A model for the gamma delta resolvase synaptic complex

The serine recombinase gamma delta resolvase performs site-specific recombination in an elaborate synaptic complex containing 12 resolvase subunits and two 114-base pair res sites. Here we present an alternative structural model for the synaptic complex. Resolvase subunits in the complex contact their neighbors in equivalent ways, using three principal interactions, one of which is a newly proposed synaptic interaction. Evidence in support of this interaction is provided by mutations at the interface that either enable resolvase to synapse two copies of site I or inhibit synapsis of complete res sites. In our model, the two crossover sites are far apart, separated by the resolvase catalytic domains bound to them. Thus, recombination would require a substantial rearrangement of resolvase subunits or domains.     

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.     

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.     

Crystal structure of an intermediate of rotating dimers within the synaptic tetramer of the G-segment invertase

The serine family of site-specific DNA recombination enzymes accomplishes strand cleavage, exchange and religation using a synaptic protein tetramer. A double-strand break intermediate in which each protein subunit is covalently linked to the target DNA substrate ensures that the recombination event will not damage the DNA. The previous structure of a tetrameric synaptic complex of γδ resolvase linked to two cleaved DNA strands had suggested a rotational mechanism of recombination in which one dimer rotates 180° about the flat exchange interface for strand exchange. Here, we report the crystal structure of a synaptic tetramer of an unliganded activated mutant (M114V) of the G-segment invertase (Gin) in which one dimer half is rotated by 26° or 154° relative to the other dimer when compared with the dimers in the synaptic complex of γδ resolvase. Modeling shows that this rotational orientation of Gin is not compatible with its being able to bind uncleaved DNA, implying that this structure represents an intermediate in the process of strand exchange. Thus, our structure provides direct evidence for the proposed rotational mechanism of site-specific recombination.     

The architecture of the gammadelta resolvase crossover site synaptic complex revealed by using constrained DNA substrates

Activated mutants of the serine recombinase, gammadelta resolvase, form a simplified recombinogenic synaptic complex containing a tetramer of resolvase and two crossover sites. We have probed the architecture of this complex by measuring the efficiency of recombination of a series of constrained DNA substrates (with phased recombination sites separated by an IHF-induced U-turn); this serves as a direct report on the topology of a productive synapse. Our data show that in the active complex, the catalytic domains from two resolvase dimers form a central core, while the DNA binding domains and the DNA lie on the outside. In addition, the crossover sites cross one another to form a local positive node. The implications of our data for the mechanism of strand exchange and the process of resolvase activation are discussed.     

Dynamics of long-range interactions on DNA: the speed of synapsis during site-specific recombination by resolvase.

A noninvasive method for monitoring communications on DNA was developed from the specificity of resolvase for the arrangement of its recombinational sites. Constraints in DNA structure, caused by interactions between distant sites, can be detected by resolvase as they arise. The method was used to follow the formation and decay of synaptic intermediates during site-specific recombination by resolvase. Synaptic complexes were formed very rapidly, at a rate limited by the initial association of the protein with DNA rather than the physical motion of DNA segments. The recombinational sites seem to encounter each other by an ordered motion, perhaps dictated by DNA supercoiling instead of random collisions, so that the first encounter produces the active complex.     

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.     

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.     

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.     

Architecture of a serine recombinase-DNA regulatory complex

An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated.     

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.     

The 43 residue DNA binding domain of gamma delta resolvase binds adjacent major and minor grooves of DNA.

The carboxyl-terminal domain of gamma delta resolvase binds to each half of the three resolvase binding sites that constitute the recombination site, res. Ethylation inhibition experiments show that the phosphate contacts made by the C-terminal DNA binding domain are similar to those made by intact resolvase, with the exception of a single phosphate at the inside end of each contact region which is contacted solely by the intact resolvase. The DNA binding domain makes essentially identical contacts to all 6 half sites, whereas the intact resolvase makes slightly different contacts to each binding site. Despite its small size, only 43 amino acid residues, the resolvase C-terminal domain interacts with an unusually large segment of DNA. Phosphate contacts extend across an adjacent major and minor groove of DNA and about one third of the circumference around the helix. The minimal binding segment, determined experimentally, is a 12 bp sequence that includes the 9 base pair inverted repeat (common to all half sites), the adjacent 3 base pairs (towards the center of the intact resolvase binding site), and phosphates at both ends.     

The catalytic residues of Tn3 resolvase

To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site (‘site I’), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.     

A DNA-binding domain swap converts the invertase gin into a resolvase

DNA resolvases and invertases are closely related, yet catalyze recombination within two distinct nucleoprotein structures termed synaptosomes and invertasomes, respectively. Different protein-protein and protein-DNA interactions guide the assembly of each type of recombinogenic complex, as well as the subsequent activation of DNA strand exchange. Here we show that invertase Gin catalyzes factor for inversion stimulation dependent inversion on isolated copies of sites I from ISXc5 res, which is typically utilized by the corresponding resolvase. The concomitant binding of Gin to sites I and III in res, however, inhibits recombination. A chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of ISXc5 resolvase, recombines two res with high efficiency. Gin must therefore contain residues proficient for both synaptosome formation and activation of strand exchange. Surprisingly, this chimera is unable to assemble a productive invertasome; a result which implies a role for the C-terminal domain in invertasome formation that goes beyond DNA binding.     

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.     

Recombination of nicked DNA knots by gamma delta resolvase suggests a variant model for the mechanism of strand exchange.

Fast and efficient recombination catalyzed by gamma delta resolvase in vitro requires negative DNA supercoiling of plasmid substrates. The current model for recombination suggests that supercoiling is required to drive DNA strand exchange within a synaptic complex by 'simple rotation' of DNA-linked resolvase promoters. Surprisingly, DNA knots are recombined efficiently in the absence of supercoiling, whereby the rate of recombination increases with the number of irreducible DNA segment crossings, or nodes, within each substrate knot. Recombination products contain three knot nodes less than substrates, suggesting that a reduction in writhe drives the reaction. However, the proposed protomer rotation model predicts that writhe is not altered during the process of strand transfer but, instead, is reduced only when a synaptic complex disassembles after strand exchange. I present evidence that recombination of knotted and of linear substrates coincides with a disassembly of synaptic complexes. The results lead to a variant model for strand exchange on non-supercoiled substrates in which a specific disassembly of the synaptic complex, triggered by a reduction in writhe, guides the cleaved DNA into the recombinant configuration.     

DNA supercoiling determines the activation energy barrier for site specific recombination by Tn21 resolvase.

A kinetic analysis of site specific recombination by Tn21 resolvase has been carried out using DNA substrates of varying superhelicities. The rates for the formation of the recombinant product increased with increasing superhelicity up to a maximum value, after which further increases in superhelicity caused no further increase in rate. The reactions with DNA of reduced superhelicity were extremely slow, yet they eventually led to virtually all of the substrate being converted to product. Hence, the level of DNA superhelicity must determine the activation energy barrier for at least one of the steps within the reaction pathway that can be rate-limiting. In the presence (but not in the absence) of Mg2+ ions, the DNA was fully saturated with resolvase whenever the protein was in stoichiometric excess over resolvase binding sites on the DNA. Thus the process affected by DNA supercoiling cannot be coupled to the binding of resolvase. Instead, the step whose rate is determined by supercoiling seems to be located within the reaction pathway after the synapse. However, these reactions may involve two forms of the synaptic complex that are converted to the recombinant product at different rates.     

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.     

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.     

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.