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.     

Purification and properties of the DNA invertase gin encoded by bacteriophage Mu

The host range of bacteriophage Mu is regulated through an invertible segment. Inversion requires the presence of two properly oriented recombination sites and a recombinational enhancer sis. The reaction is catalyzed by the Mu-encoded DNA invertase Gin and a host factor termed factors for inversion stimulation (FISs). We present a novel purification scheme for Gin. Purified Gin alone catalyzes the inversion reaction at very low efficiency recombining less than 0.8% of substrate molecules. When supplemented with FIS substrates containing the recombinational enhancer are recombined efficiently. Stoichiometric amounts of Gin are required for recombination.     

Gin mutants that can be suppressed by a Fis-independent mutation.

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.     

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.     

Intermediates in Hin-mediated DNA inversion: a role for Fis and the recombinational enhancer in the strand exchange reaction.

The site-specific inversion reaction controlling flagellin synthesis in Salmonella involves the function of three proteins: Hin, Fis and HU. The DNA substrate must be supercoiled and contain a recombinational enhancer sequence in addition to the two recombination sites. Using mutant substrates or modified reaction conditions, large amounts of complexes can be generated which are recognized by double-stranded breaks within both recombination sites upon quenching. The cleaved molecules contain 2-bp staggered cuts within the central dinucleotide of the recombination site. Hin is covalently associated with the 5' end while the protruding 3' end contains a free hydoxyl. We demonstrate that complexes generated in the presence of an active enhancer are intermediates that have advanced past the major rate limiting step(s) of the reaction. In the absence of a functional enhancer, Hin is also able to assemble and catalyze site-specific cleavages within the two recombination sites. However, these complexes are kinetically distinct from the complexes assembled with a functional enhancer and cannot generate inversion without an active enhancer. The results suggest that strand exchange leading to inversion is mediated by double-stranded cleavage of DNA at both recombination sites followed by the rotation of strands to position the DNA into the recombinant configuration. The role of the enhancer and DNA supercoiling in these reactions is discussed.     

Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu

G inversion in bacteriophage Mu requires the product of the DNA invertase gene gin and an Escherichia coli host factor termed FIS (factor for inversion stimulation). A recombination substrate must contain two recombination sites, arranged as inverted repeats, and a recombinational enhancer sequence termed sis. FIS has been purified to homogeneity. The purified protein has a relative molecular weight of 12,000 when analyzed under denaturing conditions. The intact protein behaves as a dimer of relative molecular weight 25,000 in gel filtration analysis. The purified protein does not possess any recombinogenic activity when assayed in the absence of the DNA-invertase Gin. In the presence of purified Gin FIS is the only additional protein required for efficient inversion. By performing gel retention assays, we show that FIS is a DNA-binding protein, which specifically binds to DNA fragments containing the recombinational enhancer sis.     

Processive recombination by the phage Mu Gin system: implications for the mechanisms of DNA strand exchange, DNA site alignment, and enhancer action

The Gin DNA invertase of bacteriophage Mu carries out processive recombination in which multiple rounds of exchange follow synaptic complex formation. The stereostructure of the knotted products determined by electron microscopy establishes critical features of site synapsis and DNA exchange. Surprisingly, the invertase knots substrates with directly repeated sites as well as those with inverted sites. The results suggest that the Gin synaptic complex contains three mutually perpendicular dyads; one is the axis of site rotation during exchange, and they cause inverted and direct site substrates to form a similar synaptic complex. The extensive knotting by Gin has implications for the energetics of recombination and shows that the enhancer for recombination is required only at an early stage, and thus may normally operate in a hit-and-run fashion.     

Recombination site selection by Tn3 resolvase: topological tests of a tracking mechanism

In vitro recombination by Tn3 resolvase of plasmids containing two directly repeated recombination (res) sites generates two singly interlinked catenated rings. This simple product catenane structure was maintained over a wide range of substrate supercoil densities and in a reaction mixture in which phage lambda Int-mediated recombination generated its characteristic multiply interlinked forms. Using substrates containing four res sites, we found that resolvase recombined neighboring res sites with high preference. This position effect implies that resolvase searches systematically along the DNA for a partner site. Intervening res sites in the opposite orientation did not prevent translocation. We analyzed the geometric arrangement of the interlocked rings after multiple recombination events in a four-site substrate and the pattern of segregation of nonspecific reporter rings catenated to the standard substrate. The results of these novel topological tests imply that the translocating enzyme may not make continuous contact with the DNA.     

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro

In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild-type recombination sites in vivo is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils.     

Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution

The Hin DNA invertase promotes a site-specific DNA recombination reaction in the Salmonella chromosome. The native Hin reaction exhibits overwhelming selectivity for promoting inversions between appropriately oriented recombination sites and requires the Fis regulatory protein, a recombinational enhancer, and a supercoiled DNA substrate. Here, we report a robust recombination reaction employing oligonucleotide substrates and a hyperactive mutant form of Hin. Synaptic complex intermediates purified by gel electrophoresis were found to contain four Hin protomers bound to two recombination sites. Each Hin protomer is associated covalently with a cleaved DNA end. The cleaved complexes can be ligated into both parental and recombinant orientations at equivalent frequencies, provided the core residues can base-pair, and are readily disassembled into separated DNA fragments bound by Hin dimers. Kinetic analyses reveal that synapsis occurs rapidly, followed by comparatively slow Hin-catalyzed DNA cleavage. Subsequent steps of the reaction, including DNA exchange and ligation, are fast. Thus, post-synaptic step(s) required for DNA cleavage limit the overall rate of the recombination reaction.     

Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer

The alternate expression of flagellin genes in Salmonella is the result of an inversion of a 996 bp segment of chromosomal DNA. We have analyzed the components of this site-specific recombination reaction in an in vitro system derived from E. coli. Efficient Hin-mediated inversion requires the 20,000 MW Hin protein and a proteinase K-sensitive host component. The supercoiled DNA substrate must contain two 26 bp recombination sites in inverted configuration and a 60 bp sequence that increases the rate of recombination over 20-fold. This recombinational enhancer can function at many different locations and consists of at least two noncontiguous sequence domains whose relative orientation, but not precise spacing, with respect to each other is important. Synthetically derived wild-type and mutant recombination sites were constructed to analyze the sequence and structural features that are important within the recombination site.     

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.     

Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.     

DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly.

Site-specific DNA inversion by the Hin recombinase requires the formation of a multicomponent nucleo-protein structure called an invertasome. In this structure, the two recombination sites bound by Hin are assembled together at the Fis-bound recombinational enhancer with the requisite looping of the intervening DNA segments. We have analyzed the role of the HU protein in invertasome assembly when the enhancer is located at variable positions close to one of the recombination sites. In the absence of HU in vitro and in hupA hupB mutant cells in vivo, invertasome assembly is very inefficient when there is < 104 bp of DNA between the enhancer and recombination site. Invertasome assembly in the presence of HU in vitro or in vivo displayed a periodicity beginning with 60 bp of intervening DNA that reflected its helical repeat. The average helical repeat for this DNA region was calculated by autocorrelation and Fourier transformation to be 11.2 bp per turn for supercoiled DNA both in the presence of HU in vitro and in hup+ cells in vivo. HU is the only protein in Escherichia coli that can promote invertasome formation with short DNA lengths between the enhancer and recombination sites. However, the presence of certain polyamines and a protein activity present in HeLa nuclear extracts can efficiently substitute for HU in invertasome assembly. These data support a model in which HU binds non-specifically to the DNA between the enhancer and recombination site to facilitate DNA looping.     

Geometric arrangements of Tn3 resolvase sites

Site-specific recombination by Tn3 resolvase normally occurs in vitro and in vivo only between directly repeated res sites on the same supercoiled DNA molecule. However, with multiply interlinked catenane substrates consisting of two DNA rings each containing a single res site, resolvase efficiently carried out intermolecular recombination. The topology of the knots produced by several rounds of this reaction proves that the DNA within the synaptic intermediate is coiled in an interwound (plectonemic) fashion rather than wrapped solenoidally around resolvase as in previously characterized supercoiled DNA-protein complexes. The synaptic intermediate can contain equivalently supercoil, catenane, or knot crossings as long as the res sites have a right-handed coiling and a particular relative orientation. The structure of the product knots and catenanes also shows the path the DNA takes during strand exchange. Intermolecular recombination within multiply linked catenanes required negative supercoiling, as does the standard intramolecular reaction.     

Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda

Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E. coli integration host factor (IHF). A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I. (1980) Cell 20, 721-729). In the present work, we have purified Int-h, the product of this variant gene. In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E. coli supplement. This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein. When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product. Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+. However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination. A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules. In the presence of IHF, Int-h favors intramolecular recombination, as does Int+. However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination.     

Topology of Xer recombination on catenanes produced by lambda integrase.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.     

Cre/lox位点特异重组系统在转基因植物中的应用

位点特异重组系统由重组酶和相应的重组酶识别位点组成,通过两者间的相互作用,实现外源基因精确整合与切除等一系列遗传操作.主要可分为Cre/lox系统、FLP/frt系统、R/RS系统和Gin/gix系统.目前,研究最充分应用最广泛的位点特异重组系统为Cre/lox系统.此系统为位点特异重组系统家族中的一员,由38.5kDCre重组酶和34bplox位点组成,最早被应用于动物转基因研究,包括基因敲除、基因激活、基因易位等.近年来,随着研究的深入,Cre/lox系统被逐步应用到植物研究中,并在诸多领域取得重大进展.本文总结归纳了Cre/lox系统在定点整合、定点切除以及叶绿体转化等方面的最新研究成果,旨在为利用Cre/lox系统构建环境安全和高效表达的植物遗传转化体系提供参考.     

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.     

Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange

The integrase encoded by the temperate phage HP1 promotes the site-specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli , and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP -containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA-bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi-protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four-stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four-stranded component was produced more rapidly than any product, and its steady-state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.