Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.     

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.     

Site-specific photo-cross-linking between lambda integrase and its DNA recombination target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.     

Regulation of site-specific recombination by the C-terminus of lambda integrase

Site-specific recombination catalyzed by bacteriophage λ integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein–protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.     

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.     

The RecE recombination pathway mediates recombination between partially homologous DNA sequences: structural analysis of recombination products

Escherichia coli generalized recombination, utilizing the RecA RecB recombination pathway, requires large stretches (70-200 bp) of complete DNA sequence homology. In contrast, we have found that the RecE pathway can promote recombination between DNA with only short stretches of homology. A plasmid containing 10 partially homologous direct repeats was linearized by digestion with specific restriction enzymes. After transformation, a RecE+ (sbcA) host was able to circularize the plasmid by recombination between partially homologous direct repeat sequences. Recombination occurred in regions of as little as 6 bp of perfect homology. Recombination was enhanced in the regions adjacent to restriction sites used to linearize the plasmid, consistent with a role of double-strand breaks in promoting recombination. A mechanism is proposed in which the 5' exonuclease, ExoVIII, produces 3' single-stranded ends from the linearized plasmid. These pair with other sequences of partial homology. Partial homologies in the sequences flanking the actual join serve to stabilize this recombination intermediate. Recombination is completed by a process of "copy and join." This recombination mechanism requires less homology to stabilize intermediates than the degree of homology needed for mechanisms involving strand invasion. Its role in nature may be to increase genomic diversity, for example, by enhancing recombination between bacteriophages and regions of the bacterial chromosome.     

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.     

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.     

Deletion of human JK segments by site-specific recombination recognizing the conserved nonamer and heptamer sequences.

Mapping and partial sequencing of the productive K chain genomic DNA of FK-001 demonstrated a 1.8-kb deletion including the JK2, JK3, JK4, and JK5 segments. This deletion occurred between the heptamer recombination signal sequence of the JK2 segment and the heptamer-like sequence located 1.8 kb downstream of the JK2 segment. The recombination reaction kept the reciprocally joined signal sequences on the chromosome and deleted the intervening DNA segment. The cloned FK-001 K chain gene was expressed efficiently in mouse myeloma cells, demonstrating that the 1.8-kb deleted region conferred no functions for gene expression.     

Bacteriophage P1 site-specific recombination. II. Recombination between loxP and the bacterial chromosome

Lipoprotein crystals of hagfish yolk platelets (Myxine glutinosa L.) are, by electron diffraction of embedded specimens, monoclinic (a > 19.8 nm, b > 8.9 nm, c > 9.0 nm, β ~ 105 °; space group C2: 2 dimers per cell). Using sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the four major protein bands in Myxine (molecular weights 30,000; 44,000: 80,000 and 130,000) correspond, with small differences, to similar bands of Xenopus yolk lipoproteins. The symmetric lipovitellin-phosvitin dimer in cyclostomes is unique and probably reflects the lack of diversification of homologous vertebrate protein species.     

In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase

We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding attB _TG1 and attP _TG1 sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of ϕC31 integrase. In this report, we expressed TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP _TG1 and attB _TG1 sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB _TG1 and attP _TG1 sites irrespective of their substrate topology. The minimal sequences of attP _TG1 and attB _TG1 sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of the serine integrase.     

Crystallization of resolvase, a repressor that also catalyzes site-specific DNA recombination

Resolvase, a site-specific recombination enzyme involved in transposition of movable elements of DNA, has been crystallized. The space group is P6₂22 (or enantiomorph $\text{P6}{}_4\text{22, a = b = 59.7}\text{A}\text{?}\text{, c = 169.4}\text{A}\text{?}$), with a monomer in the crystallographic asymmetric unit.     

Characterization of site-specific recombination by the integrase MJ1 from enterococcal bacteriophage phiFC1

Bacteriophage phiFC1 integrase (MJ1) was previously shown to perform a site-specific recombination between a phage attachment site (attP) and a host attachment site (attB) in its host, Enterococcus faecalis, and also in a non-host bacterium, Escherichia coli. Here, we investigated biochemical features of MJ1 integrase. First, MJ1 integrase could perform in vitro recombination between attP and attB in the absence of additional factors. Second, MJ1 integrase interacted with att sites. Electrophoretic mobility shift assays and DNase I footprinting revealed that MJ1 integrase could efficiently bind to all the att sites and that MJ1 integrase recognized relatively short sequences (approximately 50 bp) containing an overlapping region within attB and attP. These results demonstrate that MJ1 integrase indeed catalyzes an integrative recombination between attP and attB, the mechanism of which might be simple and unidirectional, as found in serine integrases.     

Cre-lox site-specific recombination between Arabidopsis and tobacco chromosomes

To create hybrid chromosomes, we tested the Cre-lox system to mediate recombination between Arabidopsis thaliana and Nicotiana tabacum chromosomes. Protoplasts of the two plants were fused to allow site-specific recombination to join a promoter from tobacco to a hygromycin resistance coding-region from Arabidopsis. The expected recombination junction was detected in hygromycin-resistant calli. Analysis of one hybrid suspension cell line revealed the presence of markers corresponding to the north arm of Arabidopsis chromosome III, but not markers from other chromosome arms. However, these markers were not detected in regenerated plants. With a second hybrid cell line we obtained a single hygromycin-resistant progeny from approximately 18 000 self-fertilized seeds of one regenerated plant. Molecular analysis of this hybrid indicated that a small portion of the north arm of Arabidopsis chromosome V is present in the tobacco genome. However, neither the recombination junction nor Arabidopsis DNA was detected in tissue from the plant grown without selection or in the subsequent generation. Thus interspecies transfer of a chromosome arm between plant cells is possible, but maintenance of the hybrid chromosome in a plant is unlikely. The feasibility of site-specific recombination between genomes of different species offers new possibilities for engineering hybrid chromosomes that may be maintained in cell culture.     

Reactions between half- and full-FLP recombination target sites. A model system for analyzing early steps in FLP protein-mediated site-specific recombination.

The FLP recombination target (FRT) can be cut in half so that only one FLP protein binding site is present (a "half site"). FLP protein binds the half sites and joins them into dimeric, asymmetric head-to-head complexes held together chiefly by strong noncovalent interactions. These complexes react with full (normal) FRT sites to generate a variety of products. Analysis of these DNA species reveals that the reaction follows a well-defined reaction pathway that generally parallels the normal reaction pathway. The system is useful in analyzing early steps in recombination, since the identity of the products in a given recombination event unambiguously pinpoints the order in which the cleavage and strand exchange reactions occur. Two conclusions are derived from the present study: (i) Formation of the dimeric head-to-head complex of half sites is a prerequisite to further steps in recombination. (ii) The identity of the base pairs at positions 6 and -6 within the FRT site has a subtle effect in directing the first strand exchange event in the reaction to predominantly one of two possible cleavage sites. In addition, results are presented that suggest that a DNA-DNA pairing intermediate involving only two base pairs of the core sequence is formed prior to the first cleavage and strand exchange. DNA-DNA interactions may therefore not be limited to the isomerization step that follows the first strand exchange.     

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.     

Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase

Background

Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.

Methodology/Principal Findings

We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.

Conclusions/Significance

The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.     

Insertion sequences in the IS1111 family that target the attC recombination sites of integron-associated gene cassettes

Members of the recently identified IS 1111 family differ from the majority of insertion sequences (IS) in that they target specific sites in an orientation-specific manner. However, the way in which target selection is achieved is not known. ISKpn4 is representative of a new subgroup of the IS 1111 family whose members are found in the attC sites (59-be) of the gene cassettes associated with integrons. The transposases of this subgroup are closely related (over 75% identity), confirming that closely related IS usually share a common target. However, among more distant relatives encoding a transposase <45% identical to those of the ISKpn4 group, one IS, ISPa25, was found that also targets attC sites. It appears that the targeting determinant of the ISKpn4 group has become associated with a transposase gene from a different group, and this allowed us to localize the region that is likely to be required for target selection to a long noncoding region found downstream of the transposase gene in all IS 1111 family members. This region may determine an RNA used to guide the IS to its specific target.