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

The 2-micron plasmid of the yeast Saccharomyces cerevisiae codes for a site-specific recombinase ('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 recombine 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 maximal 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.     

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

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.     

DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of the FLP protein and two inverted recombination sites on the plasmid. The minimal fully functional substrate for in-vitro recombination in this system consists of two FLP protein binding sites separated by an eight base-pair spacer sequence. We have used site-directed mutagenesis to generate every possible mutation (36 in all) within 11 base-pairs of one FLP protein binding site and the base-pair immediately flanking it. The base-pairs within the binding site can be separated into three classes on the basis of these results. Thirty of the 36 sequence changes, including all three at seven different positions (class I) produce a negligible or modest effect on FLP protein-promoted recombination. In particular, most transition mutations are well-tolerated in this system. In only one case do all three possible mutations produce large effects (class II). At three positions, clustered near the site at which DNA is cleaved by FLP protein, one of the two possible transversions produces a large effect on recombination, while the other two changes produce modest effects (class III). For seven mutants for which FLP protein binding was measured, a direct correlation between decreases in recombination activity and in binding was observed. Positive effects on the reaction potential of mutant sites are observed when the other FLP binding site in a single recombination site is unaltered or when the second recombination site in a reaction is wild-type. This suggests a functional interaction between FLP binding sites both in cis and in trans. When two mutant recombination sites (each with 1 altered FLP binding site) are recombined, the relative orientation of the mutations (parallel or antiparallel) has no effect on the result. These results provide an extensive substrate catalog to complement future studies in this system.     

Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination

When the FLP recombination target (FRT) is cut in half so that only one FLP protein-binding site is present, FLP protein forms a complex in which two such sites are linked head to head. Although held together exclusively by noncovalent interactions, this complex survives electrophoresis in an agarose gel and exhibits a half-life that can be measured in hours. Characterization of this complex indicates that a very stable, asymmetric dimeric complex of FLP protein monomers bound to the FRT is a likely early intermediate in FLP-mediated site-specific recombination. The apparent asymmetry is a property of the protein components of the complex. Even though the DNA components form a perfect palindrome, only one of the two possible DNA cleavage steps takes place in the course of complex formation. Formation of this complex does not occur with half-FRT site DNA substrates that preclude head to head monomer contact or when a FLP mutant protein is used that binds the FRT site but cannot cleave it. Trimeric and tetrameric complexes are also observed, the latter at very low frequency. These results are discussed in terms of an expanded model for early events in FLP-mediated site-specific recombination.     

Recombination within the yeast plasmid 2mu circle is site-specific

The multicopy yeast plasmid, 2mu circle, encodes a specialized recombination system. It contains two regions, each 599 bp in length, that are precise inverted repeats of each other and between which recombination occurs readily. In addition, this recombination requires the product of a 2mu circle gene, designated FLP. By examining the products of FLP-mediated recombination of plasmids containing single insertions within one of the repeated regions, we show that this recombination occurs only at a specific site within the repeat. This result was confirmed from analysis of the ability of plasmids containing various deletions within one of the repeated regions to serve as substrates for FLP-mediated recombination. These experiments limit the recombination site to a sequence of less than 65 bp. In addition, by mutational analysis of the recombination potential of a hybrid plasmid containing the entire 2mu circle genome, we have shown that FLP is only the 2mu circle gene necessary for this site-specific recombination. Finally, we describe a sensitive assay for recombination between the repeated sequences of 2mu circle; using it, we demonstrate that even in the absence of FLP gene product, recombination between the repeats occurs at a low but detectable level during meiosis.     

FLP recombinase of the 2 microns circle plasmid of Saccharomyces cerevisiae bends its DNA target. Isolation of FLP mutants defective in DNA bending

The FLP recombinase is encoded by the yeast plasmid 2 microns circle and catalyses a site-specific recombination reaction that results in inversion of a segment of the 2 micron plasmid. We describe a method for the isolation of inactivating mutations in the FLP gene. The analysis of the recombination and binding activity of defective FLP proteins in vitro resulted in the identification of two classes of mutations: those that completely abolish FLP function by interfering with DNA binding and others that block recombination after the binding step. We have shown that FLP-mediated recombination is accompanied by bending of the DNA target and that mutations in the FLP recombinase that block bending also eliminate recombination.     

Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation.

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.     

Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of plasmid-encoded FLP protein and two recombination sites on the plasmid. The recombination site possesses a specific orientation, which is determined by an asymmetric 8-base pair spacer sequence separating two 13-base pair inverted repeats. The outcome or directionality of site-specific recombination is defined by the alignment of two sites in the same orientation during the reaction. Sites containing point mutations or 1-base pair insertions or deletions within the spacer generally undergo recombination with unaltered sites at reduced levels. In contrast, recombination between the two identical mutant sites (where homology is restored) proceeds efficiently in all cases. Sites containing spacer sequences of 10 base pairs or more are nonfunctional under all conditions. A recombination site in which 5 base pairs are changed to yield an entirely symmetrical spacer sequence again recombines efficiently, but only with an identical site. This reaction, in addition, produces a variety of new products which can only result from random alignment of the two sites undergoing recombination, i.e. the reaction no longer exhibits directionality. These and other results demonstrate that both the efficiency and directionality of site-specific recombination is dependent upon homology between spacer sequences of the two recombining sites. This further implies that critical DNA-DNA interactions between the spacer region of the two sites involved in the reaction occur at some stage during site-specific recombination in this system. The specific spacer sequence itself appears to be unimportant as long as homology is maintained; thus, these sequences are probably not involved in recognition by FLP protein.     

Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.

Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies. We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies. We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites. Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.     

Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.

The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid. In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo. We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it. This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski, Cell 40:795-803, 1985; J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA 82:7270-7274, 1985). Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines. However, mutations outside the core are not similarly suppressed. We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate. Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency. The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.     

The FLP protein of the 2-micron plasmid of yeast. Inter- and intramolecular reactions

We have studied the mechanism of reaction of the FLP protein of the yeast 2-micron plasmid on linear substrates. The products of the reaction are dependent upon the concentration of FLP protein. At low concentrations of FLP, products resulting from intramolecular recombination between two FLP target sites accumulate. At higher concentrations of FLP, intermolecular recombination results in the accumulation of products which are larger than the starting substrate. At higher concentrations still, FLP-promoted recombination is inhibited. Potassium chloride (0.15 M) inhibits the intermolecular reaction and also prevents the inhibition of FLP-mediated recombination caused by high concentrations of FLP protein. We present a model that explains these findings.     

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.     

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.     

FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes.

The ability to place a series of gene constructs at a specific site in the genome opens new possibilities for the experimental examination of gene expression and chromosomal position effects. We report that the FLP- FRT site-specific recombination system of the yeast 2mu plasmid can be used to integrate DNA at a chromosomal FRT target site in Drosophila. The technique we used was to first integrate an FRT- flanked gene by standard P element-mediated transformation. FLP was then used to excise the FRT- flanked donor DNA and screen for FLP-mediated re-integration at an FRT target at a different chromosome location. Such events were recovered from up to 5% of the crosses used to screen for mobilization and are easily detectable by altered linkage of a white reporter gene or by the generation of a white + gene upon integration.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

FLP protein of 2 mu circle plasmid of yeast induces multiple bends in the FLP recognition target site

The FLP recombinase of the 2 mu plasmid of Saccharomyces cerevisiae binds to a target containing three 13 base-pair symmetry elements called a, b and c. The symmetry elements b and c are in direct orientation while the a element is in inverted orientation with respect to b and c on the opposite side of an eight base-pair core region. Each symmetry element acts as a binding site for the FLP protein. The FLP protein can form three different complexes with the FLP recognition target (FRT site) according to the number of elements within the site that are occupied by the FLP protein. Binding of FLP to the FRT site induces DNA bending. We have measured the angles of bends caused by the binding of the FLP protein to full and partial FRT sites. We find that FLP induces three types of bend in the FRT-containing DNA. The type I bend is approximately 60 degrees and results from a molecule of FLP bound to one symmetry element. The type II bend is greater than 144 degrees and results from FLP molecules bound to symmetry elements a and b. The type III bend is approximately 65 degrees and results from FLP proteins bound to symmetry elements b and c. Certain FLP proteins that are defective in recombination can generate the type I and type III bends but are impaired in their ability to induce the type II bend. We discuss the role of bending in FLP-mediated recombination.     

Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.     

Somatic Reversion of Chromosomal Position Effects in Drosophila Melanogaster

A transgene was inserted at several different chromosomal sites in Drosophila melanogaster, where its expression was subject to genomic position effects. Quantitative position effects and variegated and constant patterned position effects were observed. We investigated the status of the affected gene in the somatic cells where it normally functions. The FLP site-specific recombinase was used to remove the gene from the chromosome and its expression was then evaluated. We show that the FLP recombinase functions in cells that have finished their developmental program of mitoses. When FLP acts on directly repeated copies of its target site (FRT), the DNA flanked by those FRTs is excised from the chromosome as a closed circle. The extrachromosomal circle is maintained in nondividing cells, and a gene located on such a circle can be expressed. We then demonstrate that a gene subject to either variegated or constant position effect can be relieved of that effect by excision of the gene from the chromosome in cells where it would otherwise be inactive. We also observed a strong inhibition of FLP-mediated recombination for target sites located near centric heterochromatin.