The Holliday junction intermediates of lambda integrative and excisive recombination respond differently to the bending proteins integration host factor and excisionase.

In lambda site-specific recombination, the integrative and excisive reactions proceed via two different Holliday junction intermediates, both of which are generated and resolved by a pair of sequentially ordered single strand exchanges. Factors affecting the directionality and efficiency of the second pair of strand exchanges were examined using artificial Holliday junctions (chi-forms). The integrative and excisive recombination intermediates respond differently to the accessory DNA bending proteins integration host factor and excisionase (Xis). These differences between the two recombination intermediates result from a different interaction pattern between proteins binding to the left (P arm) and right (P' arm) of the crossover region. The effect of Xis protein on the directionality of resolution, i.e. the choice of which strands are exchanged, is consistent with a role in promoting the second strand exchange during excision. Proteins binding to the left of the crossover region (P arm) primarily influence the directionality of resolution, while proteins binding to the right (P' arm) have a greater effect on the overall efficiency of resolution. Together, the effect of proteins binding to sites in the P and P' arms is to greatly enhance resolution of the two different Holliday intermediates and to favor resolution in the 'forward' direction for both integrative and excisive recombination.     

Interactions between lambda Int molecules bound to sites in the region of strand exchange are required for efficient Holliday junction resolution

lambda Site-specific recombination proceeds via two sequential single-strand exchanges that first generate and then resolve a Holliday recombination intermediate. The resolution of artificial Holliday junctions (chi-forms) is well suited to studying the mechanisms involved in reciprocal strand exchange because the linear products of this reaction are stable and easily quantitated. To study the interactions between Int molecules bound at the sites of strand exchange, artificial Holliday junctions containing only the seven base-pair overlap region and the four core-type Int binding sites were used as a model system. In vitro resolution of these structures yields products of both top- and bottom-strand exchange. An abortive product resulting from simultaneous cleavage of the top and bottom strands also occurs at low frequency. Inactivation of one of the four Int binding sites by multiple base substitutions does not significantly affect the efficiency of resolution but has a dramatic effect on the directionality, i.e. the choice of top- or bottom-strand exchange. When any two of the four core-type sites are similarly inactivated, strand exchange is very inefficient and the amount of aberrant cleavage is somewhat greater than for the Holliday junction with four intact Int binding sites. Analysis of the resolution products of Holliday junctions with various combinations of defective Int binding sites leads to the following conclusions: (1) three functional core-type Int binding sites are necessary and sufficient for a strand exchange; (2) the Int molecules that are partners in a strand exchange interact with Int bound to a "cross-core" site that is not directly involved in carrying out the reaction; (3) Int molecules bound to the core-type sites interact in a way that reduces the occurrence of abortive double-strand cleavage events.     

Dissecting the resolution reaction of lambda integrase using suicide Holliday junction substrates.

A reciprocal strand exchange between two DNA helices generates the crossed-strand intermediate, or Holliday junction, which is common to many pathways of homologous and site-specific recombination. The Int family of recombinases are unique in their ability to both make and resolve Holliday junctions. Previous experiments utilizing 'synthetic' att site Holliday junctions to study the mechanisms associated with the cleavage, transfer and ligation of DNA strands have been confined to studying reciprocal strand exchanges (a pair of temporally overlapping strand cleavages). To circumvent this limitation, we have designed synthetic suicide Holliday junctions that make it possible to monitor individual DNA strand cleavage events. These substrates contain a pre-existing nick in the vicinity of the Int binding site; when Int introduces a second nick into these substrates, the 5'OH nucleophile required for ligation (in either the forward or reverse reaction) is lost by diffusion, thus trapping the covalent protein-DNA intermediate. The results indicate that resolution (involving two partner Ints) is stimulated by additional 'cross-core' Ints as a result of enhanced cleavage rates, and not as a result of enhanced co-ordination of cleavage. Several models for the role of the 'cross-core' Ints during resolution are discussed, as well as the usefulness of these substrates for studying additional aspects of the Holliday junction resolution reaction.     

The isomeric preference of Holliday junctions influences resolution bias by lambda integrase.

Lambda site-specific recombination proceeds by a pair of sequential strand exchanges that first generate and then resolve a Holliday junction intermediate. A family of synthetic Holliday junctions with the branch point constrained to the center of the 7 bp overlap region was used to show that resolution of the top strands and resolution of the bottom strands are symmetrical but stereochemically distinct processes. Lambda integrase is sensitive to isomeric structure, preferentially resolving the pair of strands that are crossed in the protein-free Holliday junction. At the branch point of stacked immobile Holliday junctions, the number of purines is preferentially maximized in the crossed (versus continuous) strands if there is an inequality of purines between strands of opposite polarity. This stacking preference was used to anticipate the resolution bias of freely mobile junctions and thereby to reinforce the conclusions with monomobile junctions. The results provide a strong indication that in the complete recombination reaction a restacking of helices occurs between the top and bottom strand exchanges.     

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

Bacteriophage lambda site-specific recombination proceeds with a defined order of strand exchanges

Previous work has established that integration of the genome of bacteriophage lambda into the chromosome of its bacterial host proceeds via two independent strand exchanges, which make and then resolve a Holliday-structure intermediate. We find that a phosphorothioate substitution at the site of exchange in one strand of a recombination site depresses the yield of Holliday structures much more than a similar substitution in the other strand. Furthermore, we show that the Holliday structures that accumulate in unblocked reactions have all been made by recombination of one particular pair of strands. We conclude that there is a strong bias in the choice of strands that initiate crossing-over. Excision, the recombination reaction that excises the integrated prophage, exhibits the same bias as integration. This proves, at least at the level of strand exchange, that excision is not the simple reversal of integration. We have altered the relative orientation of parts of the phage attachment site, attP, to demonstrate that the strand-exchange bias is determined not by the local environment around the point of exchange in the core of attP but by more distant elements in its arms. This suggests that the order of the strand exchanges is dictated by an asymmetry in the way that the nucleosome-like structure that forms at attP brings the bacterial site, attB, into juxtaposition prior to strand exchange. Finally, we use the altered attP to show that homology between attP and attB is most critical when it is adjacent to the point of strand exchange.     

The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination

Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.     

The Effect of Attachment Site Mutations on Strand Exchange in Bacteriophage λ Site-Specific Recombination

Recombination of phage λ attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination,, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.     

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.     

Site-specific recombination in human cells catalyzed by phage lambda integrase mutants

Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells.     

Sequential strand exchange by XerC and XerD during site-specific recombination at dif

Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges.     

Directional resolution of synthetic holliday structures by the Cre recombinase.

The Cre recombinase of bacteriophage P1 cleaves its target site, loxP, in a defined order. Recombination is initiated on one pair of strands to form a Holliday intermediate, which is then resolved by cleavage and exchange of the other pair of strands to yield recombinant products. To investigate the influence of the loxP sequence on the directionality of resolution, we constructed synthetic Holliday (chi) structures containing either wild-type or mutant lox sites. We found that Cre preferentially resolved the synthetic wild-type chi structures on a particular pair of strands. The bias in the direction of resolution was dictated by the asymmetric loxP sequence since the resolution bias was abolished with symmetric lox sites. Systematic substitutions of the loxP site revealed that the bases immediately 5' to the scissile phosphodiester bonds were primarily responsible for the directionality of resolution. Interchanging these base pairs was sufficient to reverse the resolution bias. The Cre-lox co-crystal structures show that Lys(86) makes a base-specific contact with guanine immediately 5' to one of the scissile phosphates. Substituting Lys(86) with alanine resulted in a reduction of the resolution bias, indicating that this amino acid is important for establishing the bias in resolution.     

Accessory factors determine the order of strand exchange in Xer recombination at psi

Xer site-specific recombination in Escherichia coli converts plasmid multimers to monomers, thereby ensuring their correct segregation at cell division. Xer recombination at the psi site of plasmid pSC101 is preferentially intramolecular, giving products of a single topology. This intramolecular selectivity is imposed by accessory proteins, which bind at psi accessory sequences and activate Xer recombination at the psi core. Strand exchange proceeds sequentially within the psi core; XerC first exchanges top strands to produce Holliday junctions, then XerD exchanges bottom strands to give final products. In this study, recombination was analysed at sites in which the psi core was inverted with respect to the accessory sequences. A plasmid containing two inverted-core psi sites recombined with a reversed order of strand exchange, but with unchanged product topology. Thus the architecture of the synapse, formed by accessory proteins binding to accessory sequences, determines the order of strand exchange at psi. This finding has important implications for the way in which accessory proteins interact with the recombinases.     

Action of site-specific recombinases XerC and XerD on tethered Holliday junctions.

In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.     

Cre induces an asymmetric DNA bend in its target loxP site

Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.     

Xer site-specific recombination. DNA strand rejoining by recombinase XerC

Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli. Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction. The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins. This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase. In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex.     

Switching catalytic activity in the XerCD site-specific recombination machine.

The tyrosine family site-specific recombinases, XerCD, function in the conversion of circular dimer replicons to monomers. In the recombining complex that contains two synapsed recombination sites and two molecules each of XerC and XerD, the DNA strand-exchange reactions are separated in time and space. XerC initiates recombination to form a Holliday junction intermediate, which undergoes a conformational change to provide a substrate for strand exchange by XerD. XerCD are two-domain proteins, whose C-terminal domains contain all of the catalytic residues. We show that XerC or XerD variants lacking their N-terminal domains are active in recombination when combined with their wild-type partner. Nevertheless, the normal pattern of catalysis is dramatically altered; strand exchange by the recombinase variant is stimulated, while that by the wild-type partner recombinase is impaired. The primary determinants for the mutant phenotype reside in the region of alpha-helix B of XerD. We propose that altered interactions within the recombining heterotetramer lead to changes in the relative concentrations of the two alternative Holliday junction substrates that are recombined by XerC or XerD, respectively.     

The bacteroides NBU1 integrase performs a homology-independent strand exchange to form a holliday junction intermediate

The Bacteroides mobilizable transposon NBU1 uses an integrase (IntN1) that is a tyrosine recombinase for its integration and excision from the host chromosome. Previously we showed that IntN1 makes 7-bp staggered cuts within the NBU1 att sites, and certain mismatches within the crossover region of the attN1 site (G(-2)C attN1) or the chromosomal target site (C(-3)G attBT1-1) enhanced the in vivo integration efficiency. Here we describe an in vitro integration system for NBU1. We used nicked substrates and a Holliday junction trapping peptide to show that NBU1 integration proceeds via formation of a Holliday junction intermediate that is formed by exchange of bottom strands. Some mismatches next to the first strand exchange site (in reactions with C(-3)G attBT1-1 or G(-2)C attN1 with their wild-type partner site) not only allowed formation of the Holliday junction intermediate but also increased the rate of recombinant formation. The second strand exchange appears to be homology-dependent. IntN1 is the only tyrosine recombinase known to catalyze a reaction that is more efficient in the presence of mismatches and where the first strand exchange is homology-independent. The possible mechanisms by which the mismatches stimulate recombination are discussed.     

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering,analytical ultracentrifugation,and NMR and FT-IR spectroscopy

Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.