Martin R. Boocock

Catalysis by site-specific recombinases

Site-specific recombination reactions bring about controlled rearrangements of DNA molecules by cutting the DNA at precise points and rejoining the ends to new partners. The recombinases that catalyse these reactions can be grouped into two families by amino acid sequence homology. We describe our current understanding of how these proteins catalyse recombination, and show how the catalytic mechanisms of the two families differ.

Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate.

The herbicide glyphosate (N‐phosphonomethy glycine) is a potent reversible inhibitor of the 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K i 1.1 μM, and uncompetitive with respect to shikimate‐3‐phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate‐3‐phosphate complex.

Chimeric recombinases with designed DNA sequence recognition

Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating “custom-built” recombinases that act at chosen natural target sequences.

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 I×site 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 Tn3 resolvase

Site-specific recombination processes in microbes bring about precise DNA rearrangements which have diverse and important biological functions. The sites and recombinase enzymes used for these processes fall into two distinct families. Here we describe how experiments with one family, exemplified by the resolution system of transposon Tn3, have provided insight into the ways in which DNA and protein interact to bring together distant recombination sites and promote strand exchange.

A Model for the γδ Resolvase Synaptic Complex

Abstract The serine recombinase γδ 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.

Sin recombinase from Staphylococcus aureus: synaptic complex architecture and transposon targeting

The Sin recombinase from Staphylococcus aureus builds a distinctive DNA‐protein synaptic complex to regulate strand exchange. Sin binds at two sites within an 86 basepair (bp) recombination site, resH. We propose that inverted motifs at the crossover site, and tandem motifs at the regulatory site, are recognized by structurally disparate Sin dimers. An essential architectural protein, Hbsu, binds at a discrete central site in resH. Positions of Hbsu‐induced DNA deformation coincide with natural targets for Tn552 integration. Remarkably, Sin has the same topological selectivity as Tn3 and γδ resolvases. Our model for the recombination synapse has at its core an assembly of four Sin dimers; Hbsu plays an architectural role that is taken by two resolvase dimers in models of the Tn3/γδ synapse.

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 × 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 × site I plasmid. Site I × 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.

Architecture of a serine recombinase-DNA regulatory complex

Summary 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.

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.