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. 1992 Dec;11(13):5079–5090. doi: 10.1002/j.1460-2075.1992.tb05615.x

Transposase-induced excision and circularization of the bacterial insertion sequence IS911.

P Polard 1, M F Prère 1, O Fayet 1, M Chandler 1
PMCID: PMC556986  PMID: 1334464

Abstract

We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type.

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Selected References

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

  1. Bainton R., Gamas P., Craig N. L. Tn7 transposition in vitro proceeds through an excised transposon intermediate generated by staggered breaks in DNA. Cell. 1991 May 31;65(5):805–816. doi: 10.1016/0092-8674(91)90388-f. [DOI] [PubMed] [Google Scholar]
  2. Benjamin H. W., Kleckner N. Excision of Tn10 from the donor site during transposition occurs by flush double-strand cleavages at the transposon termini. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4648–4652. doi: 10.1073/pnas.89.10.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benjamin H. W., Kleckner N. Intramolecular transposition by Tn10. Cell. 1989 Oct 20;59(2):373–383. doi: 10.1016/0092-8674(89)90298-5. [DOI] [PubMed] [Google Scholar]
  4. Berg D. E. Structural requirement for IS50-mediated gene transposition. Proc Natl Acad Sci U S A. 1983 Feb;80(3):792–796. doi: 10.1073/pnas.80.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bushman F. D., Craigie R. Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1339–1343. doi: 10.1073/pnas.88.4.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cam K., Béjar S., Gil D., Bouché J. P. Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start. Nucleic Acids Res. 1988 Jul 25;16(14A):6327–6338. doi: 10.1093/nar/16.14.6327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  9. Chandler M., Galas D. J. Cointegrate formation mediated by Tn9. II. Activity of IS1 is modulated by external DNA sequences. J Mol Biol. 1983 Oct 15;170(1):61–91. doi: 10.1016/s0022-2836(83)80227-7. [DOI] [PubMed] [Google Scholar]
  10. Craigie R., Fujiwara T., Bushman F. The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell. 1990 Aug 24;62(4):829–837. doi: 10.1016/0092-8674(90)90126-y. [DOI] [PubMed] [Google Scholar]
  11. Craigie R., Mizuuchi K. Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu. Cell. 1987 Nov 6;51(3):493–501. doi: 10.1016/0092-8674(87)90645-3. [DOI] [PubMed] [Google Scholar]
  12. Derbyshire K. M., Kramer M., Grindley N. D. Role of instability in the cis action of the insertion sequence IS903 transposase. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4048–4052. doi: 10.1073/pnas.87.11.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eichinger D. J., Boeke J. D. A specific terminal structure is required for Ty1 transposition. Genes Dev. 1990 Mar;4(3):324–330. doi: 10.1101/gad.4.3.324. [DOI] [PubMed] [Google Scholar]
  14. Escoubas J. M., Prère M. F., Fayet O., Salvignol I., Galas D., Zerbib D., Chandler M. Translational control of transposition activity of the bacterial insertion sequence IS1. EMBO J. 1991 Mar;10(3):705–712. doi: 10.1002/j.1460-2075.1991.tb08000.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fayet O., Ramond P., Polard P., Prère M. F., Chandler M. Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol Microbiol. 1990 Oct;4(10):1771–1777. doi: 10.1111/j.1365-2958.1990.tb00555.x. [DOI] [PubMed] [Google Scholar]
  16. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  17. Fujiwara T., Craigie R. Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration. Proc Natl Acad Sci U S A. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fujiwara T., Mizuuchi K. Retroviral DNA integration: structure of an integration intermediate. Cell. 1988 Aug 12;54(4):497–504. doi: 10.1016/0092-8674(88)90071-2. [DOI] [PubMed] [Google Scholar]
  19. Galas D. J., Chandler M. Structure and stability of Tn9-mediated cointegrates. Evidence for two pathways of transposition. J Mol Biol. 1982 Jan 15;154(2):245–272. doi: 10.1016/0022-2836(82)90063-8. [DOI] [PubMed] [Google Scholar]
  20. Iost I., Guillerez J., Dreyfus M. Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. J Bacteriol. 1992 Jan;174(2):619–622. doi: 10.1128/jb.174.2.619-622.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Katz R. A., Merkel G., Kulkosky J., Leis J., Skalka A. M. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. doi: 10.1016/0092-8674(90)90290-u. [DOI] [PubMed] [Google Scholar]
  22. Katzman M., Katz R. A., Skalka A. M., Leis J. The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration. J Virol. 1989 Dec;63(12):5319–5327. doi: 10.1128/jvi.63.12.5319-5327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Khan E., Mack J. P., Katz R. A., Kulkosky J., Skalka A. M. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. doi: 10.1093/nar/19.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kleckner N. Regulation of transposition in bacteria. Annu Rev Cell Biol. 1990;6:297–327. doi: 10.1146/annurev.cb.06.110190.001501. [DOI] [PubMed] [Google Scholar]
  25. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. LaFemina R. L., Callahan P. L., Cordingley M. G. Substrate specificity of recombinant human immunodeficiency virus integrase protein. J Virol. 1991 Oct;65(10):5624–5630. doi: 10.1128/jvi.65.10.5624-5630.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Machida C., Machida Y. Regulation of IS1 transposition by the insA gene product. J Mol Biol. 1989 Aug 20;208(4):567–574. doi: 10.1016/0022-2836(89)90148-4. [DOI] [PubMed] [Google Scholar]
  28. Morisato D., Kleckner N. Tn10 transposition and circle formation in vitro. Cell. 1987 Oct 9;51(1):101–111. doi: 10.1016/0092-8674(87)90014-6. [DOI] [PubMed] [Google Scholar]
  29. Morisato D., Kleckner N. Transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo. Cell. 1984 Nov;39(1):181–190. doi: 10.1016/0092-8674(84)90204-6. [DOI] [PubMed] [Google Scholar]
  30. O'Connor M. B., Malamy M. H. Role of the F factor oriV1 region in recA-independent illegitimate recombination. Stable replicon fusions of the F derivative pOX38 and pBR322-related plasmids. J Mol Biol. 1984 May 25;175(3):263–284. doi: 10.1016/0022-2836(84)90348-6. [DOI] [PubMed] [Google Scholar]
  31. Ohtsubo H., Ohtsubo E. Nucleotide sequence of an insertion element, IS1. Proc Natl Acad Sci U S A. 1978 Feb;75(2):615–619. doi: 10.1073/pnas.75.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Polard P., Prère M. F., Chandler M., Fayet O. Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911. J Mol Biol. 1991 Dec 5;222(3):465–477. doi: 10.1016/0022-2836(91)90490-w. [DOI] [PubMed] [Google Scholar]
  33. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  34. Prère M. F., Chandler M., Fayet O. Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences. J Bacteriol. 1990 Jul;172(7):4090–4099. doi: 10.1128/jb.172.7.4090-4099.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reimmann C., Moore R., Little S., Savioz A., Willetts N. S., Haas D. Genetic structure, function and regulation of the transposable element IS21. Mol Gen Genet. 1989 Feb;215(3):416–424. doi: 10.1007/BF00427038. [DOI] [PubMed] [Google Scholar]
  36. Sekine Y., Ohtsubo E. Frameshifting is required for production of the transposase encoded by insertion sequence 1. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4609–4613. doi: 10.1073/pnas.86.12.4609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sherman P. A., Dickson M. L., Fyfe J. A. Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions. J Virol. 1992 Jun;66(6):3593–3601. doi: 10.1128/jvi.66.6.3593-3601.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spielmann-Ryser J., Moser M., Kast P., Weber H. Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli. Mol Gen Genet. 1991 May;226(3):441–448. doi: 10.1007/BF00260657. [DOI] [PubMed] [Google Scholar]
  39. Surette M. G., Buch S. J., Chaconas G. Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA. Cell. 1987 Apr 24;49(2):253–262. doi: 10.1016/0092-8674(87)90566-6. [DOI] [PubMed] [Google Scholar]
  40. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  41. Vink C., van Gent D. C., Elgersma Y., Plasterk R. H. Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage. J Virol. 1991 Sep;65(9):4636–4644. doi: 10.1128/jvi.65.9.4636-4644.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vögele K., Schwartz E., Welz C., Schiltz E., Rak B. High-level ribosomal frameshifting directs the synthesis of IS150 gene products. Nucleic Acids Res. 1991 Aug 25;19(16):4377–4385. doi: 10.1093/nar/19.16.4377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Weinert T. A., Derbyshire K. M., Hughson F. M., Grindley N. D. Replicative and conservative transpositional recombination of insertion sequences. Cold Spring Harb Symp Quant Biol. 1984;49:251–260. doi: 10.1101/sqb.1984.049.01.029. [DOI] [PubMed] [Google Scholar]
  44. Zerbib D., Jakowec M., Prentki P., Galas D. J., Chandler M. Expression of proteins essential for IS1 transposition: specific binding of InsA to the ends of IS1. EMBO J. 1987 Oct;6(10):3163–3169. doi: 10.1002/j.1460-2075.1987.tb02627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zerbib D., Polard P., Escoubas J. M., Galas D., Chandler M. The regulatory role of the IS1-encoded InsA protein in transposition. Mol Microbiol. 1990 Mar;4(3):471–477. doi: 10.1111/j.1365-2958.1990.tb00613.x. [DOI] [PubMed] [Google Scholar]
  46. Zerbib D., Prentki P., Gamas P., Freund E., Galas D. J., Chandler M. Functional organization of the ends of IS1: specific binding site for an IS 1-encoded protein. Mol Microbiol. 1990 Sep;4(9):1477–1486. [PubMed] [Google Scholar]
  47. Zhou C., Abaigar L., Jong A. Y. A protocol for using T7 DNA polymerase in oligonucleotide site-directed mutagenesis. Biotechniques. 1990 May;8(5):503–503. [PubMed] [Google Scholar]

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