Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination

Bacteriophage P1 encodes its own site-specific recombination system consisting of a site at which recombination takes place called loxP and a recombinase called Cre. A number of lambda and plasmid substrates containing two loxP sites have been constructed. Using these substrates we have shown both in vivo and in vitro that a fully functional loxP site is composed of no more than 60 bp. In vitro, when an extract containing Cre is used, recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs efficiently. The most surprising result from the in vitro studies is that 50% of the products of recombination between loxP sites on a supercoiled DNA substrate are present as free supercoiled circles. The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule.     

Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein

Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein. The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region. A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry. This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro. This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled. The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA. We have determined that these nicks occur in both the wild-type and the mutant sites. The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive. We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase.     

Phage P1 Cre-loxP site-specific recombination. Effects of DNA supercoiling on catenation and knotting of recombinant products

Bacteriophage P1 contains a site-specific recombination system consisting of a site, loxP, and a recombinase protein Cre. We have shown that with purified Cre protein we can carry out recombination between two loxP sites in vitro. When that recombination occurs between two sites in direct orientation on the same DNA molecule, we observed the production of free and catenated circular molecules. In this paper we show that recombination between sites in opposite orientation leads to both knotted and unknotted circular products. We also demonstrate that the production of catenanes and knots is influenced by two factors: (1) supercoiling in the DNA substrate, supercoiled DNA substrates yield significantly more catenated and knotted products than nicked circular substrates; and (2) mutations in the loxP site, a class of mutations have been isolated that carry out recombination but result in a distribution of products in which the ratio of catenanes to free circles is increased over that observed with a wild-type site. A more detailed analysis of the products from recombination between wild-type sites indicates: (1) that the catenanes or knots produced by recombination are both simple and complex; (2) that the ratio of free products to catenanes is independent of the distance between the two directly repeated loxP sites; and (3) that for DNA substrates with four loxP sites significant recombination between non-adjacent sites occurs to give free circular products. These observations provide insights into how two loxP sites are brought together during recombination.     

Linking-number changes in the DNA substrate during Cre-mediated loxP site-specific recombination

We have examined the linking-number changes that occur during phage P1 Cre-mediated recombination in vitro between two loxP sites. Such recombination reactions can be divided into three types: intramolecular inversion, in which recombination occurs between two loxP sites in opposite orientations on the same DNA substrate; intramolecular excision, where recombination occurs between two loxP sites that are in the same orientation on the DNA substrate; and intermolecular recombination, which occurs between two loxP sites on separate DNA molecules. Our results indicate that inversion changes the linking number of the substrate DNA by two topological turns. With a negatively supercoiled substrate, the product is changed by +2 turns. A relaxed substrate yields products that have been changed by either +2 or -2 turns. For intermolecular reactions, the sum of the linking numbers of each of the two starting circles is equal to the linking number of the dimer circle generated by recombination, and no change occurs in linking number. For intramolecular excision reactions, the data are most consistent, with no change in linking number during recombination. These results are discussed in terms of models for alignment and synapsis of the recombining sites and the mechanism of strand exchange.     

Topological selectivity of a hybrid site-specific recombination system with elements from Tn3 res/resolvase and bacteriophage P1 loxP/Cre.

In order to investigate the functions of the parts of the Tn 3 recombination site res, we created hybrid recombination sites by placing the loxP site for Cre recombinase adjacent to the "accessory" resolvase-binding sites II and III of res. The efficiency and product topology of in vitro recombination by Cre between two of these hybrid sites were affected by the addition of Tn 3 resolvase. The effects of resolvase addition were dependent on the relative orientation and spacing of the elements of the hybrid sites. Substrates with sites II and III of res close to loxP gave specific catenated or knotted products (four-noded catenane, three-noded knot) when resolvase and Cre were added together. The product topological complexity increased when the length of the spacer DNA segment between loxP and res site II was increased. Similar resolvase-induced effects on Cre recombination product topology were observed in reactions of substrates with loxP sites adjacent to full res sites. The results demonstrate that the res accessory sites are sufficient to impose topological selectivity on recombination, and imply that intertwining of two sets of accessory sites defines the simple catenane product topology in normal resolvase-mediated recombination. They are also consistent with current models for the mechanism of catalysis by Cre.     

Protein-induced local DNA bends regulate global topology of recombination products

The tyrosine family of recombinases produces two smaller DNA circles when acting on circular DNA harboring two recombination sites in head-to-tail orientation. If the substrate is supercoiled, these circles can be unlinked or form multiply linked catenanes. The topological complexity of the products varies strongly even for similar recombination systems. This dependence has been solved here. Our computer simulation of the synapsis showed that the bend angles, phi, created in isolated recombination sites by protein binding before assembly of the full complex, determine the product topology. To verify the validity of this theoretical finding we measured the values of phi for Cre/loxP and Flp/FRT systems. The measurement was based on cyclization of the protein-bound short DNA fragments in solution. Despite the striking similarity of the synapses for these recombinases, action of Cre on head-to-tail target sites produces mainly unlinked circles, while that of Flp yields multiply linked catenanes. In full agreement with theoretical expectations we found that the values of phi for these systems are very different, close to 35 degrees and 80 degrees, respectively. Our findings have general implications in how small protein machines acting locally on large DNA molecules exploit statistical properties of their substrates to bring about directed global changes in topology.     

Altered directionality in the Cre-LoxP site-specific recombination pathway

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.     

A mutational analysis of the bacteriophage P1 recombinase Cre

Bacteriophage P1 encodes a 38,600 Mr site-specific recombinase, Cre, that is responsible for reciprocal recombination between sites on the P1 DNA called loxP. Using in vitro mutagenesis 67 cre mutants representing a total of 37 unique changes have been characterized. The mutations result in a wide variety of phenotypes as judged by the varying ability of each mutant Cre protein to excise a lacZ gene located between two loxP sites in vivo. Although the mutations are found throughout the entire cre gene, almost half are located near the carboxyl terminus of the protein, suggesting a region critical for recombinase function. DNA binding assays using partially purified mutant proteins indicate that mutations in two widely separated regions of the protein each result in loss of heparin-resistant complexes between Cre and a loxP site. These results suggest that Cre may contain two separate domains, both of which are involved in binding to loxP.     

The role of the loxP spacer region in P1 site-specific recombination.

The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction. The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region. Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region. We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites. There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites. When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.     

Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites

The bacteriophage P1-encoded recombinase Cre forms a simple DNA-protein complex at the specific recognition site loxP. Furthermore, Cre is able to mediate a synaptic union of two loxP sites. When two loxP sites are on the same linear DNA molecule, Cre binds the two sites together to form a circular protein-DNA complex. These complexes can be resolved into a linear DNA molecule and a closed circular DNA molecule, the end products of site-specific recombination.     

Communication between accessory factors and the Cre recombinase at hybrid psi-loxP sites

By placing loxP adjacent to the accessory sequences from the Xer/psi multimer resolution system, we have imposed topological selectivity and specificity on Cre/loxP recombination. In this hybrid recombination system, the Xer accessory protein PepA binds to psi accessory sequences, interwraps them, and brings the loxP sites together such that the product of recombination is a four-node catenane. Here, we investigate communication between PepA and Cre by varying the distance between loxP and the accessory sequences, and by altering the orientation of loxP. The yield of four-node catenane and the efficiency of recombination in the presence of PepA varied with the helical phase of loxP with respect to the accessory sequences. When the orientation of loxP was reversed, or when half a helical turn was added between the accessory sequences and loxP, PepA reversed the preferred order of strand exchange by Cre at loxP. The results imply that PepA and the accessory sequences define precisely the geometry of the synapse formed by the loxP sites, and that this overcomes the innate preference of Cre to initiate recombination on the bottom strand of loxP. Further analysis of our results demonstrates that PepA can stimulate strand exchange by Cre in two distinct synaptic complexes, with the C-terminal domains of Cre facing either towards or away from PepA. Thus, no specific PepA-recombinase interaction is required, and correct juxtaposition of the loxP sites is sufficient to activate Cre in this system.     

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.     

Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome.

The cre gene of coliphage P1 encodes a 38 kDa protein which efficiently promotes both intra- and intermolecular recombination at specific 34 bp sites called loxP. To demonstrate that the Cre protein can promote DNA recombination at loxP sites resident on a mammalian chromosome, a mouse cell line was constructed containing two directly repeated loxP sites flanking a 2.5 kb yeast DNA fragment and inserted between the SV40 promoter and the neo structural gene to disrupt expression of the neo gene. Expression of the cre gene in this cell line results in excision of the intervening yeast DNA and thus permits sufficient expression of the neo gene to allow cell growth in high concentrations of G418. Southern analysis indicated that Cre-mediated excision occurred at the loxP sites. In the absence of the cre gene such excisive events are quite rare. Cre-mediated recombination should thus be quite useful in effecting a variety of genomic rearrangements in eukaryotic cells.     

Cre重组酶结构与功能的研究进展

Cre/loxP定位重组系统来源于噬菌体P₁,由Cre重组酶和loxP位点两部分组成。在Cre重组酶的介导下,设定的DNA片段可以被切除,可以发生倒位,亦可造成定点的整合。由于其作用方式高效简单,Cre/loxP定位重组系统已在特定基因的删除、基因功能的鉴定、外源基因的整合、基因捕获及染色体工程等方面得到了有效的利用,在转基因的酵母、植物、昆虫、哺乳动物的体内外DNA重组方面成为一个有力的工具。这里就Cre重组酶的结构、功能及该定位重组系统的应用等方面的研究进行了综述。     

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

In Cre-loxP recombination system, Cre recombinase binds cooperatively to two 13bp inverted repeats in a 34bp loxP and catalyzes strand exchange in the 8bp spacer region. Up to date, spacer sequences within the recombined loxP sites derived from two loxP sties that have different 8bp spacer regions have never been analyzed. In the present study, we analyzed the spacer sequences within the recombined products, resulted from intramolecular recombination between heterologous loxP sites including M2, M3, M7, M11, and 2272 in vivo and in vitro. From the analyses, it was found that loxP sites with aberrant 8bp spacers can be generated from Cre-mediated recombination between heterologous loxP sites at significantly high frequency, proposing the possibility that recombination between heterologous loxP sites would have not undergone typical formula of Cre-loxP recombination.     

Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex

Cre recombinase catalyzes the cleavage and religation of DNA at loxP sites. The enzyme is a homotetramer in its functional state, and the symmetry of the protein complex enforces a pseudo-palindromic symmetry upon the loxP sequence. The Cre-lox system is a powerful tool for many researchers. However, broader application of the system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. As a first step toward achieving recombination at arbitrary asymmetric target sites, we have broken the symmetry of the Cre tetramer assembly. Using a combination of computational and rational protein design, we have engineered an alternative interface between Cre monomers that is functional yet incompatible with the wild-type interface. Wild-type and engineered interface halves can be mixed to create two distinct Cre mutants, neither of which are functional in isolation, but which can form an active heterotetramer when combined. When these distinct mutants possess different DNA specificities, control over complex assembly directly discourages recombination at unwanted half-site combinations, enhancing the specificity of asymmetric site recombination. The engineered Cre mutants exhibit this assembly pattern in a variety of contexts, including mammalian cells.     

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.     

转基因植物高效删除标记基因的实用型双元转化载体

利用雌激素诱导的Cre/loxPDNA重组系统,构建用于转基因植物删除标记基因的实用新型载体,将所有非目的基因放于两同向loxP位点间,多克隆位点位于两loxP位点外,可插入目的基因,载体设计非常实用。在插入目的基因GUS后转化烟草,雌激素诱导,分析诱导前后DNA的重组情况、不同时间基因转录水平,以及目的基因在诱导前后的表达。结果显示,诱导后染色体DNA实现了高效重组,非目的基因片段消失,目的基因GUS在诱导后表达正常。     

利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法     

Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site.

A general approach for isolating large nested deletions in P1 artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) by retrofitting with a loxP site-containing Tn10 mini-transposon is described. Cre-mediated recombination between the loxP site existing in these clones and one introduced by transposition leads to deletions and inversions of the DNA between these sites. Large deletions are selectively recovered by transducing the retrofitted PAC or BAC clones with P1 phage. The requirement that both loxP sites in the cointegrate be packaged into a P1 head ensures that only large deletions are rescued. PCR analyses identified these deletions as products of legitimate recombination between loxP sites mediated by Cre protein. BACs produce deletions much more efficiently than PACs although the former cannot be induced to greater than unit copy in cells. Mammalian cell-responsive antibiotic resistance markers are introduced as part of the transposon into genomic clone deletions for subsequent functional analysis. Most importantly, the loxP site retrofitting and P1 transduction can be performed in the same bacterial host containing these clones directly isolated from PAC or BAC libraries. These procedures should facilitate physical and functional mapping of genes and regulatory elements in these large plasmids.