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

B. J. Andrews, M. McLeod, James Broach, P. D. Sadowski

Research output: Contribution to journalArticle

26 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2482-2489
Number of pages8
JournalMolecular and cellular biology
Volume6
Issue number7
DOIs
StatePublished - Jan 1 1986

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Genetic Recombination
Saccharomyces cerevisiae
Plasmids
Base Pairing
Mutation
Proteins
Deoxyribonucleases
Proteolysis
FLP recombinase
Carrier Proteins
DNA
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

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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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Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences. / Andrews, B. J.; McLeod, M.; Broach, James; Sadowski, P. D.

In: Molecular and cellular biology, Vol. 6, No. 7, 01.01.1986, p. 2482-2489.

Research output: Contribution to journalArticle

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