Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Sep;177(17):5070–5077. doi: 10.1128/jb.177.17.5070-5077.1995

Mutagenesis of the IS1 transposase: importance of a His-Arg-Tyr triad for activity.

M C Serre 1, C Turlan 1, M Bortolin 1, M Chandler 1
PMCID: PMC177286  PMID: 7665486

Abstract

Inspection of the primary sequence of the IS1 transposase suggested that it carries residues which are characteristic of the active site of integrases of the bacteriophage lambda family (Int). In particular, these include a highly conserved triad: His-Arg-Tyr. The properties of mutants made at each of these positions were investigated in vivo. The results of several different assays confirm that each is important for transposase activity. Moreover, as in the case of members of the Int family, different mutations of the His residue exhibited different effects. In a particular, His-to-Leu mutation resulted in complete inactivation whereas the equivalent His-to-Gln mutation retained low but significant levels of activity.

Full Text

The Full Text of this article is available as a PDF (313.1 KB).

Selected References

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

  1. Abremski K. E., Hoess R. H. Evidence for a second conserved arginine residue in the integrase family of recombination proteins. Protein Eng. 1992 Jan;5(1):87–91. doi: 10.1093/protein/5.1.87. [DOI] [PubMed] [Google Scholar]
  2. Argos P., Landy A., Abremski K., Egan J. B., Haggard-Ljungquist E., Hoess R. H., Kahn M. L., Kalionis B., Narayana S. V., Pierson L. S., 3rd The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 1986 Feb;5(2):433–440. doi: 10.1002/j.1460-2075.1986.tb04229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker T. A., Luo L. Identification of residues in the Mu transposase essential for catalysis. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6654–6658. doi: 10.1073/pnas.91.14.6654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chan P. T., Lebowitz J. Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1. Nucleic Acids Res. 1982 Nov 25;10(22):7295–7311. doi: 10.1093/nar/10.22.7295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Churchward G., Belin D., Nagamine Y. A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984 Nov;31(1-3):165–171. doi: 10.1016/0378-1119(84)90207-5. [DOI] [PubMed] [Google Scholar]
  7. Doak T. G., Doerder F. P., Jahn C. L., Herrick G. A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):942–946. doi: 10.1073/pnas.91.3.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elespuru R. K., Yarmolinsky M. B. A colorimetric assay of lysogenic induction designed for screening potential carcinogenic and carcinostatic agents. Environ Mutagen. 1979;1(1):65–78. doi: 10.1002/em.2860010113. [DOI] [PubMed] [Google Scholar]
  9. Escoubas J. M., Lane D., Chandler M. Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition? J Bacteriol. 1994 Sep;176(18):5864–5867. doi: 10.1128/jb.176.18.5864-5867.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Han Y. W., Gumport R. I., Gardner J. F. Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site. EMBO J. 1993 Dec;12(12):4577–4584. doi: 10.1002/j.1460-2075.1993.tb06146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Han Y. W., Gumport R. I., Gardner J. F. Mapping the functional domains of bacteriophage lambda integrase protein. J Mol Biol. 1994 Jan 21;235(3):908–925. doi: 10.1006/jmbi.1994.1048. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Huisman O., Kleckner N. A new generalizable test for detection of mutations affecting Tn10 transposition. Genetics. 1987 Jun;116(2):185–189. doi: 10.1093/genetics/116.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim K., Namgoong S. Y., Jayaram M., Harshey R. M. Step-arrest mutants of phage Mu transposase. Implications in DNA-protein assembly, Mu end cleavage, and strand transfer. J Biol Chem. 1995 Jan 20;270(3):1472–1479. doi: 10.1074/jbc.270.3.1472. [DOI] [PubMed] [Google Scholar]
  17. Krebs M. P., Reznikoff W. S. Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant. Gene. 1988 Mar 31;63(2):277–285. doi: 10.1016/0378-1119(88)90531-8. [DOI] [PubMed] [Google Scholar]
  18. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  19. Lane D., Cavaillé J., Chandler M. Induction of the SOS response by IS1 transposase. J Mol Biol. 1994 Sep 30;242(4):339–350. doi: 10.1006/jmbi.1994.1585. [DOI] [PubMed] [Google Scholar]
  20. Lee J., Serre M. C., Yang S. H., Whang I., Araki H., Oshima Y., Jayaram M. Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family. J Mol Biol. 1992 Dec 20;228(4):1091–1103. doi: 10.1016/0022-2836(92)90317-d. [DOI] [PubMed] [Google Scholar]
  21. Leung P. C., Harshey R. M. Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains. J Mol Biol. 1991 May 20;219(2):189–199. doi: 10.1016/0022-2836(91)90561-j. [DOI] [PubMed] [Google Scholar]
  22. Lüthi K., Moser M., Ryser J., Weber H. Evidence for a role of translational frameshifting in the expression of transposition activity of the bacterial insertion element IS1. Gene. 1990 Mar 30;88(1):15–20. doi: 10.1016/0378-1119(90)90054-u. [DOI] [PubMed] [Google Scholar]
  23. Machida C., Machida Y., Ohtsubo E. Both inverted repeat sequences located at the ends of IS1 provide promoter functions. J Mol Biol. 1984 Aug 5;177(2):247–267. doi: 10.1016/0022-2836(84)90455-8. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Mendiola M. V., de la Cruz F. IS91 transposase is related to the rolling-circle-type replication proteins of the pUB110 family of plasmids. Nucleic Acids Res. 1992 Jul 11;20(13):3521–3521. doi: 10.1093/nar/20.13.3521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  27. Mizuuchi K., Adzuma K. Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism. Cell. 1991 Jul 12;66(1):129–140. doi: 10.1016/0092-8674(91)90145-o. [DOI] [PubMed] [Google Scholar]
  28. Mizuuchi K. Transpositional recombination: mechanistic insights from studies of mu and other elements. Annu Rev Biochem. 1992;61:1011–1051. doi: 10.1146/annurev.bi.61.070192.005051. [DOI] [PubMed] [Google Scholar]
  29. Parsons R. L., Evans B. R., Zheng L., Jayaram M. Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis. J Biol Chem. 1990 Mar 15;265(8):4527–4533. [PubMed] [Google Scholar]
  30. Parsons R. L., Prasad P. V., Harshey R. M., Jayaram M. Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination. Mol Cell Biol. 1988 Aug;8(8):3303–3310. doi: 10.1128/mcb.8.8.3303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  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. Reif H. J., Saedler H. IS1 is involved in deletion formation in the gal region of E. coli K12. Mol Gen Genet. 1975;137(1):17–28. doi: 10.1007/BF00332538. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Rowland S. J., Dyke K. G. Tn552, a novel transposable element from Staphylococcus aureus. Mol Microbiol. 1990 Jun;4(6):961–975. doi: 10.1111/j.1365-2958.1990.tb00669.x. [DOI] [PubMed] [Google Scholar]
  36. Rådström P., Sköld O., Swedberg G., Flensburg J., Roy P. H., Sundström L. Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol. 1994 Jun;176(11):3257–3268. doi: 10.1128/jb.176.11.3257-3268.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Topal M. D., Conrad M. Changing endonuclease EcoRII Tyr308 to Phe abolishes cleavage but not recognition: possible homology with the Int-family of recombinases. Nucleic Acids Res. 1993 Jun 11;21(11):2599–2603. doi: 10.1093/nar/21.11.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wierzbicki A., Kendall M., Abremski K., Hoess R. A mutational analysis of the bacteriophage P1 recombinase Cre. J Mol Biol. 1987 Jun 20;195(4):785–794. doi: 10.1016/0022-2836(87)90484-0. [DOI] [PubMed] [Google Scholar]
  41. Zerbib D., Gamas P., Chandler M., Prentki P., Bass S., Galas D. Specificity of insertion of IS1. J Mol Biol. 1985 Oct 5;185(3):517–524. doi: 10.1016/0022-2836(85)90068-3. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES