Control of Directionality in Streptomyces Phage φBT1 Integrase-Mediated Site-Specific Recombination

Streptomyces phage φBT1 integrates its genome into the attB site of the host chromosome with the attP site to generate attL and attR. The φBT1 integrase belongs to the large serine recombinase subfamily which directly binds to target sites to initiate double strand breakage and exchange. A recombination directionality factor (RDF) is commonly required for switching integration to excision. Here we report the characterization of the RDF protein for φBT1 recombination. The RDF, is a phage-encoded gp3 gene product (28 KDa), which allows efficient active excision between attL and attR, and inhibits integration between attB and attP; Gp3 can also catalyze topological relaxation with the integrase of supercoiled plasmids containing a single excision site. Further study showed that Gp3 could form a dimer and interact with the integrase whether it bound to the substrate or not. The synapse formation of attL or attR alone with integrase and Gp3 showed that synapsis did not discriminate between the two sites, indicating that complementarity of central dinucleotides is the sole determinant of outcome in correct excision synapses. Furthermore, both in vitro and in vivo evidence support that the RDFs of φBT1 and φC31 were fully exchangeable, despite the low amino acid sequence identity of the two integrases.     

Attachment site recognition and regulation of directionality by the serine integrases

Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.     

Efficient reversal of phiC31 integrase recombination in mammalian cells

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR. To date, use of phiC31 integrase has been limited to attP × attB recombination. The factor needed for the reverse reaction – the excisionase or recombination directionality factor (RDF) – was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL–attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.     

The mechanism of ?C31 integrase directionality: experimental analysis and computational modelling

Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the ‘reverse’ reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase–DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination.     

Highly efficient in vitro site-specific recombination system based on streptomyces phage phiBT1 integrase

The Streptomyces phage BT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra- and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.     

DNA binding and synapsis by the large C-terminal domain of ϕC31 integrase

The integrase (Int) from phage ϕC31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL × attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein–protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD–CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL × attR. hCTD^E449K was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.     

Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination

The Streptomyces phage C31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5′ ends. Rotation of one pair of half sites by 180° relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by C31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.     

Sequences in attB that affect the ability of phiC31 integrase to synapse and to activate DNA cleavage

Phage integrases are required for recombination of the phage genome with the host chromosome either to establish or exit from the lysogenic state. ϕC31 integrase is a member of the serine recombinase family of site-specific recombinases. In the absence of any accessory factors integrase is unidirectional, catalysing the integration reaction between the phage and host attachment sites, attP × attB to generate the hybrid sites, attL and attR. The basis for this directionality is due to selective synapsis of attP and attB sites. Here we show that mutations in attB can block the integration reaction at different stages. Mutations at positions distal to the crossover site inhibit recombination by destabilizing the synapse with attP without significantly affecting DNA-binding affinity. These data are consistent with the proposal that integrase adopts a specific conformation on binding to attB that permits synapsis with attP. Other attB mutants with changes close to the crossover site are able to form a stable synapse but cleavage of the substrates is prevented. These mutants indicate that there is a post-synaptic DNA recognition event that results in activation of DNA cleavage.     

A second site-specific recombination event in the lambdoid bacteriophage HK022

 An in vitro site-specific recombination reaction of the lambdoid phage HK022 has revealed two supercoiled products that proved to be Holliday intermediates. One of them is the Holliday intermediate which has resulted from an attP×attB reaction. The other is an intermediate which has resulted from a recombination reaction between attP and the attL site of the product from the first reaction. The preferential attL×attP over attR×attP reaction was confirmed in vitro and in vivo by challenging attP sites with attL and attR sites. The biased attP×attL over attP×attR reaction in phage HK022 is discussed. Received: 25 April 1996 / Accepted: 11 July 1996     

A diversity of serine phage integrases mediate site-specific recombination in mammalian cells

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, φFC1, and φRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of ∼ 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, φFC1, and φRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.     

Repetitive,Marker-Free,Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB_min), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB_min site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps.     

Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Summary pSE211 fromSaccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination betweenattP andattB, the plasmid and chromosomal attachment sites. Integration depends on the presence ofint, an open reading frame (ORF) that lies adjacent toattP and encodes the putative integrase. Immediately upstream ofint liesxis (formerly calledorf2) which encodes a basic protein that is thought to exhibit DNA binding.xis andint were cloned in various combinations in pUC18 and expressed constitutively inEscherichia coli from thelac promoter.attP andattB were cloned inStreptomyces orE. coli plasmids containing kanamycin resistance (Km^R) or chloramphenicol resistance (Cm^R) markers. Stable Km^R Cm^R cointegrates formed byattP ×attB orattP ×attP recombination (integration) were obtained inE. coli hosts that expressedint. Co-integrates were not found in hosts expressingint+xis. Excision (intraplasmidatt site recombination) was examined by constructing plasmids carryingattL andattR or twoattP sites separating Cm^R from Km^R and by following segregation of the markers in various hosts. BothattL ×attR andattP ×attP excision depended on bothxis andint inE. coli. pSE211att site integration and excision were not affected by a deletion inhimA, the gene encoding a subunit of integration host factor.     

The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication

Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth. However, the inability to construct a Δ47 deletion mutant of Bxb1 suggests that gp47 provides a second function that is required for lytic growth, although the possibility of an essential cis-acting site cannot be excluded. Characterization of a mutant prophage of mycobacteriophage L5 in which gene 54 - a homologue of Bxb1 gene 47 - is deleted shows that it also is defective in induced lytic growth, and exhibits a strong defect in DNA replication. Bxb1 gp47 and its relatives are also unusual in containing conserved motifs associated with a phosphoesterase function, although we have not been able to show robust phosphoesterase activity of the proteins, and amino acid substitutions with the conserved motifs do not interfere with RDF activity. We therefore propose that Bxb1 gp47 and its relatives provide an important function in phage DNA replication that has been co-opted by the integration machinery of the serine-integrases to control the directionality of recombination.     

Large serine recombinase domain structure and attachment site binding

Abstract

Large serine recombinases (LSRs) catalyze the movement of DNA elements into and out of bacterial chromosomes using site-specific recombination between short DNA “attachment sites”. The LSRs that function as bacteriophage integrases carry out integration between attachment sites in the phage (attP) and in the host (attB). This process is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded recombination directionality factor, nor does recombination typically occur between other pairings of attachment sites. Although the mechanics of strand exchange are reasonably well understood through studies of the closely related resolvase and invertase serine recombinases, many of the fundamental aspects of the LSR reactions have until recently remained poorly understood on a structural level. In this review, we discuss the results of several years worth of biochemical and molecular genetic studies of LSRs in light of recently described structural models of LSR–DNA complexes. The focus is understanding LSR domain structure, how LSRs bind to the attP and attB attachment sites, and the differences between attP-binding and attB-binding modes. The simplicity, site-selectivity and strong directionality of the LSRs has led to their use as important tools in a number of genetic engineering applications in a wide variety of organisms. Given the important potential role of LSR enzymes in genetic engineering and gene therapy, understanding the structure and DNA-binding properties of LSRs is of fundamental importance for those seeking to enhance or alter specificity and functionality in these systems.     

Analysis of the Site-Specific Integration System of the <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> Phage μ1/6

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3′ end of a putative tRNA^Thr gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.     

A phage protein that binds φC31 integrase to switch its directionality

The serine integrase, Int, from the Streptomyces phage φC31 mediates the integration and excision of the phage genome into and out of the host chromosome. Integrases usually require a recombination directionality factor (RDF) or Xis to control integration and excision and, as φC31 Int only mediates integration in the absence of other phage proteins, we sought to identify a φC31 RDF. Here we report that the φC31 early protein, gp3 activated attL x attR recombination and inhibited attP x attB recombination. Gp3 binds to Int in solution and when Int is bound to the attachment sites. Kinetic analysis of the excision reaction suggested that gp3 modifies the interactions between Int and the substrates to form an active recombinase. In the presence of gp3, Int assembles an excision synaptic complex and the accumulation of the integration complex is inhibited. The structure of the excision synaptic complex, like that of the hyperactive mutant of Int, IntE449K, appeared to be biased towards one that favours the production of correctly joined products. The functional properties of φC31 gp3 resemble those of the evolutionarily unrelated RDF from phage Bxb1, suggesting that these two RDFs have arisen through convergent evolution.     

Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes

Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10^1–3 in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.     

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobacillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP?×?attB recombination event. In a cell-free extract of E. coli at 42°?C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trans between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro.     

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobacillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP × attB recombination event. In a cell-free extract of E. coli at 42° C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trans between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro. Received: 22 July 1998 / Accepted: 4 June 1999