Structural basis for topological regulation of Tn3 resolvase.

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Site-specific DNA recombinases alter the connectivity of DNA by recognizing specific DNA sequences, then cutting the DNA strands and pasting them together in a new configuration. Such enzymes play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful biotechnology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. However, others have evolved elaborate mechanisms to ensure that only one of multiple possible recombination products is produced. Tn3 resolvase has long been known to be regulated by DNA topology­that is, it will cut and reconnect two target sequences only if they lie on the same DNA molecule, and if they are in the proper relative orientation. This study presents new structural and biochemical data that lead to a new, detailed model of the large protein­DNA complex formed by Tn3 resolvase and its cognate sites. This 3D model illustrates how DNA topology can be harnessed to regulate the activity of a recombinase and provides a basis for engineering Tn3 resolvase and related recombination systems as genome editing tools.

The protein-protein interactions required for assembly of the Tn3 resolution synapse.

The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis ("Bart"). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific "R" interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.

Snapshots of a molecular swivel in action.

Members of the serine family of site-specific recombinases exchange DNA strands via 180° rotation about a central protein-protein interface. Modeling of this process has been hampered by the lack of structures in more than one rotational state for any individual serine recombinase. Here we report crystal structures of the catalytic domains of four constitutively active mutants of the serine recombinase Sin, providing snapshots of rotational states not previously visualized for Sin, including two seen in the same crystal. Normal mode analysis predicted that each tetramer's lowest frequency mode (i.e. most accessible large-scale motion) mimics rotation: two protomers rotate as a pair with respect to the other two. Our analyses also suggest that rotation is not a rigid body movement around a single symmetry axis but instead uses multiple pivot points and entails internal motions within each subunit.

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.

Arginine as a general acid catalyst in serine recombinase-mediated DNA cleavage.

Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3'O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3' phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3'O leaving group and is the prime candidate for the general acid that protonates the 3'O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.

Roles of two large serine recombinases in mobilizing the methicillin-resistance cassette SCCmec.

Methicillin-resistant Staphylococcus aureus (MRSA) emerged via acquisition of a mobile element, staphylococcal cassette chromosome mec (SCCmec). Integration and excision of SCCmec is mediated by an unusual site-specific recombination system. Most variants of SCCmec encode two recombinases, CcrA and CcrB, that belong to the large serine family. Since CcrA and CcrB are always found together, we sought to address their specific roles. We show here that CcrA and CcrB can carry out both excisive and integrative recombination in Escherichia coli in the absence of any host-specific or SCCmec-encoded cofactors. CcrA and CcrB are promiscuous in their substrate choice: they act on many non-canonical pairs of recombination sites in addition to the canonical ones, which may explain tandem insertions into the SCCmec attachment site. Moreover, CcrB is always required, but CcrA is only required if one of the four half-sites is present. Recombinational activity correlates with DNA binding: CcrA recognizes only that half-site, which overlaps a conserved coding frame on the host chromosome. Therefore, we propose that CcrA serves as a specificity factor that emerged through modular evolution to enable recognition of a bacterial recombination site that is not an inverted repeat.

Intermediates in serine recombinase-mediated site-specific recombination.

Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer-dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.

Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.

The resolvase Sin regulates DNA strand exchange by assembling an elaborate interwound synaptosome containing catalytic and regulatory Sin tetramers, and an architectural DNA-bending protein. The crystal structure of the regulatory tetramer was recently solved, providing new insights into the structural basis for regulation. Here we describe the selection and characterization of two classes of Sin mutations that, respectively, bypass or disrupt the functions of the regulatory tetramer. Activating mutations, which allow the catalytic tetramer to assemble and function independently at site I (the crossover site), were found at approximately 20% of residues in the N-terminal domain. The most strongly activating mutation (Q115R) stabilized a catalytically active synaptic tetramer in vitro. The positions of these mutations suggest that they act by destabilizing the conformation of the ground-state site I-bound dimers, or by stabilizing the altered conformation of the active catalytic tetramer. Mutations that block activation by the regulatory tetramer mapped to just two residues, F52 and R54, supporting a functional role for a previously reported crystallographic dimer-dimer interface. We suggest how F52/R54 contacts between regulatory and catalytic subunits might promote assembly of the active catalytic tetramer within the synaptosome.

Orchestrating serine resolvases.

A remarkable feature of the serine resolvases is their regulation: the wild-type enzymes will catalyse intra- but not inter-molecular recombination, can sense the relative orientation of their sites and can exchange strands directionally, despite the fact that there is no net release of chemical bond energy. The key to this regulation is that they are only active within a large intertwined complex called the 'synaptosome'. Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a 'topological filter'. Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. The regulatory portion of this complex contains additional copies of the recombinase and sometimes other DNA-bending proteins. We are using a combination of X-ray crystallography, biochemistry and genetics to model the full synaptic complex and to understand how the regulatory portion activates the crossover-site-bound recombinases.

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 x site I recombination promoted by these variants can be as fast as res x 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.

Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system.

Many natural DNA site-specific recombination systems achieve directionality and/or selectivity by making recombinants with a specific DNA topology. This property requires that the DNA architecture of the synapse and the mechanism of strand exchange are both under strict control. Previously we reported that Tn3 resolvase-mediated synapsis of the accessory binding sites from the Tn3 recombination site res can impose topological selectivity on Cre/loxP recombination. Here, we show that the topology of these reactions is profoundly affected by subtle changes in the hybrid recombination site les. Reversing the orientation of loxP relative to the res accessory sequence, or adding 4 bp to the DNA between loxP and the accessory sequence, can switch between two-noded and four-noded catenane products. By analysing Holliday junction intermediates, we show that the innate bias in the order of strand exchanges at loxP is maintained despite the changes in topology. We conclude that a specific synaptic structure formed by resolvase and the res accessory sequences permits Cre to align the adjoining loxP sites in several distinct ways, and that resolvase-mediated intertwining of the accessory sequences may be less than has been assumed previously.

Synapsis and strand exchange in the resolution and DNA inversion reactions catalysed by the beta recombinase.

In the presence of a sequence-independent chromatin-associated protein, such as Hbsu or HMGB, the beta recombinase catalyses resolution between two directly oriented recombination sites (six sites) and both resolution and DNA inversion between two inversely oriented six sites. Assembly of the synaptic complex requires binding of the beta recombinase to the six sites and the presence of Hbsu. Whether resolution or inversion will take place depends on the relative orientation of the two six sites, the level of DNA supercoiling and the amounts of Hbsu. In this work, the topologies of the products of the resolution and inversion reactions were analysed. The resolution reaction generated mainly singly catenated DNA circles, while DNA inversion gave rise to unknotted circles and small amounts of DNA molecules containing 3- or 5-noded knots. In spite of the distinctive features of the beta system, the topology of synapsis and strand exchange during the resolution reaction is similar to that of Tn3 and gammadelta resolvases. The ability of the beta recombinase to catalyse both inversion and resolution reactions probably reflects different possible architectures of the synaptic complex, which to a large extent depends on Hbsu.

Interactions of protein complexes on supercoiled DNA: the mechanism of selective synapsis by Tn3 resolvase.

"Looping" interactions of distant sites on DNA molecules, mediated by DNA-binding proteins, feature in many regulated genetic processes. We used plasmids containing up to six res recombination sites for Tn3 resolvase to analyse looping interactions (synapsis) in this system. We observed that in plasmids with four or more res sites, certain pairs of sites recombine faster than others. The relative rates of recombination depend on the number, relative orientation, and arrangement of the sites. To account for the differences in rate, we propose that pairing interactions between resolvase-bound res sites are in a state of rapid flux, leading to configurations in which the maximum number of sites within each supercoiled substrate molecule are synapsed in a topologically simple arrangement. Recombination rates reflect the steady state concentrations of these synapse configurations. Our results are at variance with models for selective synapsis that rely on ordered motions within supercoiled DNA, "slithering" or "tracking", but are compatible with models that call for reversible synapsis of pairs of sites by random collision, followed by formation of an interwound productive synapse.

Synapsis of Tn3 recombination sites: unpaired sites destabilize synapses by a partner exchange mechanism.

Catalysis of site-specific recombination is preceded by the formation of a synapse comprising two DNA sites and multiple subunits of the recombinase, together with other "accessory" proteins in some cases. We investigated the stability of synapses of Tn3 resolvase-bound res recombination sites, in plasmids containing either two or three res sites. Although synapses are long-lived in plasmids with just two res sites, persisting for tens of minutes, a synapse of any two sites is relatively short-lived in plasmids with three res sites. The three alternative pairwise synapses that can be formed in three-res plasmids re-assort rapidly relative to the rate of recombination. We propose a "partner exchange" mechanism for this re-assortment, involving direct attack on a synapse by an unpaired res site. This mechanism reconciles studies on selective synapsis in multi-res substrates, which imply rapid interchange of synaptic pairings, with studies indicating that synapses of two Tn3res sites are stable.

A proposed mechanism for IS607-family serine transposases.

BACKGROUND: The transposases encoded by the IS607 family of mobile elements are unusual serine recombinases with an inverted domain order and minimal specificity for target DNA. RESULTS: Structural genomics groups have determined three crystal structures of the catalytic domains of IS607 family transposases. The dimers formed by these catalytic domains are very different from those seen for other serine recombinases and include interactions that usually only occur upon formation of a synaptic tetramer. CONCLUSIONS: Based on these structures, we propose a model for how IS607-family transposases could form a synaptic tetramer. The model suggests that, unlike other serine recombinases, these enzymes carry out sequence-specific DNA binding and catalysis in trans: the DNA binding and catalytic domains of each subunit are proposed to interact with different DNA duplexes. The model also suggests an explanation for the minimal target DNA specificity.

Structural basis for catalytic activation of a serine recombinase.

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.

Sin resolvase catalytic activity and oligomerization state are tightly coupled.

Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3'-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.

DNA bending in the Sin recombination synapse: functional replacement of HU by IHF.

The serine recombinase Sin requires a non-specific DNA-bending protein such as Hbsu for activity at its recombination site resH. Hbsu, and Sin subunits bound at site II of resH, together regulate recombination, ensuring selectivity for directly repeated resH sites by specifying assembly of an intertwined synapse. To investigate the role of the DNA-bending protein in defining the architecture of the synapse, we constructed a chimaeric recombination site (resF) which allows Hbsu to be substituted by IHF, binding specifically between site I (the crossover site) and site II. Two Sin dimers and one IHF dimer can bind together to the closely adjoining sites in resF, forming folded complexes. The precise position of the IHF site within the site I-site II spacer determines the conformation of these complexes, and also the reactivity of the resF sites in recombination assays. The data suggest that a sharp bend with a specific geometry is required in the spacer DNA, to bring the Sin dimers at sites I and II together in the correct relative orientation for synapse assembly and regulation, consistent with our model for a highly condensed synapse in which Hbsu/IHF has a purely architectural function.

Implications of structures of synaptic tetramers of gamma delta resolvase for the mechanism of recombination.

The structures of two mutants of the site-specific recombinase, gammadelta resolvase, that form activated tetramers have been determined. One, at 3.5-A resolution, forms a synaptic intermediate of resolvase that is covalently linked to two cleaved DNAs, whereas the other is of an unliganded structure determined at 2.1-A resolution. Comparisons of the four known tetrameric resolvase structures show that the subunits interact through the formation of a common core of four helices. The N-terminal halves of these helices superimpose well on each other, whereas the orientations of their C termini are more variable. The catalytic domains of resolvase in the unliganded structure are arranged asymmetrically, demonstrating that their positions can move substantially while preserving the four-helix core that forms the tetramer. These results suggest that the precleavage synaptic tetramer of gammadelta resolvase, whose structure is not known, may be formed by a similar four-helix core, but differ in the relative orientations of its catalytic and DNA-binding domains.

Regulation of Sin recombinase by accessory proteins.

Sin recombinase from Staphylococcus aureus acts selectively on directly repeated resH sites, assembling an intertwined synapse in which exactly three supercoils are trapped between the points of strand exchange. Resolution requires the two Sin binding sites in resH (site I, where strand exchange occurs, and site II) and a non-specific DNA-bending protein (e.g. Hbsu). We show that a single amino acid substitution in Sin (I100T) is sufficient to relax the normal requirements for site II and Hbsu. Using this hyperactive protein, and the variant recombination site resH(AT), we investigate the roles of site II and Hbsu in synapsis and strand exchange. We conclude that Sin bound at site II, and Hbsu, act together to control site I alignment and the topology of the synapse, and to stimulate strand exchange.