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Nucleic Acids Research, 1986, Vol. 14, No. 12 4787-4802
© 1986

Articles

The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro

Gerald Proteau, Deborah Sidenberg and Paul Sadowski

Department of Medical Genetics, University of Toronto Toronto, M5S 1A8, Canada

Received April 9, 1986. Revised May 21, 1986. Accepted May 21, 1986.

The 2–micron piiraid of tho yeast Saccharomyces cerevisiae codes for a site-specific reconbinase (‘FLP’) that efficiently catalyses recombination across the plasmid's two 599 bp repeats both in vivo and in vitro. We have used the partially purified FLP protein to define the minimal duplex DNA sequence required for intra- and intermolecular recombination in vitro. Previous DNase footprinting experiments had shown that FLP protected 50 bp of DNA around the recombination site. We made BAL31 deletions and synthetic FLP sites to show that the minimal length of the site that was able to reconbine with a wild-type site was 22 bp. The site consists of two 7 bp inverted repeats surrounding an 8 bp core region. We also showed that the deleted sites recombined with themselves and that one of three 13 bp repeated elements within the FLP target sequence was not necessary for efficient recombination in vitro. Mutants lacking this redundant 13 bp element required a lower amount of FLP recombinase to achieve naxinal yield of recombination than the wild type site. Finally, we discuss the structure of the FLP site in relation to the proposed function of FLP recombination in copy number amplification of the 2–micron plasmid in vivo.


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