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.     

Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease.

The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.     

Analysis of inhibitors of the site-specific recombination reaction mediated by Tn3 resolvase

The Tn3-encoded resolvase protein promotes a site-specific recombination reaction between two directly repeated copies of the recombination site res. Several inhibitors that block this event in vitro have been isolated. In this study four of these inhibitors were tested on various steps in the recombination reaction. Two inhibitors. A9387 and A1062, inhibit resolvase binding to the res site. Further, DNase I footprinting revealed that at certain concentrations of A9387 and A1062, resolvase was preferentially bound to site I of res, the site containing the recombinational crossover point. The two other inhibitors, A20812 and A21960, do not affect resolvase binding and bending of the DNA but inhibit synapse formation between resolvase and two directly repeated res sites.     

Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.     

Inhibitor analysis of synapsis by resolvase

Previously, we isolated several inhibitors that block the site-specific recombination reaction mediated by the Tn3-encoded resolvase protein. One class of inhibitors blocks resolvase binding to the recombination (res) sitc, and a second class inhibits synapse formation between resolvase and two directly repeated res sites. In this report, we identify an inhibitor, A20832, that does not inhibit resolvase binding to res, as measured by filter binding, or synapse formation. Inhibition of resolvase-promoted site-specific recombination by A20832 occurs postsynaptically at strand cleavage. DNase I analysis in the presence of A20832 indicates that only site I of res is bound by resolvase.     

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 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.     

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.     

Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site

Resolvase, the product of the tnpR gene of the transposable element gamma delta, mediates a site-specific recombination between two copies of the element directly repeated on the same replicon. The resolution site, res, at which resolvase acts lies in the intercistronic region between the tnpA and tnpR genes. We have studied this site-specific recombination in vitro. In the absence of Mg2+, a resolvase-res complex is formed, which contains DNA molecules that have been cleaved at res. Our data suggest that in this complex resolvase is covalently attached to the 5' ends of the cleaved DNA, leaving free 3' hydroxyl groups. DNA cleavage is stimulated by the interaction of two res sites on the same substrate molecule and appears to be an intermediate step in normal res site recombination. We show that the DNA is cut within a region previously identified as containing the crossover point at the palindromic sequence 5'- (see formula in text) to generate 3' extensions of two bases.     

Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity

Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.     

Site-specific recombination by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase

The resolvases from the transposons Tn3 and Tn21 are homologous proteins but they possess distinct specificities for the DNA sequence at their respective res sites. The DNA binding domain of resolvase contains an amino acid sequence that can be aligned with the helix-turn-helix motif of other DNA binding proteins. Mutations in the gene for Tn21 resolvase were made by replacing the section of DNA that codes for the helix-turn-helix with synthetic oligonucleotides. Each mutation substituted one amino acid in Tn21 resolvase with either the corresponding residue from Tn3 resolvase or a residue that lacks hydrogen bonding functions. The ability of these proteins to mediate recombination between res sites from either Tn21 or Tn3 was measured in vivo and in vitro. With one exception, where a glutamate residue had been replaced by leucine, the activity of these mutants was similar to that of wild-type Tn21 resolvase. A further mutation was made in which the complete recognition helix of Tn21 resolvase was replaced with that from Tn3 resolvase. This protein retained activity in recombining Tn21 res sites, though at a reduced level relative to wild-type; the reduction can be assigned entirely to weakened binding to this DNA. Neither this mutant nor any other derivative of Tn21 resolvase had any detectable activity for recombination between res sites from Tn3. The exchange of this section of amino acid sequence between the two resolvases is therefore insufficient to alter the DNA sequence specificity for recombination.     

Isolation and characterization of the Tn3 resolvase synaptic intermediate.

We have isolated in quantitative yield the synaptic intermediate formed during site-specific recombination by Tn3 resolvase and characterized it by restriction endonuclease mapping, electron microscopy and topological methods. The intermediate accumulates at low reaction temperatures and is stabilized by crosslinking of the resolvase protomers with glutaraldehyde. The DNA-resolvase complex that maintains the structure of the intermediate (the synaptosome) is approximately 100 A in diameter, forms specifically at resolution (res) sites, and requires two res sites in a supercoiled DNA molecule. Resolvase bound to individual res sites protects approximately -0.5 supercoil per site from relaxation by a topoisomerase, whereas the formation of the synaptosome protects -3 supercoils and condenses the associated DNA to a supercoil density 2.5 times that of the non-complexed substrate. Although recombination requires two directly repeated res sites, both direct and inverted sites form synaptosomes. We conclude that the specificity of recombination is achieved by a three-stage recognition system: binding of resolvase to separate sites, formation of the synaptosome and determination of site orientation from within the complex.     

Site-specific recombination and topoisomerization by Tn21 resolvase: role of metal ions.

The resolvase from the transposon Tn21 catalyses site-specific recombination between the two res sites on its DNA substrate both in the absence and presence of Mg2+ ions. This contrasts with reports on the resolvase from gamma-delta (Tn1000) and on other recombinational proteins that are homologous to Tn21 resolvase but which need Mg2+ for their activity. Magnesium ions could enhance the activity of Tn21 resolvase, as did a number of other cations but some metal ions such as Ni2+ inhibit recombination. The metal ions are not directly involved in the catalytic process but probably affect recombination by altering the conformation of the DNA. Tn21 resolvase relaxes its DNA substrate in the presence and in the absence of Mg2+, and also in ionic conditions that inhibit recombination. Hence, the topoisomerization reflects an activity of resolvase that is distinct from recombination. However, the two activities are functions of the same DNA-protein complex. The complex contains about 6 molecules of the resolvase dimer per molecule of DNA.     

Synapsis and catalysis by activated Tn3 resolvase mutants

The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.     

Specificity of DNA recognition in the nucleoprotein complex for site-specific recombination by Tn21 resolvase.

Resolvases from Tn3-like transposons catalyse site-specific recombination at res sites. Each res site has 3 binding sites for resolvase, I, II, and III. The res sites in Tn3 and Tn21 have similar structures at I and II but they differ at III. Mutagenesis of the Tn21 res site showed that sub-site III is essential for recombination though the sequences in III that are recognized by Tn21 resolvase are positioned differently from the equivalent sequences in the Tn3 site. The deletion of III caused a 1,000-fold drop in the rate of recombination. But other mutations at III, changing 3 or 4 consecutive base pairs, caused only 1.5- to 4-fold decreases in rate, even when the mutations were in target sequences for this helix-turn-helix protein. The reason why Tn21 resolvase has similar activities at a number of different DNA sequences may be due to the multiplicity of protein-protein and protein-DNA interactions in its recombinogenic complex. This lack of precision may be a general feature of nucleoprotein complexes.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.     

The gamma delta resolvase bends the res site into a recombinogenic complex.

We have characterized complexes between the gamma delta resolvase and its recombination site, res, using both a gel retardation assay and DNase I cleavage. The mobility of resolvase-res complexes in polyacrylamide gels is sensitive to the location of res within the DNA fragment and is at a minimum when res is at its center. This behavior is characteristic of a protein-dependent bend. By the same assay we have found that bends are induced upon the binding of resolvase to each of the three individual binding sites that constitute res. In the wild-type res, the centers of binding sites I and II are 53 bp apart and the central section of the intersite DNA is sensitive to DNase I cleavage. We find that insertions of 10 or 21 bp (one or two turns of the DNA helix) have no discernible effect on the ability of res to recombine or to form complexes with resolvase. However, insertions of short segment (e.g. 6 or 17 bp) equivalent to nonintegral numbers of helical turns, inhibit recombination and prevent the formation of the normally compact resolvase-res complex. Complexes of resolvase with res containing 10 or 21 bp insertions exhibit a pattern of enhanced and suppressed DNase I cleavages that suggest that the intersite segment is curved. This curvature requires both that site I and II are appropriately spaced, and that site III is also present and occupied.     

Cooperativity mutants of the gamma delta resolvase identify an essential interdimer interaction

gamma delta resolvase, a transposon-encoded site-specific recombinase, catalyzes the resolution of the cointegrate intermediate of gamma delta transposition. The recombination reaction involves the formation of a catalytic nucleoprotein complex whose structure is determined by specific protein-DNA and protein-protein interactions. We have isolated many resolvase mutants and have identified four that are unable to mediate a subclass of higher order protein-protein interactions necessary for recombination. This mutant phenotype is characterized by an inability to catalyze recombination, a loss of cooperative binding to res DNA, and a failure to induce looping out of the DNA between two resolvase binding sites within res. The amino acid side chains identified by the cooperativity mutants cluster on a surface of the protein that mediates an interaction between resolvase dimers in a crystallographic tetramer. We have therefore identified a region of resolvase that mediates an interdimer protein-protein interaction necessary for the formation of the recombinogenic synaptic intermediate.     

Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta resolvase subunits.

The packing arrangement of the 12 subunits of intact gamma delta resolvase in the unit cell of a hexagonal crystal form suggests a model for site-specific recombination that involves a DNA-mediated synaptic intermediate. The crystal structure has been determined by molecular replacement and partially refined at 2.8/3.5 A resolution. Although the small DNA-binding domain is disordered in these crystals, packing considerations show that only a small region of space in the crystal could accommodate a domain of its size. A family of related models for a synaptic complex between two DNA duplexes and 12 monomers that are arranged as situated in the crystal is consistent with the known topology of the complex and the distances between the three resolvase dimer-binding sites per DNA; further, these models place the two DNA recombination sites in contact with each other between two resolvase dimers, implying that strand exchange is accomplished through direct DNA-DNA interaction. A major role postulated, then, for the resolvase protein assembly is to stabilize a res DNA structure that is close to the topological transition state of the reaction.