Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Mar 1;91(5):1843–1847. doi: 10.1073/pnas.91.5.1843

Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae.

S P Moore 1, D J Garfinkel 1
PMCID: PMC43260  PMID: 8127892

Abstract

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands. To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast. The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal amino acid codons. Inclusion of a polyhistidine tag facilitates purification of recombinant IN by metal chelate chromatography. Recombinant Ty1 IN is active in an in vitro assay with short double-stranded oligonucleotide substrates and has biochemical properties similar to those observed with Ty1 virus-like particles. The full-length Ty1 IN produced in yeast should be useful for further biochemical, genetic, and structural analyses of Ty1 integration and for comparative analyses with retroviral IN proteins.

Full text

PDF
1847

Images in this article

1845Image
on p.1845
1845Image
on p.1845
1845Image
on p.1845
1846Image
on p.1846
1846Image
on p.1846

Selected References

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

  1. Adams S. E., Mellor J., Gull K., Sim R. B., Tuite M. F., Kingsman S. M., Kingsman A. J. The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins. Cell. 1987 Apr 10;49(1):111–119. doi: 10.1016/0092-8674(87)90761-6. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., Eichinger D., Castrillon D., Fink G. R. The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1. Mol Cell Biol. 1988 Apr;8(4):1432–1442. doi: 10.1128/mcb.8.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chapman K. B., Byström A. S., Boeke J. D. Initiator methionine tRNA is essential for Ty1 transposition in yeast. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3236–3240. doi: 10.1073/pnas.89.8.3236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Craigie R., Mizuuchi K., Bushman F. D., Engelman A. A rapid in vitro assay for HIV DNA integration. Nucleic Acids Res. 1991 May 25;19(10):2729–2734. doi: 10.1093/nar/19.10.2729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Curcio M. J., Garfinkel D. J. Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing. Mol Cell Biol. 1992 Jun;12(6):2813–2825. doi: 10.1128/mcb.12.6.2813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drelich M., Wilhelm R., Mous J. Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. Virology. 1992 Jun;188(2):459–468. doi: 10.1016/0042-6822(92)90499-f. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Eichinger D. J., Boeke J. D. The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition. Cell. 1988 Sep 23;54(7):955–966. doi: 10.1016/0092-8674(88)90110-9. [DOI] [PubMed] [Google Scholar]
  12. Garfinkel D. J., Boeke J. D., Fink G. R. Ty element transposition: reverse transcriptase and virus-like particles. Cell. 1985 Sep;42(2):507–517. doi: 10.1016/0092-8674(85)90108-4. [DOI] [PubMed] [Google Scholar]
  13. Garfinkel D. J., Hedge A. M., Youngren S. D., Copeland T. D. Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1. J Virol. 1991 Sep;65(9):4573–4581. doi: 10.1128/jvi.65.9.4573-4581.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garfinkel D. J., Mastrangelo M. F., Sanders N. J., Shafer B. K., Strathern J. N. Transposon tagging using Ty elements in yeast. Genetics. 1988 Sep;120(1):95–108. doi: 10.1093/genetics/120.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamilton R., Watanabe C. K., de Boer H. A. Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs. Nucleic Acids Res. 1987 Apr 24;15(8):3581–3593. doi: 10.1093/nar/15.8.3581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson A. W., Kolodner R. D. Strand exchange protein 1 from Saccharomyces cerevisiae. A novel multifunctional protein that contains DNA strand exchange and exonuclease activities. J Biol Chem. 1991 Jul 25;266(21):14046–14054. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Leavitt A. D., Rose R. B., Varmus H. E. Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae. J Virol. 1992 Apr;66(4):2359–2368. doi: 10.1128/jvi.66.4.2359-2368.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maroux S., Baratti J., Desnuelle P. Purification and specificity of porcine enterokinase. J Biol Chem. 1971 Aug 25;246(16):5031–5039. [PubMed] [Google Scholar]
  24. Mellor J., Malim M. H., Gull K., Tuite M. F., McCready S., Dibbayawan T., Kingsman S. M., Kingsman A. J. Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast. Nature. 1985 Dec 12;318(6046):583–586. doi: 10.1038/318583a0. [DOI] [PubMed] [Google Scholar]
  25. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):263–273. doi: 10.1101/sqb.1986.051.01.032. [DOI] [PubMed] [Google Scholar]
  26. Mumm S. R., Grandgenett D. P. Defining nucleic acid-binding properties of avian retrovirus integrase by deletion analysis. J Virol. 1991 Mar;65(3):1160–1167. doi: 10.1128/jvi.65.3.1160-1167.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Sherman P. A., Fyfe J. A. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119–5123. doi: 10.1073/pnas.87.13.5119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Terry R., Soltis D. A., Katzman M., Cobrinik D., Leis J., Skalka A. M. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. J Virol. 1988 Jul;62(7):2358–2365. doi: 10.1128/jvi.62.7.2358-2365.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vink C., Oude Groeneger A. M., Plasterk R. H. Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein. Nucleic Acids Res. 1993 Mar 25;21(6):1419–1425. doi: 10.1093/nar/21.6.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Woerner A. M., Klutch M., Levin J. G., Marcus-Sekura C. J. Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein. AIDS Res Hum Retroviruses. 1992 Feb;8(2):297–304. doi: 10.1089/aid.1992.8.297. [DOI] [PubMed] [Google Scholar]
  33. Woerner A. M., Marcus-Sekura C. J. Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis. Nucleic Acids Res. 1993 Jul 25;21(15):3507–3511. doi: 10.1093/nar/21.15.3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Youngren S. D., Boeke J. D., Sanders N. J., Garfinkel D. J. Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition. Mol Cell Biol. 1988 Apr;8(4):1421–1431. doi: 10.1128/mcb.8.4.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van Gent D. C., Elgersma Y., Bolk M. W., Vink C., Plasterk R. H. DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2. Nucleic Acids Res. 1991 Jul 25;19(14):3821–3827. doi: 10.1093/nar/19.14.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES