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.     

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.     

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.     

The topological mechanism of phage lambda integrase.

Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int.     

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.     

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.     

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.     

Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants.

The Gin recombination system of phage Mu mediates inversion of the DNA sequence between two sites (gix). In addition to Gin protein and gix sites, recombination requires an enhancer bound by the host factor FIS. We analyzed mutants of Gin that function in the absence of the enhancer and FIS and mediate deletion and intermolecular fusion in addition to inversion. The linking number changes caused by inversion imply that mutant Gin alone can form the same synaptic complex and can use the same strand exchange mechanism as the complete wild-type system. However, the linking number changes also reveal that unlike wild-type Gin, mutant Gin can recombine through more than one synaptic complex and can relax DNA in the absence of synapsis. This expanded repertoire allows mutant Gin to mediate DNA rearrangements not performed by wild-type Gin. Because mutant Gin, but not wild-type Gin, unwinds gix site DNA upon binding, we postulate that FIS and the enhancer function with (-) supercoiling to promote this unwinding with wild-type Gin. The analysis of the topological changes during DNA fusion shows that both the parallel gix site configuration and the right-handed rotation of the sites during exchange of wild-type Gin are a result of the (-) supercoiling of the substrate and the number of entrapped supercoils in the synaptic complex.     

Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein

Bacteriophage P1 encodes a site-specific recombination system that consists of a site (loxP) at which recombination occurs and a gene, cre, whose protein product is essential for recombination. The loxP-Cre recombination event can be studied in greater detail by the use of an in vitro system that efficiently carries out recombination between two loxP sites. This paper presents a purification and characterization of the Cre protein (Mr = 35,000), which is the only protein required for the in vitro reaction. No high energy cofactors are needed. The purified Cre protein binds to loxP-containing DNA and makes complexes that are resistant to heparin. Cre efficiently converts 70% of the DNA substrate to products and appears to act stoichiometrically. The action of Cre on a loxP2 supercoiled substrate containing two directly repeated loxP sites results in product molecules that are topologically unlinked. Several models to account for the ability of Cre to produce free supercoiled products are discussed.     

Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses

Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.     

A new combination of mutated loxPs in a vector for construction of phage antibody libraries

In the construction of large antibody libraries by in vivo recombination, two non-homogeneous loxP sites are required for the exchange of Vgenes between phagemids to create many new VH-VL combinations.The mutated loxP511 was designed not to recombine with the wild-type loxP (loxPwt) in early studies and a combination of the two has been used to construct antibody libraries. But recent reports have shown that recombination occurs between loxPwt and loxP511. This suggests that the combinational use of loxP511 and loxPwt might lead to the loss of the V gene diversity of antibody libraries. Therefore, it is necessary to find a new combination of loxPs to avoid the excision recombination in the antibody library. In this study,we found that the excision recombination between loxP511 and loxP2272, another mutated loxP sequence,was undetectable within one phagemid, while the excision recombination between loxP511 and loxPwt occurred at a frequency of 40%, higher than that reported previously. Furthermore, the in vivo recombination of different phagemids with loxP511 and loxP2272 showed that the V gene exchange was efficiently mediated to produce new VH-VL combinations. It was concluded that the loxP511 and loxP2272 combination was more favorable for reducing the excision recombination and constructing large phage antibody libraries with high diversity.     

Site-specific recombination of bacteriophage lambda. The change in topological linking number associated with exchange of DNA strands

The changes in supercoiling that accompany site-specific recombination have been measured. In each experiment, the substrate was a circle that contained two attachment sites oriented as an inverted repeat; recombination between the sites inverts one segment of the circle with respect to the other. Using conditions developed in the accompanying work, a measurable amount of the recombinant is in the form of unknotted, simple circles. The difference between the topological linking number of this product relative to that of the substrate can be determined directly from the change in mobility during agarose gel electrophoresis. With partially supercoiled substrates, both integrative and excisive recombination are characterized by a unique change in linking number, a relaxation of two topological turns. For excisive recombination, it has been possible to study closed circular substrates that lack supercoils. In this case, changes in linking number of both +2 and -2 are observed. These results are used to evaluate various proposals for synapsis and strand exchange in bacteriophage lambda site-specific recombination.     

Site-specific recombination in eukaryotic cells mediated by mutant lambda integrases: implications for synaptic complex formation and the reactivity of episomal DNA segments

Mutant lambda integrases catalyze site-specific DNA recombination in the absence of accessory factors IHF, XIS, and negative DNA supercoiling. Here we investigate the effects that a human cellular environment exerts on these reactions in order to (i) gain further insights into mechanistic aspects of recombination in eukaryotic cells and (ii) to further develop the Int system for biotechnological applications. First, we compared intra- and intermolecular integrative as well as excisive recombination pathways on episomal substrates after co-transfection with recombinase expression vectors. Our results demonstrate that, within 24 hours after transfection, intermolecular recombination by mutant integrase is at least as efficient as intramolecular recombination. Second, a significant intermolecular recombination activity was observed between two copies of a recombination site containing only the 21 bp comprising core-type DNA sequence. This basic activity was stimulated several-fold when arm-type DNA sequences were present in addition to core sites. Therefore, one recombination pathway in human cells involves mutant integrases bound solely at core sites, which is reminiscent of the Flp/FRT and Cre/loxP pathways. The stimulatory effect of arm-type sequences could be explained by an increase in integrase concentration in the vicinity of core sites. We show, in addition, that an N-terminal truncated mutant integrase exhibited only a very weak recombinogenic activity in a eukaryotic background. This result strengthens a functional role for the N-terminal domain in recombination in addition to its arm-type DNA-binding activity. Finally, we demonstrate that low level integrative recombination by wild-type integrase is stimulated when purified integration host factor is co-transfected. This corroborates our previous conclusion that sufficient amounts of eukaryotic protein co-factors, which could functionally replace IHF, are not present in human cells. It also provides a potential means to control site-specific recombination in eukaryotic cells.     

Geometry of site alignment during int family recombination: antiparallel synapsis by the Flp recombinase

The Flp site-specific recombinase functions in the copy number amplification of the yeast 2 microm plasmid. The recombination reaction is catalyzed by four monomers of Flp bound to two separate, but identical, recombination sites (FRT sites) and occurs in two sequential pairs of strand exchanges. The relative orientation of the two recombination sites during synapsis was examined. Topoisomerase relaxation and nick ligation were used to detect topological nodes introduced by the synapse prior to the chemical steps of recombination. A single negative supercoil was found to be trapped by Flp in substrates with inverted FRT sites whereas no trapped supercoils were observed with direct repeats. The topology of products resulting from Flp-mediated recombination adjacent to a well characterised synapse, that of Tn3 resolvase/res, was analyzed. The deletion and inversion reactions yielded the four noded catenane and the three noded knot, respectively, as the simplest and the most abundant products. The linking number change introduced by the Flp-mediated inversion reaction was determined to be +/-2. The most parsimonious explanation of these results is that Flp aligns its recombination sites with antiparallel geometry. The majority of synapses appear to occur without entrapment of additional random plectonemic DNA supercoils between the sites and no additional crossings are introduced as a result of the chemical steps of recombination.     

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.     

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.     

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.     

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.     

Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71

The Cre/loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP-flanked DNA segments. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type-specific cre-transgenes in vivo.     

Preferential recombination between GC clusters in yeast mitochondrial DNA.

Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome.