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. 1986 Jul;6(7):2482–2489. doi: 10.1128/mcb.6.7.2482

Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

B J Andrews, M McLeod, J Broach, P D Sadowski
PMCID: PMC367802  PMID: 3537720

Abstract

The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.

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Selected References

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

  1. Abremski K., Hoess R. Phage P1 Cre-loxP site-specific recombination. Effects of DNA supercoiling on catenation and knotting of recombinant products. J Mol Biol. 1985 Jul 20;184(2):211–220. doi: 10.1016/0022-2836(85)90374-2. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Babineau D., Vetter D., Andrews B. J., Gronostajski R. M., Proteau G. A., Beatty L. G., Sadowski P. D. The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene. J Biol Chem. 1985 Oct 5;260(22):12313–12319. [PubMed] [Google Scholar]
  4. Bauer C. E., Hesse S. D., Gardner J. F., Gumport R. I. DNA interactions during bacteriophage lambda site-specific recombination. Cold Spring Harb Symp Quant Biol. 1984;49:699–705. doi: 10.1101/sqb.1984.049.01.079. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cox M. M. The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4223–4227. doi: 10.1073/pnas.80.14.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gronostajski R. M., Sadowski P. D. Determination of DNA sequences essential for FLP-mediated recombination by a novel method. J Biol Chem. 1985 Oct 5;260(22):12320–12327. [PubMed] [Google Scholar]
  8. Gronostajski R. M., Sadowski P. D. The FLP protein of the 2-micron plasmid of yeast. Inter- and intramolecular reactions. J Biol Chem. 1985 Oct 5;260(22):12328–12335. [PubMed] [Google Scholar]
  9. 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]
  10. Hartley J. L., Donelson J. E. Nucleotide sequence of the yeast plasmid. Nature. 1980 Aug 28;286(5776):860–865. doi: 10.1038/286860a0. [DOI] [PubMed] [Google Scholar]
  11. Jayaram M. Two-micrometer circle site-specific recombination: the minimal substrate and the possible role of flanking sequences. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5875–5879. doi: 10.1073/pnas.82.17.5875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnson R. C., Simon M. I. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer. Cell. 1985 Jul;41(3):781–791. doi: 10.1016/s0092-8674(85)80059-3. [DOI] [PubMed] [Google Scholar]
  13. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  14. McLeod M., Volkert F., Broach J. Components of the site-specific recombination system encoded by the yeast plasmid 2-micron circle. Cold Spring Harb Symp Quant Biol. 1984;49:779–787. doi: 10.1101/sqb.1984.049.01.088. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Mizuuchi K., Weisberg R., Enquist L., Mizuuchi M., Buraczynska M., Foeller C., Hsu P. L., Ross W., Landy A. Structure and function of the phage lambda att site: size, int-binding sites, and location of the crossover point. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):429–437. doi: 10.1101/sqb.1981.045.01.057. [DOI] [PubMed] [Google Scholar]
  17. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  18. Sadowski P. D., Lee D. D., Andrews B. J., Babineau D., Beatty L., Morse M. J., Proteau G., Vetter D. In vitro systems for genetic recombination of the DNAs of bacteriophage T7 and yeast 2-micron circle. Cold Spring Harb Symp Quant Biol. 1984;49:789–796. doi: 10.1101/sqb.1984.049.01.089. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Vetter D., Andrews B. J., Roberts-Beatty L., Sadowski P. D. Site-specific recombination of yeast 2-micron DNA in vitro. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7284–7288. doi: 10.1073/pnas.80.23.7284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  22. Weisberg R. A., Enquist L. W., Foeller C., Landy A. Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda. J Mol Biol. 1983 Oct 25;170(2):319–342. doi: 10.1016/s0022-2836(83)80151-x. [DOI] [PubMed] [Google Scholar]
  23. de Massy B., Studier F. W., Dorgai L., Appelbaum E., Weisberg R. A. Enzymes and sites of genetic recombination: studies with gene-3 endonuclease of phage T7 and with site-affinity mutants of phage lambda. Cold Spring Harb Symp Quant Biol. 1984;49:715–726. doi: 10.1101/sqb.1984.049.01.081. [DOI] [PubMed] [Google Scholar]

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