FLP Recognition Target Sites Research Articles

The Flp recombinase cleaves Holliday junctions in trans.

The Flp site-specific recombinase is encoded by the 2 micrometers plasmid Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday-like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges. Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3' phosphoryl group at the cleavage site. It has previously been shown that Flp efficiently resolves synthetic, Holliday-like (chi) structures to linear products. In this paper, we examined whether resolution of chi structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked chi structures to show that Flp resolves chi structures by trans cleavage. We propose a model for Flp-mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.

Cleavage-dependent Ligation by the FLP Recombinase: CHARACTERIZATION OF A MUTANT FLP PROTEIN WITH AN ALTERATION IN A CATALYTIC AMINO ACID

The FLP recombinase of the 2 microM plasmid of Saccharomyces cerevisiae belongs to the integrase family of recombinases whose members have in common four absolutely conserved residues (Arg-191, His-305, Arg-308, and Tyr-343). We have studied the mutant protein FLP R308K in which the arginine residue at position 308 has been replaced by lysine. Although FLP R308K was previously reported to be defective in ligation of certain substrates (Pan, G., Luetke, K., and Sadowski, P.D., Mol. Cell. Biol. 13, 3167-3175, 1993b), we show in this work that the protein is able to ligate those substrates that it can cleave (cleavage-dependent ligation activity). FLP R308K is defective in in vitro recombination and in strand exchange. It is able to carry out strand exchange at one of the two cleavage sites of the FLP recognition target site (FRT site), but is defective in strand exchange at the other cleavage site. These results are consistent with a model in which wild-type FLP initiates recombination only at one of the two cleavage sites. FLP R308K may be defective in the initiation of recombination.

The Role of DNA Bending in Flp-mediated Site-specific Recombination

The Flp recombinase of the 2 μm plasmid of Saccharomyces cerevisiaebinds to a recognition target site, induces DNA bending and catalyses DNA cleavage and strand exchange to bring about recombination. The minimal Flp recognition target site contains two Flp binding sequences flanking an 8 bp core region; binding of Flp results in the formation of two Flp:DNA complexes (complexes I and II). Binding of a Flp monomer to a single symmetry element generates a DNA bend of about 60 ° (a type I bend), whereas binding of two Flp monomers to the FRT site generates a DNA bend of>144 ° (a type II bend). We have used circular permutation analysis to locate the centre of the type I and type II DNA bends induced by Flp, and the Flp peptides P27 (27 kDa; amino acid residues 124 to 346) and P32 (32 kDa; amino acid residues 124 to 423). The location of the centre of the type I bend depends upon whether the substrate contains one or two Flp binding elements. When the substrate contains one symmetry element, the centre of the type I bend induced by Flp is located at the core-distal end of the belement. However, it is located at the core-proximal end of the belement when the substrate contains two Flp-binding elements. The P27 and P32 peptides, which lack the NH 2-terminal 13 kDa region of Flp, do not show this behavior. We deduce that the 13 kDa region of Flp is critical for the positioning of the type I bend centre on a minimal Flp recognition site. We propose a model in which a single molecule of Flp interactions with two symmetry elements to account for these results. The centre of the type II bend induced by Flp is in the middle of the core region. We used ligation-defective Flp proteins to determine the location of the type II bend centres in complexes where either the top or bottom strand was cleaved. The bend centres of such complexes depend upon which strand is cleaved. We propose a model which associates the position of Flp-induced type II bends with a defined order of strand exchanges in the recombination reaction.

Homology Requirements for Ligation and Strand Exchange by the FLP Recombinase

The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae belongs to the integrase family whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. Ligation takes place when the 5'-OH generated during the cleavage step attacks the phosphotyrosine bond and reforms a phosphodiester bond. When the incoming 5'-OH is from the partner duplex, strand exchange occurs. The FLP recognition target (FRT) contains two inverted 13-base pair (bp) FLP binding sequences that surround an 8-bp core region. It has been shown that heterology in the core regions of the recombinase FLP recognition target sites can dramatically impair recombination. Therefore, it was of interest to study the homology requirements of the core sequence for FLP-mediated ligation. Using nicked duplex substrates containing mismatches in the core sequence, we have demonstrated that the FLP ligation reaction can tolerate mismatches at all positions in the 8-bp core except the position immediately adjacent to the cleavage site. Using half-FRT substrates that contain a single-stranded core sequence, we showed that 4 base pairs adjacent to the cleavage site in the core are required for FLP to execute ligation with a single-stranded oligonucleotide. FLP is also able to ligate the protruding single strand on a half-FRT site to the opposite strand to form a hairpin. We have studied the effect of the base composition of the protruding 8-nucleotide single strand upon the efficiency of hairpin ligation. These studies revealed the importance of intrastrand complementarity in the formation of hairpin by FLP. Hence we conclude that the homology in the position adjacent to the cleavage site is most important, and the degree of the homology required is dependent on the nature of the ligation assay.

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.

Resolution of Immobile χ Structures by the FLP Recombinase of 2 μm Plasmid

FLP is a conservative site-specific recombinase that is encoded by the 2 μm plasmid of the yeast, Saccharomyces cerevisiae. FLP is member of the integrase family of recombinases that mediate the recombination reaction through a Holliday intermediate. The FLP recognition target (FRT) sites lie within two 599 bp inverted repeats of the 2 μm plasmid. The minimal target contains two inverted FLP binding sites (13 bp) that surround an 8 bp core region. FLP nicks the top and the bottom strands of the FRT site at the margins of the core and these nicks are thought to be the sites of strand exchange. Hence, recombination generates heteroduplex DNA in the core region. It is known that heterology between the core regions of two FRT sites inhibits their ability to engage in recombination. It is possible that two homologous cores arc required to allow the junction of the Holliday intermediate to branch migrate through the core during resolution. If so, an immobile Holliday junction point should inhibit the recombination activity of FLP in the same manner as a heterology between the cores of two double-stranded FRT sites. In order to test this prediction, we generated synthetic Holliday structures specific for FLP that had the junction immobilised at representative points within the FRT core. We used either sequence heterologies or nicked strands in order to immobilise the junction. We found that immobilisation of a Holliday junction within the core region did not inhibit resolution of the Holliday structure by FLP. Hence, homology is not required for the resolution of the Holliday intermediate but rather, for an earlier step in the reaction.

Interaction of the NH2- and COOH-terminal domains of the FLP recombinase with the FLP recognition target sequence.

The FLP protein that is encoded by the 2-microns plasmid of yeast Saccharomyces cerevisiae is a 45-kDa site-specific recombinase that belongs to the Int family of recombination proteins. FLP catalyzes a recombination event within the plasmid by binding specifically to each of three 13-base pair (bp) symmetry elements of the FLP recognition target (FRT). We have shown previously that partial proteolysis of the FLP protein by proteinase K resulted in a COOH-terminal fragment of size 32 kDa (P32) and an NH2-terminal fragment of 13 kDa (P13). In this study we have used footprinting with dimethyl sulfate to show that P32 binds specifically to the outer 9 bp of the 13 bp symmetry element. Binding of P13 alone to the FRT site was not detectable in this assay. However, when P13 and P32 were incubated together with the FRT site, protection of the remaining 4-bp region of the symmetry element was observed. To confirm these results we used bromodeoxyuridine (BrdU)-dependent UV cross-linking. P32 became cross-linked to the substrate that contained BrdU substitutions in the outer 9 bp of a 13-bp symmetry element, but not to one with the BrdU substitutions in the inner 4 bp. Reciprocally P13 cross-linked to the latter substrate but not the former. Cross-linking was both BrdU and ultraviolet light-dependent. This study indicates that the COOH-terminal domain (P32) of FLP recognizes the outer 9 bp of the 13-bp symmetry element, whereas its NH2-terminal domain (P13) is needed for protection of the inner 4 bp of each symmetry element.

Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci.

Using the FLP/FRT system for site-specific recombination and the wild-type recognition site (FRT) in conjunction with certain mutant FRT sites, it becomes possible to provoke, with high yield, a double-reciprocal crossover event in cultured mammalian cells. It is demonstrated that this technology enables a targeting of expression cassettes to appropriate chromosomal reference sites in the recipient cell to improve the concepts of reverse genetics. The design of mutant FRT sites promoting such a process will be delineated. Our results show that the five spacer mutations tested are functional as the wild type but differ in the extent of their cross-recombination, which has to be minimized for their simultaneous usage.

The FLP protein contacts both major and minor grooves of its recognition target sequence.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.

Identification of the DNA-binding domain of the FLP recombinase

We have subjected the FLP protein of the 2-micron plasmid to partial proteolysis by proteinase K and have found that FLP can be digested into two major proteinase K-resistant peptides of 21 and 13 kDa, respectively. The 21-kDa peptide contains a site-specific DNA-binding domain that binds to the FLP recognition target (FRT) site with an affinity similar to that observed for the native FLP protein. This peptide can induce DNA bending upon binding to a DNA fragment containing the FRT site, but the angle of the bend (approximately 24 degrees) is smaller in magnitude than that induced by the native FLP protein (60 degrees). The additional DNA bending induced by the interaction between two native FLP molecules bound to the FRT site is not observed with the 21-kDa DNA-binding peptide. Amino-terminal sequencing has been used to map this peptide to an internal region of FLP that begins at residue Leu-148. It is likely that the DNA-binding peptide includes the catalytic site of the FLP protein.

FLP protein of 2 μ 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.

Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

FLP-FRT mediated intrachromosomal recombination on a tandemly duplicated YEp integrant at the ILV2 locus of chromosome XIII in Saccharomyces cerevisiae.

A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-delta 1 locus in a [cir (o)] background. The 1.5 kb BglII deletion of ilv2-delta 1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid. Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2 microns site-specific recombinase (FLP) recognition target (FRT). Isogenic [cir (o)] and [cir+] diploids formed by crossing the [cir (o)] TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers. [cir (o)] diploids showed no plasmid marker loss and maintained the TD structure. In the absence of selection pressure, the [cir+] diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al. 1988). Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated. Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment. These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.

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.

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.