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. 1997 May 15;16(10):2646–2655. doi: 10.1093/emboj/16.10.2646

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture.

M S Junop 1, D B Haniford 1
PMCID: PMC1169875  PMID: 9184211

Abstract

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.

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Selected References

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

  1. Arber W. Mechanisms in microbial evolution. J Struct Biol. 1990 Jul-Sep;104(1-3):107–111. doi: 10.1016/1047-8477(90)90064-j. [DOI] [PubMed] [Google Scholar]
  2. Bainton R. J., Kubo K. M., Feng J. N., Craig N. L. Tn7 transposition: target DNA recognition is mediated by multiple Tn7-encoded proteins in a purified in vitro system. Cell. 1993 Mar 26;72(6):931–943. doi: 10.1016/0092-8674(93)90581-a. [DOI] [PubMed] [Google Scholar]
  3. Bender J., Kleckner N. IS10 transposase mutations that specifically alter target site recognition. EMBO J. 1992 Feb;11(2):741–750. doi: 10.1002/j.1460-2075.1992.tb05107.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bolland S., Kleckner N. The three chemical steps of Tn10/IS10 transposition involve repeated utilization of a single active site. Cell. 1996 Jan 26;84(2):223–233. doi: 10.1016/s0092-8674(00)80977-0. [DOI] [PubMed] [Google Scholar]
  6. Chalmers R. M., Kleckner N. IS10/Tn10 transposition efficiently accommodates diverse transposon end configurations. EMBO J. 1996 Sep 16;15(18):5112–5122. [PMC free article] [PubMed] [Google Scholar]
  7. Chalmers R. M., Kleckner N. Tn10/IS10 transposase purification, activation, and in vitro reaction. J Biol Chem. 1994 Mar 18;269(11):8029–8035. [PubMed] [Google Scholar]
  8. Hallet B., Rezsöhazy R., Mahillon J., Delcour J. IS231A insertion specificity: consensus sequence and DNA bending at the target site. Mol Microbiol. 1994 Oct;14(1):131–139. doi: 10.1111/j.1365-2958.1994.tb01273.x. [DOI] [PubMed] [Google Scholar]
  9. Halling S. M., Kleckner N. A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity. Cell. 1982 Jan;28(1):155–163. doi: 10.1016/0092-8674(82)90385-3. [DOI] [PubMed] [Google Scholar]
  10. Haniford D. B., Chelouche A. R., Kleckner N. A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment. Cell. 1989 Oct 20;59(2):385–394. doi: 10.1016/0092-8674(89)90299-7. [DOI] [PubMed] [Google Scholar]
  11. Jeltsch A., Maschke H., Selent U., Wenz C., Köhler E., Connolly B. A., Thorogood H., Pingoud A. DNA binding specificity of the EcoRV restriction endonuclease is increased by Mg2+ binding to a metal ion binding site distinct from the catalytic center of the enzyme. Biochemistry. 1995 May 9;34(18):6239–6246. doi: 10.1021/bi00018a028. [DOI] [PubMed] [Google Scholar]
  12. Jeltsch A., Wenz C., Stahl F., Pingoud A. Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme. EMBO J. 1996 Sep 16;15(18):5104–5111. [PMC free article] [PubMed] [Google Scholar]
  13. Junop M. S., Haniford D. B. Multiple roles for divalent metal ions in DNA transposition: distinct stages of Tn10 transposition have different Mg2+ requirements. EMBO J. 1996 May 15;15(10):2547–2555. [PMC free article] [PubMed] [Google Scholar]
  14. Junop M. S., Hockman D., Haniford D. B. Intragenic suppression of integration-defective IS10 transposase mutants. Genetics. 1994 Jun;137(2):343–352. doi: 10.1093/genetics/137.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kennedy A. K., Haniford D. B. Isolation and characterization of IS10 transposase separation of function mutants: identification of amino acid residues in transposase that are important for active site function and the stability of transposition intermediates. J Mol Biol. 1996 Mar 1;256(3):533–547. doi: 10.1006/jmbi.1996.0106. [DOI] [PubMed] [Google Scholar]
  16. Kleckner N., Chalmers R. M., Kwon D., Sakai J., Bolland S. Tn10 and IS10 transposition and chromosome rearrangements: mechanism and regulation in vivo and in vitro. Curr Top Microbiol Immunol. 1996;204:49–82. doi: 10.1007/978-3-642-79795-8_3. [DOI] [PubMed] [Google Scholar]
  17. Kostrewa D., Winkler F. K. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. Biochemistry. 1995 Jan 17;34(2):683–696. doi: 10.1021/bi00002a036. [DOI] [PubMed] [Google Scholar]
  18. Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lee S. Y., Butler D., Kleckner N. Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7876–7880. doi: 10.1073/pnas.84.22.7876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lesser D. R., Kurpiewski M. R., Jen-Jacobson L. The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. Science. 1990 Nov 9;250(4982):776–786. doi: 10.1126/science.2237428. [DOI] [PubMed] [Google Scholar]
  22. Polard P., Chandler M. Bacterial transposases and retroviral integrases. Mol Microbiol. 1995 Jan;15(1):13–23. doi: 10.1111/j.1365-2958.1995.tb02217.x. [DOI] [PubMed] [Google Scholar]
  23. Rezsöhazy R., Hallet B., Delcour J., Mahillon J. The IS4 family of insertion sequences: evidence for a conserved transposase motif. Mol Microbiol. 1993 Sep;9(6):1283–1295. doi: 10.1111/j.1365-2958.1993.tb01258.x. [DOI] [PubMed] [Google Scholar]
  24. Sandmeyer S. B., Hansen L. J., Chalker D. L. Integration specificity of retrotransposons and retroviruses. Annu Rev Genet. 1990;24:491–518. doi: 10.1146/annurev.ge.24.120190.002423. [DOI] [PubMed] [Google Scholar]
  25. Vipond I. B., Baldwin G. S., Halford S. E. Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases. Biochemistry. 1995 Jan 17;34(2):697–704. doi: 10.1021/bi00002a037. [DOI] [PubMed] [Google Scholar]

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