Cleavage-dependent Ligation by the FLP Recombinase

doi:10.1074/jbc.270.39.23044

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Volume 270, Number 39, Issue of September 29, pp. 23044-23054, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, March 23, 1995; and in revised form, July 20, 1995)

Xu-Dong Zhu (§), , Paul D. Sadowski (¶)

From the Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

ABSTRACT

The FLP recombinase of the 2 µM 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 We have studied the mutant protein FLP R308K in which the arginine residue at position 308 has been replaced by lysine.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.

INTRODUCTION

Most strains of the yeast Saccharomyces cerevisiae harbor 50-100 copies of an autonomously replicating plasmid, the 2 µM circle (Broach and Hicks, 1980). This plasmid is 6318 bp ()in length and contains two identical 599-bp inverted repeats that separate a small and large unique region. One of the open reading frames of the plasmid encodes a site-specific recombinase called ``FLP'' that promotes reciprocal recombination across the inverted repeats. The result is that the yeast contains approximately equal amounts of two isoforms, the A and B forms of the plasmid.

The targets of the FLP protein are the two FLP recognition target sites (FRT) that are within the 599-bp inverted repeats. The FRT sites consist of three 13-bp symmetry elements to which the FLP protein binds in a site-specific manner (see Fig. 1; Andrews et al.(1985, 1987)). Two of the three symmetry elements (a and b) are in inverted orientation surrounding an 8-bp core region. The third symmetry element (c), which is dispensable for recombination both in vivo and in vitro (Jayaram, 1985; Gronostajski and Sadowski, 1985; Proteau et al., 1986), is in direct orientation with symmetry element b.

Figure 1: The FLP recognition target site. The sequence of the FRT site is composed of three 13-bp symmetry elements (horizontal arrows labeled a, b, and c) surrounding an asymmetrical 8-bp core (open box). FLP-mediated cleavage sites are indicated by two small vertical arrows.

The FLP protein promotes efficient recombination in vitro, and the reaction has been studied extensively. After binding specifically to the symmetry elements, FLP induces an acute bend in the FRT site (Schwartz and Sadowski, 1989, 1990), cleaves the top or bottom strands of the FRT site (see vertical arrows, Fig. 1), and promotes the reciprocal exchange of a pair of strands to form a Holliday-like intermediate (Holliday, 1964; Meyer-Leon et al., 1988, 1990; Jayaram et al., 1988) which is then resolved by a second pair of strand exchanges to form reciprocally recombinant molecules (Dixon and Sadowski, 1993, 1994).

The FLP protein is a member of the integrase family of site-specific recombinases whose members have in common four absolutely conserved residues (arginine 191, histidine 305, arginine 308, and tyrosine 343, where numbers refer to the amino acid residues of FLP; Argos et al.(1986), Abremski and Hoess(1992)). These residues are thought to underlie a common catalytic mechanism of strand cleavage and ligation that is shared by all members of the integrase family.

Strand cleavage is brought about by a nucleophilic attack of tyrosine 343 upon the scissile phosphodiester bonds of the FRT site (Jayaram, 1994). This results in the covalent attachment of FLP to the 3`-phosphoryl group at the site of the nick through a phosphotyrosine linkage (Gronostajski and Sadowski, 1985; Evans et al., 1990) and the formation of a free 5`-OH group. Residues Arg-191, His-305, and Arg-308 have also been implicated in the cleavage step of the reaction, presumably in activation of the scissile phosphodiester bond (Parsons et al., 1988, 1990). After swapping of strands with a partner FRT site that has been similarly nicked, strand ligation occurs, which is essentially a reversal of the cleavage step. The 5`-OH group promotes a nucleophilic attack on the phosphotyrosine bond resulting in the re-establishment of the continuity of the phosphodiester backbone and liberation of free FLP protein. The energy of the original phosphodiester bond is conserved in the phosphotyrosine linkage, and the reaction requires no external energy source such as ATP (Pan and Sadowski, 1992; Pan et al., 1993a). Residues Arg-191, His-305, and Arg-308 have also been implicated in the ligation step, but ligation does not require the participation of Tyr-343 (Pan and Sadowski, 1992; Kulpa et al., 1993).

Half-FRT sites and certain mutant proteins have been used to provide evidence that FLP cleaves the FRT site in trans (see Fig. 2; Chen et al.(1992, 1993), Lee and Jayaram (1993), Yang and Jayaram(1994)). This means that the nucleophilic tyrosine that causes breakage of the phosphodiester bond at a cleavage site is not contributed by a FLP monomer bound next to the site; rather it is donated by a FLP monomer bound elsewhere in the synaptic complex. There are, in principle, three different configurations that are possible for cleavage in trans (Fig. 2). These are trans-horizontal, trans-vertical, and trans-diagonal. Although Chen et al.(1992) provided evidence that favored the trans-diagonal mode, Lee et al.(1994) have recently published evidence supporting the trans-horizontal mode of cleavage.

Figure 2: trans-Cleavage by FLP recombinase on a full-FRT site. Two FRT sites are aligned in parallel with FLP molecules (1-4) bound to the a and b symmetry elements. Arms of FLP molecules indicate the tyrosine nucleophiles. Donors of the tyrosine nucleophiles are situated trans-diagonal (i), trans-vertical (ii), or trans-horizontal (iii). From Sadowski(1995) with permission of publisher.

While DNA cleavage by FLP was found to occur in trans, FLP-mediated ligation occurs in cis (Pan et al., 1993b), i.e. the FLP monomer bound adjacent to the site of cleavage carries out ligation at that site. FLP was found to be able to execute strand ligation independently of its ability to cleave and covalently attach to the DNA (Pan and Sadowski, 1992).

In this study, we have investigated the defects in FLP R308K, a mutant protein in which arginine 308 has been changed to lysine. We have developed a novel assay to show cleavage-dependent ligation activity for FLP R308K. We have also shown that this protein is defective in recombination although it is able to cleave a linear FRT site. The protein is competent for ligation but appears to have a defect in the activation of a half-FRT site for cleavage. We provide evidence that FLP R308K executes trans-horizontal cleavage and discuss a model for an ordered strand exchange by FLP.

MATERIALS AND METHODS

FLP Preparations

Wild-type FLP protein (>90% pure) was a Sephacryl S300 fraction, purified as described previously (Pan et al., 1991). Mutant FLP proteins were either partially purified (15-50% pure, Bio-Rex 70 fractions) or highly purified (>90% pure). The highly purified preparations were obtained after chromatography on Bio-Rex 70, Sephacryl S300, and fast protein liquid chromatography Mono S columns (Pan et al., 1991). Experiments using partially purified and highly purified proteins gave identical results. The concentration of protein was estimated using the method of Bradford (1976). FLP plasmids that encoded the FLP proteins R308K, H305L, and Y343F were obtained from M. Jayaram. (R308K means that the arginine normally present at position 308 has been replaced by a lysine. The same notation is applied to other FLP mutant proteins.) The FLP gene bearing the R191K mutation was isolated in our laboratory (Friesen and Sadowski, 1992).

Synthetic Substrates

Duplex half-FRT sites or nicked full-FRT sites were prepared by annealing the appropriate oligonucleotides in 5 mM MgCl (2)

, 100 mM NaCl as described previously (Pan et al., 1993a). Where appropriate, the 5` termini of oligonucleotides were either labeled with [

P]ATP or phosphorylated with cold ATP by T4 polynucleotide kinase (New England Biolabs) and then annealed to complementary oligonucleotides as described. The oligonucleotide bearing a 3`-phosphotyrosine residue was synthesized at the Biotechnology Research Institute, Montreal, Quebec, Canada, as described previously (Pan et al., 1993a). Other oligonucleotides were synthesized by the Carbohydrate Research Center, Faculty of Medicine, University of Toronto.

The oligonucleotides are listed in Table 1. Table 2summarizes the assembled substrates.

Ligation Assays

The assays were carried out essentially as described previously (Pan and Sadowski, 1992). Approximately 0.02 pmol of each substrate was incubated with

5 pmol of FLP protein in a 30-µl reaction at room temperature. After a 60-min incubation, the reaction was stopped by addition of 7 µg of proteinase K and SDS to 0.01% and then analyzed on an 8% denaturing polyacrylamide gel. Ligation products were quantitated using a Molecular Dynamics PhosphorImager.

Assay of Covalent Attachment of Protein to DNA

The FLP proteins were reacted with the various substrates as described above. After a 60-min incubation, the reactions were stopped by adding sample buffer to achieve final concentrations of 10% glycerol, 3% SDS, 60 mM Tris-Cl (pH 6.8), and 5% beta

-mercaptoethanol. The samples were then boiled for 5 min and analyzed on a 15% SDS-polyacrylamide gel (Laemmli, 1970; Amin et al., 1991; Pan et al., 1993a).

In Vitro Recombination Assay

The assay was done essentially as described by Amin et al.(1991) and Pan et al. (1993b). FLP catalyzes recombination between a plasmid pLB112-generated FRT site (Beatty and Sadowski, 1988) and a synthetic FRT site (FS15, Table 2), producing two recombinant molecules with different sizes which can be analyzed on an 8% denaturing gel. A 100-bp EcoRI-HindIII fragment of pLB112 plasmid containing the FRT site was 3`-labeled using [ alpha

P]dATP and reverse transcriptase. Approximately 0.02 pmol of the labeled fragment and

0.10 pmol of the unlabeled synthetic FRT site were incubated with 5 pmol of FLP for 60 min at room temperature in a 30-µl reaction mixture containing 50 mM Tris-Cl (pH 7.4), 33 mM NaCl, 1 mM EDTA, and 3 µg of sonicated and denatured calf thymus DNA. The reactions were terminated by the addition of 7 µg of proteinase K and 0.01% (w/v) SDS. After 30 min at 37 °C, the samples were extracted once with phenol-chloroform, and DNA was precipitated with ethanol. Products were then analyzed on an 8% denaturing gel.

Complementation Assays to Demonstrate Defects of FLP R308K

Each half-site (

0.02 pmol of molecules) was preincubated with the appropriate mutant FLP protein (

0.03 pmol) for 15 min at room temperature in a 30-µl reaction mixture as described previously (Pan et al., 1993b), and a 5-fold excess of unlabeled half-site was then added prior to mixing of the reactions. The complementing reaction mixtures were mixed and incubated for an additional 45 min. Reactions were terminated by addition of 7 µg of proteinase K and 0.01% (w/v) SDS and incubated at 37 °C for 30 min. The reactions were analyzed on an 8% denaturing polyacrylamide gel.

RESULTS

Ligation Activity of R308K Depends on Its Cleavage Ability

We have observed previously that a nicked FRT site that bears a 3`-phosphotyrosine and a 5`-OH at the nick is an effective substrate for FLP-mediated ligation that occurs independently of strand cleavage (Pan and Sadowski, 1992; Pan et al., 1993a). This ligation assay was used to examine the ligation ability of various mutant FLP proteins. FLP R308K was unable to ligate this substrate (Pan et al., 1993b). However, studies of Serre and Jayaram(1992) revealed that FLP R308K was able to perform half-site strand transfer almost as well as the wild-type FLP protein, suggesting that R308K is proficient in ligation. In the half-site transfer assay, FLP R308K must cleave the half-site substrate before carrying out ligation, whereas, in our assay, no cleavage step was thought to be required.

To resolve the apparent discrepancy in the activities of FLP R308K, we employed an activated half-site substrate containing a 3`-PO (4) -tyrosine to examine the reactivity of R308K using an assay that measures transfer and ligation of a 5`-hydroxyl strand to a labeled half-site (Fig. 3). As shown in Fig. 3, we found that FLP R308K exhibited less than 10% of the activity of wild-type FLP where the recipient substrate contained a 3`-PO (4) -tyrosine-bearing strand (HS1). Consistent with the data of Serre and Jayaram(1992), when the half-site substrate contained three T residues adjacent to the FLP cleavage site (HS2), R308K carried out strand transfer and ligation as well as wild-type FLP.

Figure 3: Half-site strand transfer mediated by FLP proteins. Each half-site contains one FLP binding symmetry element (represented by an arrow) and a single-stranded core. Half-sites (HS1 and HS2) are 5`-end-labeled with P (asterisks), and their top strands are 5`-phosphorylated to block intramolecular hairpin ligation. The reactions were carried out with wild-type FLP and FLP R308K as described under ``Materials and Methods.'' The proteins and substrates of each reaction are shown above the lanes. After a 60-min incubation at room temperature with the protein as indicated, the reactions were terminated as described under ``Materials and Methods,'' and the samples were analyzed on an 8% denaturing polyacrylamide gel. P and S represent products and substrates, respectively.

Since strand transfer of substrate (HS2) requires cleavage and loss of the three T residues (Serre and Jayaram, 1992), ()the results suggest that the difference in behavior of FLP R308K toward the two substrates (HS1 and HS2 of Fig. 3) was due to the ability of the protein to cleave substrate HS2 more efficiently than substrate HS1.

The FLP protein cleaves the FRT site and covalently attaches to the 3`-phosphoryl group via tyrosine 343 (Gronostajski and Sadowski, 1985; Evans et al., 1990). The cleavage activity of FLP proteins can be sensitively measured using a substrate that contains extra 3`-end nucleotides at the site of cleavage in the FRT site. FLP efficiently cleaves the extra nucleotides and covalently attaches to the substrate. The covalent FLP-FRT complex can be detected on a SDS-polyacrylamide gel (Pan et al., 1993a).

To examine the ability of FLP R308K to cleave and covalently attach to nicked full-FRT substrates, we analyzed the reactions by using SDS-polyacrylamide gel electrophoresis. Wild-type FLP protein could cleave and covalently attach to the 3`-PO (4) of a substrate that contained three extra nucleotides (FS1) as effectively as it attached to a nicked full-FRT site bearing a 3`-phosphotyrosine at the site of the nick (FS2, lanes 2 versus 7 of Fig. 4A). In contrast, FLP R308K was barely able to cleave and covalently attach to the nicked full-FRT site bearing a 3`-phosphotyrosine at the site of the nick (FS2), whereas it effectively attached to the 3`-PO (4) of the substrate that contained extra 3`-nucleotides (FS1, lanes 8 versus 3 of Fig. 4A). As shown by a denaturing polyacrylamide gel (Fig. 4B), FLP R308K carried out efficient ligation on the substrate (FS1) that it was able to cleave. This implies that ligation activity of FLP R308K may be linked to its cleavage activity. Therefore, FLP R308K differs from other mutant proteins such as FLP Y343F whose ligation activity is totally independent of its ability to cleave the FRT site (Pan and Sadowski, 1992).

Figure 4: A, covalent attachment of FLP proteins to nicked substrates containing either extra 3`-nucleotides TTT (FS1) or 3`-phosphotyrosine (FS2). Substrates FS1 and FS2 contain two symmetry elements (shown by arrows) and a nick at the a cleavage site. Substrates were 5`-end-labeled with P (asterisks). Reactions were carried out as described under ``Materials and Methods.'' Products were analyzed on a 15% SDS-polyacrylamide gel. The substrates and proteins of each reaction are given above the lanes. Cov and S refers to covalent DNA-protein complexes and substrates, respectively. B, ligation activity of FLP proteins on the nicked substrate FS1. Reactions were done as described under ``Materials and Methods,'' and ligation products were analyzed on an 8% denaturing polyacrylamide gel. Lane 1 contained only the substrate (no FLP protein). Lanes 2-5 contained the substrate and the FLP proteins as shown above the lanes. LP and S refer to ligation products and substrates, respectively. HP is likely a hairpin product resulting from cleavage of the top strand and ligation to the labeled bottom strand. CL refers to the cleaved substrate which was incompletely digested with proteinase K.

We also studied two other mutant FLP proteins that bear alterations in the catalytic tetrad of conserved active-site residues in order to compare their activities with FLP R308K. FLP H305L behaved similarly to FLP R308K in that it could efficiently attach to the nicked FRT substrate bearing three extra nucleotides (FS1, lane 4 of Fig. 4A). However, FLP R191K, a protein that cleaves a full non-nicked FRT site substrate efficiently (Friesen and Sadowski, 1992), cleaves the three extra nucleotides of the nicked full-FRT substrate (FS1) less efficiently than FLP R308K does (lane 5 of Fig. 4A). FLP H305L and FLP R191K also fail to cleave the 3`-phosphotyrosine of the nicked full-FRT substrate (FS2, lanes 9 and 10 of Fig. 4A). Unlike FLP R308K, both FLP H305L and R191K have defects in ligation activity (lanes 4 and 5 of Fig. 4B), in spite of their ability to cleave the nicked FRT site bearing the three extra nucleotides (FS1, lanes 4 and 5 of Fig. 4A).

R308K Has an Altered Cleavage Activity but Shows a Normal Ligation Activity

The results presented above show that the defect of R308K in ligation is likely due to its inability to cleave a substrate containing a 3`-phosphotyrosine residue and suggest that the cleavage pocket of R308K is different from that of wild-type FLP. To further understand the defect of FLP R308K, we examined its cleavage and ligation activities on a series of nicked full-site substrates that contain extra 3`-nucleotides linked to the symmetry element a. Analyses using SDS-polyacrylamide gels (Fig. 5) showed that wild-type FLP could form covalent intermediates with all the substrates tested. However, FLP R308K was scarcely able to cleave substrates containing one, two, or three extra A residues or one extra T residue, but it was able to form covalent intermediates with substrates that have extra 3` sequences of TT, TTT, TTTC, TTTCT, and TTTCTAGA at the cleavage site of the bottom strand. This means that FLP R308K could only cleave those substrates in which the extra nucleotides were complementary to the opposite strand of the core region. In addition, the observation that R308K does not cleave the substrate containing one extra T suggests that the mutant protein requires at least two complementary residues to cleave the substrate.

Figure 5: A-D, covalent attachment of FLP proteins to nicked substrates bearing various extra 3`-nucleotides. All substrates contained two symmetry elements (arrows) and a nick at the a cleavage site and were 5`-end-labeled with P (asterisks). Reactions were carried out as described under ``Materials and Methods.'' FLP-mediated cleavage activity was measured as formation of DNA-protein complexes on 15% SDS-polyacrylamide gels. The proteins and substrates of each reaction are illustrated above the lanes. Cov and S refer to covalent DNA-protein complexes and substrates, respectively.

To test the importance of base pair complementarity in cleavage by FLP R308K, we changed the wild-type core sequence to 5`-TCTAGTTT-3`, 3`-AGATCAAA-5`, and examined the cleavage ability of R308K on the same series of substrates. We observed that FLP R308K was now able to cleave substrates bearing 3` termini of AA, AAA, but not TT, TTT (data not shown). Because the AA or AAA termini were complementary to the top strand of the altered core, complementarity rather than nucleotide composition of the 3` terminus was important for cleavage. Again, R308K did not cleave the substrate with one extra nucleotide A although the nucleotide A is complementary to the top strand of the core (data not shown). Therefore, at least 2 base pairs of complementarity adjacent to the cleavage site in the core region are required for cleavage by FLP R308K.

We also studied the ligation of these substrates (Fig. 6). Wild-type FLP could seal the nick in all substrates, and mutant R308K could seal only those substrates that it could cleave. Thus, the ligation ability of R308K is actually as efficient as that of wild-type FLP, and this implies that the arginine residue at position 308 may not be directly involved in the chemistry of the ligation reaction. Alternatively, the lysine at position 308 may substitute for the arginine in the ligation reaction.

Figure 6: A-C, ligation activity of FLP proteins on nicked substrates containing various extra 3`-nucleotides. Substrates used were the same as in Fig. 5. Reactions were carried out as described under ``Materials and Methods.'' Ligation products were analyzed on 8% denaturing polyacrylamide gels.

These assays have also been done with FLP proteins R191K and H305L to compare their activities with FLP R308K. As seen on SDS-polyacrylamide gels (Fig. 5), the defects of the R191K and H305L proteins in cleavage are similar to those of R308K. However, the R191K and H305L proteins failed to ligate efficiently the substrates that they were able to cleave (Fig. 6), suggesting that these amino acid substitutions directly affect the FLP ligation pocket.

Strand Exchange Activity of R308K Is Normal at the aCleavage Site, but Defective at thebCleavage Site

The experiment described above showed that FLP R308K has an altered cleavage activity, but that its ligation activity was normal. Since FLP R308K was able to cleave a linear FRT site (Fig. 7, lane 3), we therefore assayed the ability of FLP R308K to promote recombination between linear FRT sites. FLP R308K exhibited no recombination, whereas recombination catalyzed by wild-type FLP was readily detectable (lanes 5 versus 6 of Fig. 7). Thus, it was of interest to examine the strand exchange activity of R308K using model substrates. We define the strand exchange as the step which takes place between the cleavage of an FRT site and the ligation of a 5`-OH of one FRT site to the 3`-PO (4)

group of another nicked FRT site. Since the ligation activity of R308K is dependent on its cleavage activity, strand exchange substrates were designed to require cleavage of one of the partner substrates.

Figure 7: Cleavage and recombination mediated by FLP proteins. A fragment containing a FRT site was generated from plasmid pLB112 (Beatty and Sadowski, 1988) by double restriction digestion with EcoRI and HindIII. The fragment (100 bp) was 3`-end-labeled with alpha -P using reverse transcriptase (indicated by asterisks). As illustrated at the top of the figure, the fragment contains three symmetry elements (horizontal arrows). The wavy lines indicate sequences derived from the vector. Two small vertical arrows indicate the cleavage sites on the top and bottom strands. Two vertical lines refer to the cleavage sites of the restriction enzyme XbaI. Substrate FS15 is a synthetic FRT site, containing two symmetry elements as illustrated. FLP-mediated recombination was carried out between the pLB112-generated fragment and the substrate FS15 as described under ``Materials and Methods.'' The reaction conditions for FLP-mediated cleavage were essentially the same as that for recombination except that the substrate FS15 was omitted. Cleavage products and recombination products were analyzed on an 8% denaturing polyacrylamide gel. Substrates and proteins are shown below and above the lanes, respectively. Ra and Rb represent recombinant products. CLa and CLb represent FLP-mediated products of cleavage at symmetry elements a and b, respectively. Xa and Xb refer to cleavage products resulting from an XbaI restriction digest of the substrate. The numbers in parentheses indicate the length of products.

As illustrated in Fig. 8A, strand exchange activity was measured at the a cleavage site between two full-site substrates. One partner was a labeled ``suicide substrate'' (Nunes-Düby et al., 1987) bearing a nick three nucleotides (TTT) away from the a cleavage site (FS11), and the other was a substrate with a nick precisely at the a cleavage site (FS12). The latter nick bore 3`-OH and 5`-OH ends to prevent religation on the same strand. In order to examine the importance of single-strandedness of the core region, strand exchange assays were also carried out between the suicide full-site substrate (FS11) and two b half-site substrates (HS3 and HS4). The two half-site substrates differed in that one had a single-stranded core (HS3) and the other had a double-stranded core (HS4). If FLP R308K was able to cleave the FS11 substrate, the trinucleotide, TTT, would diffuse away, leaving a three-nucleotide gap which would prevent ligation of the 5`-OH end of the suicide substrate. Hence, strand exchange with a 5`-OH end from another molecule (FS12) would be detected as the appearance of a 48-nucleotide product on a denaturing gel.

Figure 8: A, FLP-mediated strand exchange at the a cleavage site. Strand exchange was carried out between two full-FRT sites or between a full-FRT site and a half-FRT site. Substrates are diagrammed on the top. Full-FRT sites (FS11 and FS12) contained two symmetry elements, and half-FRT sites (HS3 and HS4) contained one FLP binding symmetry element. Substrate FS11 bore a nick on the bottom strand three nucleotides away from the a cleavage site. Substrate FS12 bore a nick at the a cleavage site. Substrates HS3 and HS4 differed in that HS3 contained a single-stranded core, whereas HS4 contained a duplex core. Substrate FS11 was 5`-end-labeled with P (asterisk). Reactions were carried out as described under ``Materials and Methods,'' and strand exchange products were analyzed on an 8% denaturing polyacrylamide gel. The proteins and substrates of each reaction are shown above the lanes. SEP and S represent strand exchange products and substrates, respectively. B, FLP-mediated strand exchange at the b cleavage site. Strand exchange assays were carried out as described in A. Substrates are illustrated on the left of the figure. These are modeled after those shown in A.

In all reactions (Fig. 8A), FLP R308K yielded a level of strand exchange products similar to wild-type FLP. This implies that the strand exchange activity of R308K is nearly normal at the a cleavage site. The same reactions were also carried out with the mutant FLP proteins R191K and H305L to compare their activities with FLP R308K. These two proteins failed to make strand exchange products, probably due to their deficiencies in ligation.

Strand exchange was then examined at the b cleavage site using similar substrates (Fig. 8B). If the top strand of FS13 substrate was cleaved by FLP R308K, the trinucleotide, TCT, would diffuse away. Strand exchange would be detected as a 55-nucleotide product on a denaturing gel.

Unlike the results obtained when strand exchange at the a cleavage site was measured (Fig. 8A), R308K showed defective strand exchange activity at the b cleavage site. When strand exchange was measured between the two full-site substrates (FS13 and FS14, lanes 2 and 3 of Fig. 8B), the level of strand exchange products was at least 3-fold less than obtained with wild-type FLP (quantitated by PhosphorImager analysis). When the a half-site substrate (HS5) containing a double-stranded core was used (lanes 4 and 5), FLP R308K exhibited a greater than 10-fold decrease in the level of strand exchange products compared to wild-type FLP. When the a half-site substrate (HS6) containing a single-stranded core was used, the strand exchange reaction was improved. FLP R308K exhibited 50% of wild-type strand exchange activity (lanes 6 and 7). This could be due to the presence of an exposed 5`-OH end that is available for strand exchange.

The observation that the single-strandedness of the core region improved strand exchange by FLP R308K at the b cleavage site implies that the melting of the core region on both substrates is important for strand exchange to occur at the b cleavage site (Fig. 8B, lanes 5 versus 7) but not for strand exchange at the a cleavage site (Fig. 8A, lanes 6 versus 7). This difference may be exaggerated by the difference in the base composition adjacent to the cleavage site (TTT at aversus TCT at b). Furthermore, the defect of strand exchange by FLP R308K at the b cleavage site may explain why FLP R308K is defective in recombination even though it is apparently competent for cleavage and ligation. The fact that R308K shows normal strand exchange at the a cleavage site but defective strand exchange at the b cleavage site suggests that the FLP protein may use a different mechanism of strand exchange at a and b cleavage sites.

FLP R308K Executes trans-Horizontal Cleavage

The data in Fig. 3showed that the labeled a half-site with a trinucleotide (TTT) in the core (HS2) can participate in FLP R308K-mediated ligation when mixed with a b half-site (HS3). However, we detected no intramolecular hairpin products when the same labeled a half-site substrate was reacted with FLP R308K alone (data not shown); such products are effectively produced by wild-type FLP. To further understand this discrepancy, we assayed strand exchange between a labeled a half-site (HS2) and a nicked full-site substrate (FS12). As shown in Fig. 9, the nick of the full-site substrate (FS12) bore a 3`-OH and 5`-OH to prevent ligation on the same strand. Cleavage and strand exchange reactions were analyzed on a SDS-polyacrylamide gel and a denaturing polyacrylamide gel, respectively.

Figure 9: FLP-mediated strand exchange and cleavage between a labeled half-FRT site and a full-FRT site. The half-FRT substrate (HS2) used is the same as in Fig. 3. The full-FRT substrate (HS12) used is the same in Fig. 8A. A, strand exchange activity of FLP proteins. Reactions were carried out as described under ``Materials and Methods.'' Aliquots were removed at 30 min (lanes 2 and 4) and 60 min (lanes 3 and 5), and the strand exchange products were analyzed on an 8% denaturing polyacrylamide gel. Lane 1 contains only the labeled substrate HS2. FLP proteins were added as indicated above the lanes. SEP and S represent strand exchange products and substrates, respectively. B, cleavage activity of FLP proteins. Substrates were the same as in A. Cleavage activity of FLP proteins was monitored on a 15% SDS-polyacrylamide gel. The reactions shown here were stopped at 60 min. Cov refers to covalent DNA-protein complexes. SEP represents strand exchange products. S refers to substrates.

FLP R308K promoted little strand exchange between the cleavable a half-site (HS2) and the nicked full-FRT site (FS12) (Fig. 9A, lanes 2 and 3). Results from the SDS-polyacrylamide gel revealed that the defective strand exchange activity of R308K was likely due to its failure to cleave the labeled half-FRT site. As seen in Fig. 9B (lanes 2 versus 3), R308K formed scarcely any DNA-protein covalent intermediates, whereas wild-type FLP formed DNA-protein covalent complexes readily.

The inability of FLP R308K to catalyze strand exchange between the a half-site and the nicked full-site (Fig. 9A, lanes 2 and 3) contrasted with its ability to promote strand exchange between the same a half-site (HS2) and a b half-site (HS3) (Fig. 3, lane 5). This suggested that it might be possible to rescue cleavage and strand exchange activity of FLP R308K by providing it with a partner b half-site. Therefore, the experiment was repeated except an unlabeled b half-site containing a 5-nucleotide protrusion on the bottom strand that was complementary to the top strand of the core region of the a half-site was included in the reaction (Fig. 10). The addition of the b half-site (HS7) bearing a 5-nucleotide protrusion promoted a marked stimulation of cleavage and strand exchange (Fig. 10A, lane 3; Fig. 10B, lane 3). This suggested that the FLP R308K protein bound to the b half-site cleaved the labeled a half-site in a trans-horizontal manner which in turn allowed strand exchange with the nicked full-FRT site to occur.

Figure 10: In vitro complementation analysis of FLP R308K and FLP Y343F. The reaction conditions were described under ``Materials and Methods.'' Small vertical lines represent unpaired nucleotides in the core region. Substrates HS2 and HS12 are the same as in Fig. 9. Substrate HS7 is a half-FRT site containing a 5-nucleotide single-stranded core (indicated by 5 small vertical lines). The FLP proteins are indicated as R (FLP R308K), Y (FLP Y343F). A, analysis of strand exchange by complementation assays. Each substrate was incubated separately for 15 min at room temperature with the protein as indicated. The substrate-protein mixtures were then combined and allowed to incubate for 45 min at room temperature. The reactions were subsequently terminated with proteinase K and SDS as described under ``Materials and Methods.'' The samples were then analyzed on an 8% denaturing polyacrylamide gel. SEP and S represent strand exchange products and substrates, respectively. B, analysis of FLP protein-DNA covalent complexes. The reactions were done essentially as in A. After a 45-min incubation at room temperature, the reactions were stopped by adding sample buffer as described under ``Materials and Methods.'' The protein-DNA covalent complexes were then analyzed on a 15% SDS-polyacrylamide gel. Reactions in lanes 1 to 8 are the same as those in A. Cov and S refer to covalent DNA-protein complexes and substrates, respectively.

To gain further evidence of trans-horizontal cleavage by FLP R308K, we analyzed complementation between FLP R308K and FLP Y343F. When Y343F was bound to the nicked full-site FS12 and to the half-site HS2, no cleavage and strand exchange were detected (Fig. 10, A and B, lane 7). However, when the reaction was supplemented with the b half-site (HS7) to which FLP R308K had been bound, strand cleavage and strand exchange were stimulated markedly (lane 8 in Fig. 10, A and B). Since FLP Y343F lacks the active site tyrosine needed for cleavage, FLP R308K must be providing the tyrosine that leads to the formation of covalent DNA-protein complexes that were seen in lane 8 of Fig. 10B. Thus, we conclude that FLP R308K executes trans-horizontal cleavage.

R308K May Be Defective in Activation of Half-sites for Cleavage

The above results showed that FLP R308K seems to carry out trans-horizontal cleavage when presented with two half-sites that have complementary nucleotides in the core region. In order to learn whether the R308K protein could also engage in trans-vertical or trans-diagonal cleavage, we carried out complementation experiments using the FLP Y343F protein. In addition to its cleavage deficiency, this protein is competent for ligation (Pan and Sadowski, 1992; Pan et al., 1993b), but is incapable of forming half-site dimers (Qian et al., 1990). These dimers are formed as a result of strong protein-protein interactions between FLP molecules that are each bound to one half-site, and their formation requires cleavage of and covalent attachment of FLP to one of the half-sites. When FLP R308K was bound to the full-site and Y343F was bound to the half-site, we were able to detect a wild-type level of strand exchange product (lane 4 of Fig. 10A) and covalent DNA-protein complexes (lane 4 of Fig. 10B), respectively. Since Y343F is incompetent for cleavage, FLP R308K must be supplying the tyrosine that leads to the formation of covalent DNA-protein complexes. When the position of the proteins on the two substrates was reversed (lane 5), little cleavage and strand exchange product was formed. This implies that when R308K is bound to the full-site, it is able to make trans-vertical or trans-diagonal interactions with the protein (FLP Y343F) that occupies the half-site which it will cleave.

These results suggest that R308K is defective in activation of the a half-site for trans-vertical or trans-diagonal cleavage (Fig. 9B, lane 2, and Fig. 10B, lane 2). However, it can nevertheless provide the nucleophilic tyrosine in trans to the a half-site that contains bound FLP Y343F (Fig. 10B, lane 4).

DISCUSSION

Cleavage-dependent Ligation Activity

Using nicked FRT substrates bearing extra 3`-nucleotides, we have developed an assay to demonstrate cleavage-dependent ligation activity of FLP R308K. This protein was defective in ligation when we used an activated half-site substrate bearing a 3`-phosphotyrosine (Pan and Sadowski, 1992; Pan et al., 1993b), because the R308K protein was unable to covalently attach to this substrate. This differs from the case of FLP Y343F where ligation occurs in the absence of cleavage. Therefore, it seems that FLP proteins may actually catalyze two different ligation activities: cleavage-dependent ligation and cleavage-independent ligation. The latter activity is disrupted by a substitution of arginine at position 308 to lysine.

The defect of FLP R308K in cleavage-independent ligation (Pan et al., 1993b) may account for its apparent ability to cleave the FRT site more efficiently than wild-type FLP (Jayaram et al., 1988; Parsons et al., 1990). FLP R191K and FLP H305L have also been shown to exhibit such hypercleavage ability (Friesen and Sadowski, 1992; Jayaram et al., 1988; Parsons et al., 1988). However, the hypercleavage activity of FLP R308K is not as marked as that of FLP R191K and FLP H305L (data not shown). This may be due to the fact that FLP R308K can promote cleavage-dependent ligation, but FLP R191K and FLP H305L fail to execute both cleavage-dependent ligation and cleavage-independent ligation ( Fig. 4and Fig. 5; Pan et al., 1993b). It is possible that amino acids Arg-191 and His-305 are directly involved in the chemistry of ligation, but that residue Arg-308 may be involved in the activation of the scissile phosphodiester bond for cleavage and of the phosphotyrosine bond for ligation. The arginine at position 308 has been shown to be important for cleavage as well. Other changes of Arg-308 have been shown to affect primarily the ability of the protein to cleave the FRT site (Parsons et al., 1990; Serre and Jayaram, 1992).

Strand Cleavage by R308K Requires Base Pair Complementarity in the Core Region

Our results showed that FLP R308K only cleaved those substrates whose extra 3`-nucleotides could pair to the top strand of the core. This effect was shown to be due to the requirement by FLP R308K for complementarity of the top strand of the core and the bottom strand of extra nucleotides by an experiment in which the sequence of the core region was reversed (cited under ``Results''; data not shown). Therefore, at least 2 base pairs of complementarity adjacent to the cleavage site are required for cleavage by FLP R308K. This may indicate that the arginine at position 308 activates or positions the scissile phosphodiester bond for cleavage.

Wild-type FLP is able to cleave substrates containing complementary or noncomplementary nucleotide protrusions. This suggests that, during evolution, FLP has retained its ability to cleave mismatched substrates. This would enable it to carry out recombination of FRT sites with mutation(s) adjacent to the cleavage site. This may have been important in the coevolution of FRT sites and FLP-like proteins (Murray et al., 1988).

FLP R308K May Initiate the First Strand Exchange at the bCleavage Site

FLP R308K was unable to recombine linear FRT sites, and no evidence of exchange of either top or bottom strands was detected on a denaturing gel (Fig. 7). However, model substrates showed that the protein was able to carry out strand exchange at the a cleavage site as well as wild-type FLP, but failed to do so at the b cleavage site. FLP R308K has been shown to be able to resolve a synthetic immobile

structure as efficiently as wild-type FLP. (

)These observations suggest that R308K may fail to form a Holliday intermediate, because it attempts to initiate the first strand exchange at the b cleavage site.

On the other hand, if the first strand exchange had occurred at the a cleavage site, R308K should have been able to complete strand exchange and form a Holliday intermediate (or structure). Since R308K was shown to be able to resolve immobile structures, one would have expected R308K to resolve the Holliday intermediate resulting from the first strand exchange at the a cleavage site, producing recombinant molecules. But no recombinant products were detected with FLP R308K (Fig. 7, lane 6). These observations are compatible with a model in which wild-type FLP initiates recombination at the a cleavage site. FLP R308K is defective in recombination because it attempts to initiate recombination at the b cleavage site, but strand exchange is abortive.

trans-Cleavage

Results from this study reveal that FLP R308K alone fails to exhibit trans-vertical or trans-diagonal cleavage between the labeled a half-site and the nicked full-site. This could be due to the fact that FLP R308K fails to activate the half-site for cleavage. Such trans-cleavage could occur when the half-site contains bound FLP Y343F. However, such half-site activation did not seem to be required for trans-horizontal cleavage by FLP R308K, because cleavage of the a half-site could be restored by addition of the b half-site to the reaction (Fig. 10, A and B). This implies that FLP R308K itself can only carry out trans-horizontal cleavage but that trans-vertical/diagonal cleavages are possible in concert with FLP Y343F.

Early results from Chen et al.(1992) seemed to favor a trans-diagonal mechanism. However, Lee et al. (1994) have recently provided evidence favoring trans-horizontal cleavage. Results from the present study suggest that FLP R308K can use more than one mode of trans cleavage. Complementation experiments between R308K and Y343F (Fig. 10, A and B, lanes 7 and 8) showed that FLP R308K executed strand cleavage in a trans-horizontal manner. But, cooperation with FLP Y343F will allow R308K to carry out trans-vertical or trans-diagonal cleavage. It is possible that wild-type FLP uses a different mode of cleavage for the initial cleavages from that used for the final (resolution) cleavages. ()Although resolution of Holliday structures by FLP follows the trans-cleavage paradigm, ()the distinction among the three modes of trans-cleavage awaits a definitive experiment.

FOOTNOTES

*

This work is supported by a grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M23380[GenBank].

§

Recipient of a Connaught Scholarship from the University of Toronto.

¶

To whom correspondence should be addressed. Tel.: 416-978-6061; Fax: 416-978-6885; sadowski{at}gene04.med.utoronto.ca.

()

The abbreviations used are: bp, base pairs; FRT, FLP recognition target.

()

X.-D. Zhu, unpublished results.

()

J. E. Dixon, unpublished results.

()

X.-H. Qian and M. M. Cox, personal communication.

()

Dixon, J. E., Shaikh, A., and Sadowski, P. D. (1995) Mol. Microbiol., in press.

ACKNOWLEDGEMENTS

We thank Barbara Funnell, Helena Friesen, Karen Luetke, Doug Kuntz, and Gagan Panigrahi for careful reading of the manuscript. We thank Donna Clary, Arkady Shaikh, and John R. Walker for preparing the FLP proteins.

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