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. 1998 Mar 1;26(5):1329–1336. doi: 10.1093/nar/26.5.1329

Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein.

X D Zhu 1, P D Sadowski 1
PMCID: PMC147392  PMID: 9469845

Abstract

Flp is a member of the integrase family of site-specific recombinases. Flp is known to be a double-stranded (ds)DNA binding protein that binds sequence specifically to the 13 bp binding elements in the FRT site (Flprecognitiontarget). We subjected a random pool of oligonucleotides to the in vitro binding site selection method and have unexpectedly recovered a series of single-stranded oligonucleotides to which Flp binds with high affinity. These single-stranded oligonucleotides differ in sequence from the duplex FRT site. The minimal length of the oligonucleotides which is active is 29 nt. This single strand-specific DNA binding activity is located in the same C-terminal 32 kDa domain of Flp in which the site-specific dsDNA binding activity resides. Competition studies suggest that the apparent affinity of Flp for single-stranded oligonucleotide is somewhat less than for a complete duplex FRT site but greater than for a single duplex 13 bp binding element. We have also shown that Cre, another member of the integrase family of site-specific recombinases, also exhibits single-stranded DNA binding similar to that of Flp.

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Selected References

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

  1. Amin A. A., Beatty L. G., Sadowski P. D. Synaptic intermediates promoted by the FLP recombinase. J Mol Biol. 1990 Jul 5;214(1):55–72. doi: 10.1016/0022-2836(90)90146-D. [DOI] [PubMed] [Google Scholar]
  2. Andrews B. J., Beatty L. G., Sadowski P. D. Isolation of intermediates in the binding of the FLP recombinase of the yeast plasmid 2-micron circle to its target sequence. J Mol Biol. 1987 Jan 20;193(2):345–358. doi: 10.1016/0022-2836(87)90223-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Bergemann A. D., Johnson E. M. The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication. Mol Cell Biol. 1992 Mar;12(3):1257–1265. doi: 10.1128/mcb.12.3.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bergemann A. D., Ma Z. W., Johnson E. M. Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Mol Cell Biol. 1992 Dec;12(12):5673–5682. doi: 10.1128/mcb.12.12.5673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blackwell T. K., Weintraub H. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science. 1990 Nov 23;250(4984):1104–1110. doi: 10.1126/science.2174572. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Broach J. R., Guarascio V. R., Jayaram M. Recombination within the yeast plasmid 2mu circle is site-specific. Cell. 1982 May;29(1):227–234. doi: 10.1016/0092-8674(82)90107-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dixon J. E., Sadowski P. D. Resolution of immobile chi structures by the FLP recombinase of 2 microns plasmid. J Mol Biol. 1994 Oct 21;243(2):199–207. doi: 10.1006/jmbi.1994.1647. [DOI] [PubMed] [Google Scholar]
  12. Dixon J. E., Sadowski P. D. Resolution of synthetic chi structures by the FLP site-specific recombinase. J Mol Biol. 1993 Dec 5;234(3):522–533. doi: 10.1006/jmbi.1993.1608. [DOI] [PubMed] [Google Scholar]
  13. Evans B. R., Chen J. W., Parsons R. L., Bauer T. K., Teplow D. B., Jayaram M. Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination. J Biol Chem. 1990 Oct 25;265(30):18504–18510. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Jayaram M., Crain K. L., Parsons R. L., Harshey R. M. Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7902–7906. doi: 10.1073/pnas.85.21.7902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. McLeod M., Craft S., Broach J. R. Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Mol Cell Biol. 1986 Oct;6(10):3357–3367. doi: 10.1128/mcb.6.10.3357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Meyer-Leon L., Huang L. C., Umlauf S. W., Cox M. M., Inman R. B. Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination. Mol Cell Biol. 1988 Sep;8(9):3784–3796. doi: 10.1128/mcb.8.9.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Meyer-Leon L., Inman R. B., Cox M. M. Characterization of Holliday structures in FLP protein-promoted site-specific recombination. Mol Cell Biol. 1990 Jan;10(1):235–242. doi: 10.1128/mcb.10.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Meyer-Leon L., Senecoff J. F., Bruckner R. C., Cox M. M. Site-specific genetic recombination promoted by the FLP protein of the yeast 2-micron plasmid in vitro. Cold Spring Harb Symp Quant Biol. 1984;49:797–804. doi: 10.1101/sqb.1984.049.01.090. [DOI] [PubMed] [Google Scholar]
  23. Pan G., Sadowski P. D. Identification of the functional domains of the FLP recombinase. Separation of the nonspecific and specific DNA-binding, cleavage, and ligation domains. J Biol Chem. 1993 Oct 25;268(30):22546–22551. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Reynolds A. E., Murray A. W., Szostak J. W. Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Oct;7(10):3566–3573. doi: 10.1128/mcb.7.10.3566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. Senecoff J. F., Bruckner R. C., Cox M. M. The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7270–7274. doi: 10.1073/pnas.82.21.7270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shaikh A. C., Sadowski P. D. The Cre recombinase cleaves the lox site in trans. J Biol Chem. 1997 Feb 28;272(9):5695–5702. doi: 10.1074/jbc.272.9.5695. [DOI] [PubMed] [Google Scholar]
  33. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  34. Virta-Pearlman V., Morris D. K., Lundblad V. Est1 has the properties of a single-stranded telomere end-binding protein. Genes Dev. 1996 Dec 15;10(24):3094–3104. doi: 10.1101/gad.10.24.3094. [DOI] [PubMed] [Google Scholar]
  35. Volkert F. C., Broach J. R. Site-specific recombination promotes plasmid amplification in yeast. Cell. 1986 Aug 15;46(4):541–550. doi: 10.1016/0092-8674(86)90879-2. [DOI] [PubMed] [Google Scholar]
  36. Zhu X. D., Pan G., Luetke K., Sadowski P. D. Homology requirements for ligation and strand exchange by the FLP recombinase. J Biol Chem. 1995 May 12;270(19):11646–11653. doi: 10.1074/jbc.270.19.11646. [DOI] [PubMed] [Google Scholar]
  37. Zhu X. D., Sadowski P. D. Cleavage-dependent ligation by the FLP recombinase. Characterization of a mutant FLP protein with an alteration in a catalytic amino acid. J Biol Chem. 1995 Sep 29;270(39):23044–23054. doi: 10.1074/jbc.270.39.23044. [DOI] [PubMed] [Google Scholar]

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