. coli DNA topoisomerase I, correlates with the expression of a catalytically active yeast enzyme that reduces the degree of negative supercoiling of intracellular DNA. We also show that approximately 130 amino acids of the amino-terminal portion of the yeast enzyme can be deleted without affecting its activity in vitro; activity of the enzyme inside E. coli, however, is more sensitive to such deletions.

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Selected References

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  • Wang JC. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. [PubMed]
  • Vosberg HP. DNA topoisomerases: enzymes that control DNA conformation. Curr Top Microbiol Immunol. 1985;114:19–102. [PubMed]
  • Wang JC. Recent studies of DNA topoisomerases. Biochim Biophys Acta. 1987 Jun 6;909(1):1–9. [PubMed]
  • Dean F, Krasnow MA, Otter R, Matzuk MM, Spengler SJ, Cozzarelli NR. Escherichia coli type-1 topoisomerases: identification, mechanism, and role in recombination. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):769–777. [PubMed]
  • Srivenugopal KS, Lockshon D, Morris DR. Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme. Biochemistry. 1984 Apr 24;23(9):1899–1906. [PubMed]
  • Been MD, Champoux JJ. Breakage of single-stranded DNA by eukaryotic type 1 topoisomerase occurs only at regions with the potential for base-pairing. J Mol Biol. 1984 Dec 15;180(3):515–531. [PubMed]
  • Champoux JJ, Dulbecco R. An activity from mammalian cells that untwists superhelical DNA--a possible swivel for DNA replication (polyoma-ethidium bromide-mouse-embryo cells-dye binding assay). Proc Natl Acad Sci U S A. 1972 Jan;69(1):143–146. [PMC free article] [PubMed]
  • Baase WA, Wang JC. An omega protein from Drosophila melanogaster. Biochemistry. 1974 Oct 8;13(21):4299–4303. [PubMed]
  • Champoux JJ. Strand breakage by the DNA untwisting enzyme results in covalent attachment of the enzyme to DNA. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3800–3804. [PMC free article] [PubMed]
  • Kirkegaard K, Wang JC. Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. J Mol Biol. 1985 Oct 5;185(3):625–637. [PubMed]
  • Depew RE, Liu LF, Wang JC. Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein. J Biol Chem. 1978 Jan 25;253(2):511–518. [PubMed]
  • Tse-Dinh YC, Wang JC. Complete nucleotide sequence of the topA gene encoding Escherichia coli DNA topoisomerase I. J Mol Biol. 1986 Oct 5;191(3):321–331. [PubMed]
  • Thrash C, Bankier AT, Barrell BG, Sternglanz R. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4374–4378. [PMC free article] [PubMed]
  • Cozzarelli NR. DNA gyrase and the supercoiling of DNA. Science. 1980 Feb 29;207(4434):953–960. [PubMed]
  • Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. [PubMed]
  • Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. [PMC free article] [PubMed]
  • DiNardo S, Voelkel KA, Sternglanz R, Reynolds AE, Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. [PubMed]
  • Pruss GJ, Manes SH, Drlica K. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell. 1982 Nov;31(1):35–42. [PubMed]
  • Raji A, Zabel DJ, Laufer CS, Depew RE. Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I. J Bacteriol. 1985 Jun;162(3):1173–1179. [PMC free article] [PubMed]
  • Goto T, Wang JC. 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. [PMC free article] [PubMed]
  • Weiss MA, Eliason JL, States DJ. Dynamic filtering by two-dimensional 1H NMR with application to phage lambda repressor. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6019–6023. [PMC free article] [PubMed]
  • Ish-Horowicz D, Burke JF. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Sternglanz R, DiNardo S, Voelkel KA, Nishimura Y, Hirota Y, Becherer K, Zumstein L, Wang JC. Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition. Proc Natl Acad Sci U S A. 1981 May;78(5):2747–2751. [PMC free article] [PubMed]
  • Zumstein L, Wang JC. Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis. J Mol Biol. 1986 Oct 5;191(3):333–340. [PubMed]
  • 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]
  • Keller W. Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4876–4880. [PMC free article] [PubMed]
  • Wang JC, Becherer K. Cloning of the gene topA encoding for DNA topoisomerase I and the physical mapping of the cysB-topA-trp region of Escherichia coli. Nucleic Acids Res. 1983 Mar 25;11(6):1773–1790. [PMC free article] [PubMed]
  • Wang JC, Peck LJ, Becherer K. DNA supercoiling and its effects on DNA structure and function. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):85–91. [PubMed]
  • Lee CH, Mizusawa H, Kakefuda T. Unwinding of double-stranded DNA helix by dehydration. Proc Natl Acad Sci U S A. 1981 May;78(5):2838–2842. [PMC free article] [PubMed]
  • Liu LF, Wang JC. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. [PMC free article] [PubMed]
  • Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed]

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