Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.     

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.     

Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site

The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering. As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets (mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first, seventh or both positions of the Flp-binding elements. The variants that recombine the double-mutant mFRT contain a subset of the mutations present in those that are active on the single-mutant mFRTs, plus additional mutations. Specificity for and discrimination between target sites, effected primarily by amino acid residues that contact DNA, can be modulated by those that do not interact with DNA or with a DNA-contacting residue. The degree of modulation can range from relaxed DNA specificity to almost completely altered specificity. Our results suggest that combined DNA shuffling and mutagenesis of libraries of Flp variants active on distinct mFRTs can yield variants that can recombine mFRTs containing combinations of the individual mutations.     

Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination

A combination of site-directed mutagenesis and amino acid sequence analysis identifies Tyr-343 of Flp recombinase as the residue that covalently attaches to DNA during the strand-cleavage step of recombination. This residue is part of the invariant His-Arg-Tyr triad of the Int family of recombinases. Tyr-343 is located in a highly protease-accessible (and hence "open") region of Flp. This placement may provide the conformational flexibility required for the dual role of Tyr-343 in recombination: nicking of the DNA strands to initiate recombination and joining of the nicked strands across partner substrates to complete recombination. In-frame insertion of a few amino acids close to Tyr-343 (and to its amino-terminal side) does not affect substrate recognition by Flp but abolishes its catalytic function.     

Directed protein replacement in recombination full sites reveals trans-horizontal DNA cleavage by Flp recombinase.

One round of site-specific recombination between two DNA partners mediated by the Flp recombinase requires the breakage and reformation of four phosphodiester bonds. The reaction is accomplished by the combined action of four Flp monomers. Within the recombination complex, what is the relative disposition of a Flp monomer with respect to the target diester that it cleaves? To address this question, we have devised a strategy for the targeted orientation of Flp monomers within full-site recombination substrates. Our experimental design is not dependent on ''altered binding specificity'' of the recombinase. Analysis of the pattern of DNA cleavage by this method reveals no evidence for DNA cleavage in cis. A Flp monomer bound to its recognition element within the full site does not cleave the scissile phosphodiester bond adjacent to it. Our results are most consistent with ''trans-horizontal cleavage''. Cleavage by Flp occurs at the scissile phosphodiester distal to it, but within the same full site. The general experimental design employed here will be of widespread utility in mechanistic analyses of nucleic acid transactions involving multimeric DNA-protein assemblies.     

Geometry of site alignment during int family recombination: antiparallel synapsis by the Flp recombinase

The Flp site-specific recombinase functions in the copy number amplification of the yeast 2 microm plasmid. The recombination reaction is catalyzed by four monomers of Flp bound to two separate, but identical, recombination sites (FRT sites) and occurs in two sequential pairs of strand exchanges. The relative orientation of the two recombination sites during synapsis was examined. Topoisomerase relaxation and nick ligation were used to detect topological nodes introduced by the synapse prior to the chemical steps of recombination. A single negative supercoil was found to be trapped by Flp in substrates with inverted FRT sites whereas no trapped supercoils were observed with direct repeats. The topology of products resulting from Flp-mediated recombination adjacent to a well characterised synapse, that of Tn3 resolvase/res, was analyzed. The deletion and inversion reactions yielded the four noded catenane and the three noded knot, respectively, as the simplest and the most abundant products. The linking number change introduced by the Flp-mediated inversion reaction was determined to be +/-2. The most parsimonious explanation of these results is that Flp aligns its recombination sites with antiparallel geometry. The majority of synapses appear to occur without entrapment of additional random plectonemic DNA supercoils between the sites and no additional crossings are introduced as a result of the chemical steps of recombination.     

Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination.

One key feature of the interaction of Flp recombinase with its target site (FRT) is the large bend introduced in the substrate as a result of protein binding. The extent of bending was found to depend on the phasing and spacing of the Flp monomers occupying the two Flp-binding elements (FBE) bordering the strand-exchange region (spacer) of the substrate. The relative mobilities of the Flp complexes formed by the two permuted substrate fragments, containing the FRT site near the end or in the middle, corresponded to a DNA bend of approx. 140 degrees when each of the two FBEs flanking the spacer was occupied by a protein monomer. The estimated bend angle was the same when the reference DNA fragment with the FRT site at the end was substituted by one with the site in the middle, but containing a 4-bp insertion within the spacer. We used a combination of wild-type Flp and Flp variants that were competent or incompetent in DNA bending, together with full, or half FRT sites, to ask whether bending is a conformational requirement for catalysis, namely cleavage and exchange of strands. We obtained the following results: in full-site (FRT) vs. full-site recombinations or in full-site vs. half-site (half FRT) recombinations, there was a large difference in the reactivity between Flp and a bending-incompetent Flp variant. This difference virtually disappeared when reactions were done with half-FRT sites. We conclude that bending is not a prerequisite for catalysis, but represents the manner in which the substrate accommodates the Flp protomer-protomer interactions that are pertinent to catalysis.     

Functional expression of the Flp recombinase in Mycobacterium bovis BCG

Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants because there are only two efficient antibiotic resistance genes available for allelic exchange experiments. Sequence-specific recombinbases such as the Flp recombinase can be used to excise resistance markers. Expression of the flp(e) gene from Saccharomyces cerevisiae is functional for this purpose in fast-growing Mycobacterium smegmatis but not in slow-growing mycobacteria such as M. bovis BCG or M. tuberculosis. We synthesized the flp(m) gene by adapting the codon usage to that preferred by M. tuberculosis. This increased the G+C content from 38% to 61%. Using the synthetic flp(m) gene, the frequency of removal of FRT-hyg-FRT cassette from the chromosome by the Flp recombinase was increased by more than 100-fold in M. smegmatis. In addition, 40% of all clones of M. bovis BCG had lost the hyg resistance cassette after transient expression of the flp(m) gene. Sequencing of the chromosomal DNA showed that excision of the FRT-hyg-FRT cassette by Flp was specific. These results show that the flp(m) encoded Flp recombinase is not only an improved genetic tool for M. smegmatis, but can also be used in slow growing mycobacteria such as M. tuberculosis for constructing unmarked mutations. Other more sophisticated applications in mycobacterial genetics would also profit from the improved Flp/FRT system.     

Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase Flp.

The Flp recombinase of Saccharomyces cerevisae can mediate strand transfer within a half-site, between two half-sites and between a half-site and a full-site. The ability of "step-arrest" mutants of Flp to partake in half-site reactions has been examined. Arg308 variants of Flp, which show little or no strand cleavage in reactions with normal full-sites, execute significant levels of strand transfer in half-site reactions. On the other hand, His305 variants of Flp, which normally accumulate the strand cleavage product from full-sites but do not complete strand transfer, yield only minute amounts of strand transfer products from half-sites. As would be predicted, the step-arrest mutants are unable to produce "normal" or "reverse" recombinants between a half-site and a full-site. The Flp protein is able to form higher-order complexes in association with a half-site. The step-arrest mutants of Flp show specific defects in forming these complexes.     

Stoichiometry of the Cre recombinase bound to the lox recombining site.

The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site. The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region. Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site. We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site.     

Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase

Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time. The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate. The conformational dynamics by which the recombination complex "isomerizes" from the Holliday-forming to the Holliday-resolving mode are not well understood. Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction. We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong resolution bias in favor of the strands designed to assume the "crossed" configuration within the unbound junction. The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends.     

RAD52-dependent and -independent homologous recombination initiated by Flp recombinase at a single FRT site flanked by direct repeats

We have devised a system for isolating yeast DNA sequences that are able to act as initiators of recombination leading to deletions in mitotically growing yeast cells. This system has allowed us to identify the FRT site of the 2μ site-specific recombinase Flp as such a sequence. We show that Flp is able to initiate recombination leading to deletions at a single FRT site in cir ^o strains. These results indicate that Flp is able to cleave a single FRT site, supporting the observation that the mechanism of cleavage by Flp is trans-horizontal. Interestingly, Flp can induce homologous recombination in both a RAD52-dependent and RAD52-independent manner. Our work provides a new system for the study of homologous recombination leading to deletions, in which the initiation step can be efficiently controlled. We discuss the possibility that Flp-induced, RAD52-independent events occur by single-strand annealing. Received: 20 July 1999 / Accepted: 27 October 1999     

Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis

The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.     

The mechanism of loading of the FLP recombinase onto its DNA target sequence

The FLP recombinase interacts with its target sequence with the formation of three distinct DNA-protein complexes. The first complex leaves neither a DNase footprint nor is the DNA protected from methylation by dimethyl sulfate. We have found, however, that the FLP protein is bound predominantly to only one of the three 13 base-pair (bp) symmetry elements. This asymmetric loading of the FLP site seems to require the presence of an adjacent directly repeated 13 bp element. We speculate that this asymmetric filling of the target site may be accompanied by the unique order of cleavage and exchange of DNA strands.     

Inhibition of Flp recombinase by the topoisomerase I-targeting drugs, camptothecin and NSC-314622

Recombinases of the lambda-Int family and type IB topoisomerases act by introducing transient single strand breaks in DNA using chemically identical reaction schemes. Recent structural data have supported the relationship between the two enzyme groups by revealing considerable similarities in the architecture of their catalytic pockets. In this study we show that the Int-type recombinase Flp is inhibited by the two structurally unrelated topoisomerase I-directed anti-cancer drugs, camptothecin (CPT) and NSC-314622. The interaction of these drugs with topoisomerase I is very specific with several single amino acid substitutions conferring drug resistance to the enzyme. Thus, the observed interaction of CPT and NSC-314622 with Flp, which is comparable to their interaction with the cleavage complex formed by topoisomerase I, strongly supports a close mechanistic and evolutionary relationship between the two enzymes. The results suggest that Flp and other Int family recombinases may provide model systems for dissecting the molecular mechanisms of topoisomerase I-directed anti-cancer therapeutic agents.     

A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity

We have developed a dual reporter screen in Escherichia coli for identifying variants of the Flp site-specific recombinase that have acquired reactivity at an altered target site (mFRT). In one reporter, the lacZα gene segment is flanked by mFRTs in direct orientation. In the other, the red fluorescence protein (RFP) gene is flanked by the native FRTs. Hence, the color of a colony on an X-gal indicator plate indicates the recombination potential of the variant Flp protein expressed in it: blue if no recombination or only FRT recombination occurs, red if only mFRT recombination occurs and white if both FRT and mFRT recombinations occur. The scheme was validated by identification and in vivo characterization of Flp variants that show either relaxed specificity (active on FRT and mFRT) or moderately shifted specificity toward mFRT. We find that alteration of Lys-82 to Met, Thr, Arg or His enables the corresponding Flp variants to recombine FRT sites as well as altered FRT sites containing a substitution of G-C by C-G at position 1 of the Flp binding element (mFRT11). In contrast, wild-type Flp has no detectable activity on mFRT11. When Lys-82 is replaced by Tyr, the resulting Flp variant shows a small but reproducible preference for mFRT11 over FRT. However, this preference for mFRT11 is nearly lost when Tyr-82 is substituted by Phe.     

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.     

The recombination rate is not increased when retroviral RNA is missing an encapsidation sequence.

Retroviruses, as a result of the presence of two identical RNA molecules in their virions, recombine at a high rate. When nonhomologous RNA is present in the dimer RNA molecules, nonhomologous recombination occurs, although the rate is only 0.1% of the rate of homologous recombination. We developed a protocol to study transduction of cellular proto-oncogenes in a single cycle of retrovirus replication. The psi sequences is a cis required sequence for packaging viral RNA into viral particles. To test if the rate of nonhomologous recombination increases about 1,000 times when the psi sequence is deleted, as reported by other, we modified vectors we used previously (J. Zhang and H. M. Temin, Science 259:234-238, 1993). Our results indicated that the recombination rate did not undergo the increase of about 1,000 times when the psi sequence of a chimeric RNA was deleted.     

The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2 % or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.