DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.     

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.     

Effect of deletion mutation on the recombination activity of Cre recombinase

Cre recombinase from bacteriophage P1 is widely used in both in vitro and in vivo DNA manipulations. Based on a structural and functional analysis, three deleted cre mutants were constructed and expressed in Escherichia coli. Mutated recombinases were purified and their recombination activities were determined in vitro. Our results revealed that the mutant with amino-terminal deletion retains the recombination activity as high as wild type Cre; however, the carboxy-terminal deletion and the middle region deletion both lead to a complete loss of the recombinase function.     

Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity

To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.     

Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.     

C-terminal interactions between the XerC and XerD site-specific recombinases

Studies of the site-specific recombinase Cre suggest a key role for interactions between the C-terminus of the protein and a region located about 30 residues from the C-terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer. By extrapolating the Cre DNA recombinase structure to the related site-specific recombinases XerC and XerD, it is predicted that the extreme C-termini of XerC and XerD interact with alpha-helix M in XerD and the equivalent region of XerC respectively. Consequently, XerC and XerD recombinases deleted for C-terminal residues, and mutated XerD proteins containing single amino acid substitutions in alphaM or in the C-terminal residues were analysed. Deletion of C-terminal residues of XerD has no measurable effect on co-operative interactions with XerC in DNA-binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co-operativity with XerD some 20-fold. Co-operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20- to 30-fold. All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired.     

Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.     

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.     

Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by Flp and Cre

The Flp and Cre recombinases are members of the integrase family of tyrosine recombinases. Each protein consists of a 13 kDa NH(2)-terminal domain and a larger COOH-terminal domain that contains the active site of the enzyme. The COOH-terminal domain also contains the major determinants for the binding specificity of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attachment of a conserved nucleophilic tyrosine residue to the 3'-phosphate group at the sites of cleavage. In order to gain further insights into the determinants of the binding specificity and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH(2)-terminal domain of Flp to the COOH-terminal domain of Cre ("Fre") and the NH(2)-terminal domain of Cre to the COOH-terminal domain of Flp ("Clp"). These chimeras have novel binding specificities in that they bind strongly to hybrid sites containing elements from both the Flp and Cre DNA targets but poorly to the native target sites.In this study we have taken advantage of the unique binding specificities of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp. We find that the COOH-terminal domain of the recombinases determines their mode of cleavage. Thus Flp and Clp cleave in trans whereas Cre and Fre cleave in cis. These results agree with the studies of Flp and with the cocrystal structure of Cre bound to its DNA target site. They disagree with our previous findings that Cre could carry out trans cleavage. We discuss the variations in the experimental approaches in order to reconcile the different results.     

Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester

DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA–3′-phosphotyrosyl–enzyme adduct and a free 5′-hydroxyl (5′-OH). Strand ligation results when the 5′-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3′-phospho-(para-nitrophenyl) oligonucleotides (3′-pNP DNAs), which mimic the natural 3′-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5′-OH strand on the 3′-pNP DNA (i.e., without a covalent protein–DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.     

Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity

The Cre-lox system is often used to manipulate sequences in mammalian genomes. We have observed that continuous expression of the Cre recombinase in cultured cells lacking exogenous lox sites caused decreased growth, cytopathic effects, and chromosomal aberrations. Cre mutants defective in DNA cleavage were not geno- or cytotoxic. A self-excising retroviral vector that incorporates a negative feedback loop to limit the duration and intensity of Cre expression avoided measurable toxicity, retained the ability to excise a target sequence flanked by lox sites, and may provide the basis of a less toxic strategy for the use of Cre or similar recombinases.     

Control of Cre recombination by regulatory elements from Xer recombination systems

Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination. We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein-protein interactions between accessory proteins and the recombinases.     

Trans complementation of variant Cre proteins for defects in cleavage and synapsis

The Cre recombinase is a member of the integrase family of conservative site-specific recombinases. These proteins share five conserved catalytic residues, one of which is a tyrosine that acts as the nucleophile to attack the scissile phosphodiester bond in the DNA target. Recombination by the Cre recombinase takes place in a supramolecular structure called a synapse that consists of four molecules of Cre bound to two DNA target sequences called lox sites. The synapse is held together by an intricate network of protein-protein interactions. They bend the two sites into square planar structure that resembles a Holliday intermediate. We have studied three mutant Cre proteins that appear to have defects in synapsis (Cre A36V, Cre T41F, and Cre G314R). We found that they were unable to carry out strand cleavage but that cleavage occurred if they were mixed with a cleavage-defective Cre protein that lacks the catalytic nucleophilic tyrosine residue. The three variant proteins could also be complemented for the formation of a novel structure ("complexV"), which may be a cleaved synaptic intermediate. We suggest that these three mutant proteins have a defect in DNA bending and discuss the relationship between bending, synapsis, and cleavage.     

High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells

DNA site-specific recombinases (SSRs) such as Cre, FLPe, and phiC31, are powerful tools for analyzing gene function in vertebrates. While the availability of multiple high-efficiency SSRs would facilitate a wide array of genomic engineering possibilities, efficient recombination in mammalian cells has only been observed with Cre recombinase. Here we report the de novo synthesis of mouse codon-optimized FLP (FLPo) and PhiC31 (PhiC31o) SSRs, which result in recombination efficiencies similar to Cre.     

Conservation of structure and function among tyrosine recombinases: homology-based modeling of the lambda integrase core-binding domain

Tyrosine recombinases participate in diverse biological processes by catalyzing recombination between specific DNA sites. Although a conserved protein fold has been described for the catalytic (CAT) domains of five recombinases, structural relationships between their core-binding (CB) domains remain unclear. Despite differences in the specificity and affinity of core-type DNA recognition, a conserved binding mechanism is suggested by the shared two-domain motif in crystal structure models of the recombinases Cre, XerD and Flp. We have found additional evidence for conservation of the CB domain fold. Comparison of XerD and Cre crystal structures showed that their CB domains are closely related; the three central α-helices of these domains are superposable to within 1.44 Å. A structure-based multiple sequence alignment containing 25 diverse CB domain sequences provided evidence for widespread conservation of both structural and functional elements in this fold. Based upon the Cre and XerD crystal structures, we employed homology modeling to construct a three-dimensional structure for the λ integrase CB domain. The model provides a conceptual framework within which many previously identified, functionally important amino acid residues were investigated. In addition, the model predicts new residues that may participate in core-type DNA binding or dimerization, thereby providing hypotheses for future genetic and biochemical experiments.     

A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells

Zinc-finger recombinases (ZFRs) represent a potentially powerful class of tools for targeted genetic engineering. These chimeric enzymes are composed of an activated catalytic domain derived from the resolvase/invertase family of serine recombinases and a custom-designed zinc-finger DNA-binding domain. The use of ZFRs, however, has been restricted by sequence requirements imposed by the recombinase catalytic domain. Here, we combine substrate specificity analysis and directed evolution to develop a diverse collection of Gin recombinase catalytic domains capable of recognizing an estimated 3.77 × 10⁷ unique DNA sequences. We show that ZFRs assembled from these engineered catalytic domains recombine user-defined DNA targets with high specificity, and that designed ZFRs integrate DNA into targeted endogenous loci in human cells. This study demonstrates the feasibility of generating customized ZFRs and the potential of ZFR technology for a diverse range of applications, including genome engineering, synthetic biology and gene therapy.     

Functional expression of the Cre recombinase in actinomycetes

Site-specific recombinases revolutionized “in vivo” genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. We have constructed a synthetic gene encoding Cre recombinase with the GC content 67.7% optimized for expression in high-GC bacteria and demonstrated this gene to be functional in Streptomyces lividans. Using the synthetic cre(a) gene, we have removed an apramycin resistance gene flanked by loxP sites from the chromosome of S. lividans with 100% efficiency. Sequencing of the chromosomal DNA part showed that excision of the apramycin cassette by Cre recombinase was specific.     

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.     

PhiC31 integrase facilitates genetic approaches combining multiple recombinases

Homologous and site-specific DNA recombination has revolutionized genetic engineering. The reliability of recombinases such as Cre and FLP has allowed scientists to design complex strategies to study gene function in mammals. However, the retention of recombination sites in the genome limits the use of Cre and FLP recombinases in subsequent modifications. Access to additional recombinases in the ES cell toolbox would enormously widen the number of possibilities to manipulate the genome. In the method presented here, we combine the use of PhiC31, a site-specific integrase, with FLP to obtain site-specific insertion and replacement in pre-inserted docking sites in the genome of mouse ES cells. This method allows for the integration of any sequence of interest in a pre-defined locus, leaving Cre recombinase available for downstream applications. The selection strategy is based on a silent selection marker activated by a plasmid-delivered promoter, making the integration system highly reliable and reducing the need for extensive molecular screens. This article describes how to create "dockable" mouse embryonic stem (ES) cell lines, integrate incoming vectors, and analyze the resulting clones. Current applications of this technology are also discussed.