Diversity in the serine recombinases

Most site-specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or lambda integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N-terminal catalytic domain and a C-terminal DNA binding domain. Phage-encoded serine recombinases (e.g. phiC31 integrase) cause integration and excision with strictly controlled directionality, and have an N-terminal catalytic domain but much longer C-terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N-terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.     

Site-specific recombination systems for the genetic manipulation of eukaryotic genomes

Site-specific recombination systems, such as the bacteriophage Cre-lox and yeast FLP-FRT systems, have become valuable tools for the rearrangement of DNA in higher eukaryotes. As a first step to expanding the repertoire of recombination tools, we screened recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe. Here, we report that seven recombination systems, four from the small serine resolvase subfamily (CinH, ParA, Tn1721, and Tn5053) and three from the large serine resolvase subfamily (Bxb1, TP901-1, and U153), can catalyze site-specific deletion in S. pombe. Those from the large serine resolvase subfamily were also capable of site-specific integration and inversion. In all cases, the recombination events were precise. Functional operation of these recombination systems in the fission yeast holds promise that they may be further developed as recombination tools for the site-specific rearrangement of plant and animal genomes.     

Characterization of the functional domains of human foamy virus integrase using chimeric integrases

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.     

Phage integrases: biology and applications

Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis. Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria. Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale. These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome. Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites. This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines. Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.     

A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.     

Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31

The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.     

Controlling tetramer formation,subunit rotation and DNA ligation during Hin-catalyzed DNA inversion

Two critical steps controlling serine recombinase activity are the remodeling of dimers into the chemically active synaptic tetramer and the regulation of subunit rotation during DNA exchange. We identify a set of hydrophobic residues within the oligomerization helix that controls these steps by the Hin DNA invertase. Phe105 and Met109 insert into hydrophobic pockets within the catalytic domain of the same subunit to stabilize the inactive dimer conformation. These rotate out of the catalytic domain in the dimer and into the subunit rotation interface of the tetramer. About half of residue 105 and 109 substitutions gain the ability to generate stable synaptic tetramers and/or promote DNA chemistry without activation by the Fis/enhancer element. Phe106 replaces Phe105 in the catalytic domain pocket to stabilize the tetramer conformation. Significantly, many of the residue 105 and 109 substitutions support subunit rotation but impair ligation, implying a defect in rotational pausing at the tetrameric conformer poised for ligation. We propose that a ratchet-like surface involving Phe105, Met109 and Leu112 within the rotation interface functions to gate the subunit rotation reaction. Hydrophobic residues are present in analogous positions in other serine recombinases and likely perform similar functions.     

The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn4451.

Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostridium perfringens and is found on the conjugative plasmid pIP401. The element undergoes spontaneous excision from multicopy plasmids in Escherichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is probably the transposition intermediate. Excision of Tn4451 is dependent upon the site-specific recombinase TnpX, which contains potential motifs associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid residues within these domains was used to show that the resolvase/invertase domain was essential for TnpX-mediated excision of Tn4451 from multicopy plasmids in E. coli. An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity. The Tn4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanked by directly repeated GA dinucleotides, and there was also a GA at the junction of the circular form, where the left and right termini of Tn4451 were fused. We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp staggered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives with altered GA dinucleotides provided experimental evidence to support the model.     

Phage integrases for the construction and manipulation of transgenic mammals

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the φC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the φC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.     

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.     

Phage TP901-1 site-specific integrase functions in human cells

We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.     

The architecture of the gammadelta resolvase crossover site synaptic complex revealed by using constrained DNA substrates

Activated mutants of the serine recombinase, gammadelta resolvase, form a simplified recombinogenic synaptic complex containing a tetramer of resolvase and two crossover sites. We have probed the architecture of this complex by measuring the efficiency of recombination of a series of constrained DNA substrates (with phased recombination sites separated by an IHF-induced U-turn); this serves as a direct report on the topology of a productive synapse. Our data show that in the active complex, the catalytic domains from two resolvase dimers form a central core, while the DNA binding domains and the DNA lie on the outside. In addition, the crossover sites cross one another to form a local positive node. The implications of our data for the mechanism of strand exchange and the process of resolvase activation are discussed.     

TPW22, a lactococcal temperate phage with a site-specific integrase closely related to Streptococcus thermophilus phage integrases

The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.     

In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase

The site-specific integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specific recombination between the phage genome and the chromosome of Streptomyces parvulus 2297. An in vivo assay using Escherichia coli cells revealed that the minimum lengths of the recombination sites attB and attP are 50-bp and 49-bp, respectively, for efficient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His(6))-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purified protein preparation retains the site-specific recombination activity which catalyzes the site-specific recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for efficient in vitro intermolecular recombination. In addition, a gel shift assay showed that His(6)-GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifically. Moreover, based on a detailed comparison analysis of amino acid sequences of serine integrases, we found the DNA binding region that is conserved in the serine recombinase containing the large C-terminal domain. Based on the results presented on this report, attachment sites needed in vitro and in vivo for site-specific recombination by the R4 integrase have been defined more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.     

Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase

Integration of retroviral cDNA is a necessary step in viral replication. The virally encoded integrase protein and DNA sequences at the ends of the linear viral cDNA are required for this reaction. Previous studies revealed that truncated forms of Rous sarcoma virus integrase containing two of the three protein domains can carry out integration reactions in vitro. Here, we describe the crystal structure at 2.5 A resolution of a fragment of the integrase of Rous sarcoma virus (residues 49-286) containing both the conserved catalytic domain and a modulatory DNA-binding domain (C domain). The catalytic domains form a symmetric dimer, but the C domains associate asymmetrically with each other and together adopt a canted conformation relative to the catalytic domains. A binding path for the viral cDNA is evident spanning both domain surfaces, allowing modeling of the larger integration complexes that are known to be active in vivo. The modeling suggests that formation of an integrase tetramer (a dimer of dimers) is necessary and sufficient for joining both viral cDNA ends at neighboring sites in the target DNA. The observed asymmetric arrangement of C domains suggests that they could form a rotationally symmetric tetramer that may be important for bridging integrase complexes at each cDNA end.     

Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.     

Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.     

The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB

Integration of the mycobacteriophage Bxb1 genome into its host chromosome is catalyzed by a serine-integrase, a member of the transposon-resolvase family of site-specific recombinases. These enzymes use a concerted mechanism of strand exchange involving double-stranded cleavages with two-base extensions, and covalent protein-DNA linkages via phosphoserine bonds. In contrast to the resolvase/invertase recombination systems--where there are strict requirements for a specific synaptic complex within which the catalytic potential of the enzyme is activated--synapsis of attP and attB by Bxb1 integrase is completely promiscuous, aligning the sites with equal proclivity in parallel and antiparallel alignments. Moreover, the catalytic potential of Bxb1 integrase is fully active in either alignment. As a consequence, the nonpalindromic central dinucleotide (5'-GT) at the center of attP and attB is the sole determinant of Bxb1 prophage orientation, and a single base pair substitution in the two sites is sufficient to eliminate orientation control.     

Structural basis for catalytic activation of a serine recombinase

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 ? crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.