Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Nov 1;25(21):4240–4249. doi: 10.1093/nar/25.21.4240

Asymmetry in Flp-mediated cleavage.

K H Luetke 1, B P Zhao 1, P D Sadowski 1
PMCID: PMC147029  PMID: 9336453

Abstract

Flp is a member of the integrase family of site-specific recombinases. Members of the integrase family mediate DNA strand cleavage via a transesterification reaction involving an active site tyrosine residue. The first step of the reaction results in covalent linkage of the protein to the 3'-phosphoryl DNA terminus, leaving a 5'-hydroxyl group at the site of the nick. We have used Flp recognition target (FRT) sites containing a 5'-bridging phosphorothioate linkage at the site of Flp cleavage to accumulate intermediates in which Flp is covalently bound at a cleavage site. We have probed these intermediates with dimethylsulfate using methylation protection and find that Flp-mediated cleavage is associated with protection of two adenine residues that are opposite the sites of cleavage and covalent attachment by Flp. Methylation interference studies showed that cleavage and covalent attachment are also accompanied by differences in the contacts of Flp with each of the two cleavage sites and with the surrounding symmetry elements. Therefore, we provide evidence that Flp-mediated cleavage and covalent attachment result in changes to the conformation of the Flp-FRT complex. These changes may be required for Flp-mediated strand exchange activity.

Full Text

The Full Text of this article is available as a PDF (296.3 KB).

Selected References

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

  1. Andrews B. J., Proteau G. A., Beatty L. G., Sadowski P. D. The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences. Cell. 1985 Apr;40(4):795–803. doi: 10.1016/0092-8674(85)90339-3. [DOI] [PubMed] [Google Scholar]
  2. Bailly C., Waring M. J., Travers A. A. Effects of base substitutions on the binding of a DNA-bending protein. J Mol Biol. 1995 Oct 13;253(1):1–7. doi: 10.1006/jmbi.1995.0530. [DOI] [PubMed] [Google Scholar]
  3. Beatty L. G., Sadowski P. D. The mechanism of loading of the FLP recombinase onto its DNA target sequence. J Mol Biol. 1988 Nov 20;204(2):283–294. doi: 10.1016/0022-2836(88)90576-1. [DOI] [PubMed] [Google Scholar]
  4. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Bruckner R. C., Cox M. M. Specific contacts between the FLP protein of the yeast 2-micron plasmid and its recombination site. J Biol Chem. 1986 Sep 5;261(25):11798–11807. [PubMed] [Google Scholar]
  6. Burgin A. B., Jr, Huizenga B. N., Nash H. A. A novel suicide substrate for DNA topoisomerases and site-specific recombinases. Nucleic Acids Res. 1995 Aug 11;23(15):2973–2979. doi: 10.1093/nar/23.15.2973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen J. W., Lee J., Jayaram M. DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands. Cell. 1992 May 15;69(4):647–658. doi: 10.1016/0092-8674(92)90228-5. [DOI] [PubMed] [Google Scholar]
  8. Friesen H., Sadowski P. D. Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation. J Mol Biol. 1992 May 20;225(2):313–326. doi: 10.1016/0022-2836(92)90924-9. [DOI] [PubMed] [Google Scholar]
  9. Futcher A. B. Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae. J Theor Biol. 1986 Mar 21;119(2):197–204. doi: 10.1016/s0022-5193(86)80074-1. [DOI] [PubMed] [Google Scholar]
  10. Gronostajski R. M., Sadowski P. D. The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage. Mol Cell Biol. 1985 Nov;5(11):3274–3279. doi: 10.1128/mcb.5.11.3274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grove A., Galeone A., Mayol L., Geiduschek E. P. Localized DNA flexibility contributes to target site selection by DNA-bending proteins. J Mol Biol. 1996 Jul 12;260(2):120–125. doi: 10.1006/jmbi.1996.0386. [DOI] [PubMed] [Google Scholar]
  12. Grove A., Galeone A., Mayol L., Geiduschek E. P. On the connection between inherent DNA flexure and preferred binding of hydroxymethyluracil-containing DNA by the type II DNA-binding protein TF1. J Mol Biol. 1996 Jul 12;260(2):196–206. doi: 10.1006/jmbi.1996.0392. [DOI] [PubMed] [Google Scholar]
  13. Guo F., Gopaul D. N., van Duyne G. D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature. 1997 Sep 4;389(6646):40–46. doi: 10.1038/37925. [DOI] [PubMed] [Google Scholar]
  14. Hoess R., Wierzbicki A., Abremski K. Isolation and characterization of intermediates in site-specific recombination. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6840–6844. doi: 10.1073/pnas.84.19.6840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jayaram M. Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms. Trends Biochem Sci. 1994 Feb;19(2):78–82. doi: 10.1016/0968-0004(94)90039-6. [DOI] [PubMed] [Google Scholar]
  16. Kitts P. A., Nash H. A. An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA. Nucleic Acids Res. 1988 Jul 25;16(14B):6839–6856. doi: 10.1093/nar/16.14.6839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kitts P. A., Nash H. A. Bacteriophage lambda site-specific recombination proceeds with a defined order of strand exchanges. J Mol Biol. 1988 Nov 5;204(1):95–107. doi: 10.1016/0022-2836(88)90602-x. [DOI] [PubMed] [Google Scholar]
  18. Kitts P. A., Nash H. A. Homology-dependent interactions in phage lambda site-specific recombination. Nature. 1987 Sep 24;329(6137):346–348. doi: 10.1038/329346a0. [DOI] [PubMed] [Google Scholar]
  19. Landy A. Mechanistic and structural complexity in the site-specific recombination pathways of Int and FLP. Curr Opin Genet Dev. 1993 Oct;3(5):699–707. doi: 10.1016/s0959-437x(05)80086-3. [DOI] [PubMed] [Google Scholar]
  20. Lee J., Jayaram M. Functional roles of individual recombinase monomers in strand breakage and strand union during site-specific DNA recombination. J Biol Chem. 1995 Sep 29;270(39):23203–23211. doi: 10.1074/jbc.270.39.23203. [DOI] [PubMed] [Google Scholar]
  21. Lee J., Jayaram M. Mechanism of site-specific recombination. Logic of assembling recombinase catalytic site from fractional active sites. J Biol Chem. 1993 Aug 15;268(23):17564–17570. [PubMed] [Google Scholar]
  22. Lee J., Whang I., Lee J., Jayaram M. Directed protein replacement in recombination full sites reveals trans-horizontal DNA cleavage by Flp recombinase. EMBO J. 1994 Nov 15;13(22):5346–5354. doi: 10.1002/j.1460-2075.1994.tb06869.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luetke K. H., Sadowski P. D. The role of DNA bending in Flp-mediated site-specific recombination. J Mol Biol. 1995 Aug 25;251(4):493–506. doi: 10.1006/jmbi.1995.0451. [DOI] [PubMed] [Google Scholar]
  24. Mag M., Lüking S., Engels J. W. Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5'-phosphorothioate linkage. Nucleic Acids Res. 1991 Apr 11;19(7):1437–1441. doi: 10.1093/nar/19.7.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nunes-Düby S. E., Matsumoto L., Landy A. Site-specific recombination intermediates trapped with suicide substrates. Cell. 1987 Aug 28;50(5):779–788. doi: 10.1016/0092-8674(87)90336-9. [DOI] [PubMed] [Google Scholar]
  26. Pan G., Luetke K., Juby C. D., Brousseau R., Sadowski P. Ligation of synthetic activated DNA substrates by site-specific recombinases and topoisomerase I. J Biol Chem. 1993 Feb 15;268(5):3683–3689. [PubMed] [Google Scholar]
  27. Pan H., Clary D., Sadowski P. D. Identification of the DNA-binding domain of the FLP recombinase. J Biol Chem. 1991 Jun 15;266(17):11347–11354. [PubMed] [Google Scholar]
  28. Panigrahi G. B., Beatty L. G., Sadowski P. D. The FLP protein contacts both major and minor grooves of its recognition target sequence. Nucleic Acids Res. 1992 Nov 25;20(22):5927–5935. doi: 10.1093/nar/20.22.5927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Proteau G., Sidenberg D., Sadowski P. The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro. Nucleic Acids Res. 1986 Jun 25;14(12):4787–4802. doi: 10.1093/nar/14.12.4787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qian X. H., Inman R. B., Cox M. M. Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination. J Biol Chem. 1990 Dec 15;265(35):21779–21788. [PubMed] [Google Scholar]
  32. Sadowski P. D. Site-specific genetic recombination: hops, flips, and flops. FASEB J. 1993 Jun;7(9):760–767. doi: 10.1096/fasebj.7.9.8392474. [DOI] [PubMed] [Google Scholar]
  33. Sadowski P. D. The Flp recombinase of the 2-microns plasmid of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol. 1995;51:53–91. [PubMed] [Google Scholar]
  34. Schwartz C. J., Sadowski P. D. FLP protein of 2 mu circle plasmid of yeast induces multiple bends in the FLP recognition target site. J Mol Biol. 1990 Nov 20;216(2):289–298. doi: 10.1016/s0022-2836(05)80320-1. [DOI] [PubMed] [Google Scholar]
  35. Schwartz C. J., Sadowski P. D. FLP recombinase of the 2 microns circle plasmid of Saccharomyces cerevisiae bends its DNA target. Isolation of FLP mutants defective in DNA bending. J Mol Biol. 1989 Feb 20;205(4):647–658. doi: 10.1016/0022-2836(89)90310-0. [DOI] [PubMed] [Google Scholar]
  36. Sekiguchi J., Shuman S. Covalent DNA binding by vaccinia topoisomerase results in unpairing of the thymine base 5' of the scissile bond. J Biol Chem. 1996 Aug 9;271(32):19436–19442. doi: 10.1074/jbc.271.32.19436. [DOI] [PubMed] [Google Scholar]
  37. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stark W. M., Boocock M. R., Sherratt D. J. Catalysis by site-specific recombinases. Trends Genet. 1992 Dec;8(12):432–439. [PubMed] [Google Scholar]
  39. Voziyanov Y., Lee J., Whang I., Lee J., Jayaram M. Analyses of the first chemical step in Flp site-specific recombination: Synapsis may not be a pre-requisite for strand cleavage. J Mol Biol. 1996 Mar 8;256(4):720–735. doi: 10.1006/jmbi.1996.0120. [DOI] [PubMed] [Google Scholar]
  40. Wissmann A., Hillen W. DNA contacts probed by modification protection and interference studies. Methods Enzymol. 1991;208:365–379. doi: 10.1016/0076-6879(91)08020-i. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES