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.     

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.     

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.     

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.     

Evolutionary perspectives on multiresistance beta-lactamase transposons.

A series of intragenic DNA probes, encoding the major part of the transposase resolvase and inverted repeats of transposons Tn3, Tn21, and Tn2501, were used in hybridization assays for homologous DNA sequences in 18 transposons studied. The tnpA and tnpR probes detected extensive homology with Tn3-like and Tn21-like elements for 11 transposons. This high degree of homology was confirmed with the 38- and 48-base-pair inverted-repeat oligonucleotide probes of Tn3, Tn21, and Tn2501. The Southern-type gel hybridization experiments localized the tnpA-homologous sequences on the physical DNA maps constructed. The genetic and physical maps of the transposons were compared, as were their nucleic acid sequence homologies. These comparisons suggested a subfamily of mobile elements distinct from but related to the Tn21 group. Based on these results, an evolutionary model is proposed and a pedigree is presented for the genesis of multiresistance beta-lactamase transposons.     

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.     

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.     

Behavior of Tn3 resolvase in solution and its interaction with res

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.     

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res 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.     

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.     

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.     

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.     

Solution structure of the Tn3 resolvase-crossover site synaptic complex

Tn3 resolvase is a site-specific DNA recombinase, which catalyzes strand exchange in a synaptic complex containing twelve resolvase subunits and two res sites. Hyperactive mutants of resolvase can form a simpler complex (X synapse) containing a resolvase tetramer and two shorter DNA segments at which strand exchange takes place (site I). We have solved the low-resolution solution structure of the purified, catalytically competent X synapse from small-angle neutron and X-ray scattering data, using methods in which the data are fitted with models constructed by rigid body transformations of a published crystallographic structure of a resolvase dimer bound to site I. Our analysis reveals that the two site I fragments are on the outside of a resolvase tetramer core and provides some information on the quaternary structure of the tetramer. We discuss implications of our structure for the architecture of the natural synaptic complex and the mechanism of strand exchange.     

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.     

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.     

tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution

We have identified a new gene, tnpM, in Tn21 that encodes the 12.6 kilodalton modulator protein. The Tn21 modulator enhances Tn21 transposition and suppresses resolution of cointegrate replicons in vivo. A putative binding site may be located in the N-terminal portion of the TnpR (resolvase) structural gene sequences. Tn501 transposition and cointegrate resolution can be regulated by the subcloned tnpM gene of Tn21 in trans-complementation experiments. Examination of the Tn501 DNA sequence also reveals a potential tnpM coding sequence upstream of the Tn501 resolvase gene. We conclude that Tn21 and Tn501 are different from Tn3 and Tn1000 both in genome organization and in regulation of transposition functions.     

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.