Resolvase

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Stephen E. Halford - One of the best experts on this subject based on the ideXlab platform.

  • Recombination by Resolvase to analyse DNA communications by the SfiI restriction endonuclease.
    The EMBO journal, 1996
    Co-Authors: Mark D. Szczelkun, Stephen E. Halford
    Abstract:

    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+.

  • Site-specific Recombination atresSites Containing DNA-binding Sequences for both Tn21 and Tn3 Resolvases
    Journal of molecular biology, 1995
    Co-Authors: Panos Soultanas, Mark Oram, Stephen E. Halford
    Abstract:

    Abstract Tn3 and *gamma;δ Resolvases catalyse site-specific recombination atressites from Tn3 but not at Tn21ressites. Tn21 Resolvase has no activity at Tn3 and acts only at Tn21 sites. In both Tn3 and Tn21,reshas three binding sites for the cognate Resolvases; the cross-over site, I; and the accessory sites II and III, from which the bound proteins may stabilize the synaptic complex by protein-protein interactions. In this study hybridressites were made by replacingb either II or III in the Tn21ressite with the equivalent sequence from Tn3. Plasmids containing either a hybrid and a wild-type Tn21ressite, or two hybrid sites, were tested for recombination. Relative to the reaction with two wild-type sites, recombination by Tn21 Resolvase was reduced by replacing II at oneressite and it was reduced further by replacing II at both loci but, in both cases, Tn21 recombination was enhanced by Tn3 or γδ Resolvases. Very few of the amino acid on the external surface of γδ Resolvases are conserved in Tn21. Moreover, mutants of γδ Resolvase with defective protein-protein interactions also enhanced Tn21 recombination at this hybrid site. The Resolvase at II thus seems not to be involved in protein-protein interactions and its main role may be to bend the DNA to the required structure. The replacement of III in the Tn21 site with Tn3 sequence also reduced recombination by Tn21 Resolvase, especially when both loci carried the alteration but, in contrast to before, Tn3 or γδ Resolvases now inhibited the Tn21 reaction. Recombination thus seems to require identical proteins at I and III, perhaps to allow for protein-protein interactions.

  • Site-specific recombination atresSites Containing DNA-binding sequences for both Tn21 Resolvase and CAP
    Journal of molecular biology, 1995
    Co-Authors: Panos Soultanas, Stephen E. Halford
    Abstract:

    Abstract The res sites, the loci for site-specific recombination by Resolvase, contain three binding sites for the protein; the cross-over site, I, and accessory sites II and III. The role of DNA bending by Resolvase was examined by replacing either II or III in the res site from Tn21 with the recognition sequence for a heterologous DNA-bending protein, CAP (the catabolite-gene activator protein from Escherichia coli ). The CAP sequence was placed at either the same position as the target sequence for Tn21 Resolvase or a different position along the DNA. The activity of Tn21 Resolvase for recombination between each hybrid and a wild-type res site was measured in the presence of CAP and cyclic AMP. When III was substituted, CAP inhibited Tn21 recombination, except when the CAP sequence was placed sufficiently far away from site II to allow Resolvase to bind non-specifically to the DNA between II and the CAP site. With the substitutions at II, the extent of Tn21 recombination in the presence of CAP varied with the position of the CAP sequence: more recombination was observed when it superimposed the target sequence for Resolvase than when it was displaced by five base-pairs. Efficient recombination by Tn21 Resolvase thus seems to demand the cognate protein at site III in res , presumably for pprotein-protein interactions in the synaptic complex, while the function of Resolvase at site II can be fulfilled, at least in part, by a heterologous DNA bend.

  • Synapsis by Tn3 Resolvase: speed and dependence on DNA supercoiling.
    Biochemical Society transactions, 1994
    Co-Authors: Mark Oram, Emma Shipstone, Stephen E. Halford
    Abstract:

    The transposon Tn3 encodes a protein known as Resolvase. During transposition, Resolvase catalyses a site-specific recombination between two directly repeated copies of the transposon DNA [I]. Resolvase acts by binding to 120 bp sites on the DNA, known as res sites, each of which contains three sub-sites called I, I1 and 111 [2]. During recombination, three Resolvase dimers bind co-operatively to each res site, forming a resolvosome. Two resolvosomes then interact to form a synaptic complex, in which the strand transfer reactions take place. Resolvases from other Tn3-like transposons such as Tn2l act in the same way. However, T d l Resolvase has no activity at Tn3 res sites and Tn3 Resolvase has no activity at T d l sites [2-41. Both Resolvases, however, have an absolute requirement for a supercoiled substrate. The recombination reactions by either Tn3 or TnZl Resolvases generally take several minutes to convert the DNA substrate to the recombinant product [5,6]. However, the synapsis of two res sites is complete within 0.5 seconds [6]. This was shown by using plasmids such as pSHl that contain two Tn3 res sites interspersed with two T d l sites [7]. On a plasmid of this type, synapsis of the res sites for one Resolvase segregates the other pair of res sites into different topological domains. This segregation inhibits recombination by the second enzyme [6] and synapsis can then be followed by quantifjing the extent of inhibition. The aims of the current work were two-fold: first, to establish the speed of synapsis using rapid reaction techniques; second, to determine the supercoiling requirement of the steps in the reaction that lead to the formation of the synaptic complex. We have used a rapid-reaction quenched-flow apparatus, constructed by Dr. C. Bagshaw, (Department of Biochemistry, University of Leicester) to measure the rate of formation of the Tn3 synaptic complex. With this apparatus, solutions of pSHl DNA and Tn3 Resolvase were loaded separately into two sample chambers, mixed together and then expelled at high velocity into a quench solution. The time between mixing and quenching was varied between 5 and 1000 milliseconds. Typical experiments used 9 nM pSHl and 270 nM Tn3 Resolvase as reactants, with 900 nM Tn21 Resolvase as the quench solution. The amounts of the products from both Tn3 and T d l recombination formed after 20 minutes were then determined. When the mixture of Tn3 Resolvase and DNA was added to Tn21 Resolvase after 200 ms, the DNA yielded almost exclusively Tn3 product: i.e., the Tn3 synaptic complex is formed in less than this time interval. Subsequent work indicated that the speed of synapsis increased if the concentration of Tn3 Resolvase was doubled. Thus, the rate-limiting step in synapsis appears to be the association of the protein with the DNA to form the resolvosome. The effects of substrate superhelicity (ao) on the rate of recombination and on the stability of the synaptic complex were also examined. Substrates that contained either more or fewer supercoils than native pSHl were produced by relaxing the plasmid with calf thymus topoisomerase I in the presence of ethidium bromide [8]. The linking differences were measured from the altered distributions of topoisomers in the treated samples compared with native pSHI, as observed on chloroquine gels. The rates of recombination by both Tn3 and T d 1 Resolvases were reduced when 00 was altered from -0.062 (native DNA) to -0.02 I . Tn21 recombination was also reduced on DNA with a a0 value of 0.038. Tn3 recombination, however, proceeded at comparable rates on DNA with 00 values of -0.038, -0.062 or -0.073. These results with Tn3 Resolvase are similar to an earlier study on the effect of supercoiling on the rate of Tn21 recombination [5], except that the T d l enzyme is more sensitive to reductions in superhelical density. The stabilities of synaptic complexes on pSHl with varying 00 values were determined by using the assay described previously [6]. With native pSHl of a. -0.062, the addition of one Resolvase before the second lead to the majority (275%) of the DNA being commited to recombination by the first Resolvase, even when the second enzyme was in large excess over the first. The commitment to the first enzyme was unaltered on DNA of a0 -0.073. However, on DNA with a0 -0.038, approximately equal amounts of each product were formed (i.e. less commitment to the first Resolvase was observed). This implies that the synaptic complexes formed on DNA of reduced superhelicity are less stable than those on native DNA. The results measuring the speed of synapsis have implications for the dynamics of supercoiled DNA molecules. For example, distant sites on supercoiled DNA may encounter each other by random diffusion [9]. However, the speed of interaction between res sites determined here is too fast to be explained by simple diffusion within a DNA molecule modelled as a dynamic worm-like coil. An alternative proposal is that the sites encounter each other by an ordered 'slithering' motion of the DNA [ 101. Further experiments are planned to explore this possibility.

  • Stereoselectivity of DNA catenane fusion by Resolvase
    Nature, 1994
    Co-Authors: W. Marshall Stark, Christian N. Parker, Stephen E. Halford, Martin R. Boocock
    Abstract:

    COMMUNICATIONS between distant sites on DNA often depend on the way in which the sites are connected1,2. For example, site-specific recombination catalysed by Tn3 Resolvase is most efficient when the 114-base-pair res recombination sites are directly repeated in the same DNA molecule3. In vitro a supercoiled plasmid substrate containing two directly repeated res sites gives a resolution product in which the two recombinant circles are topologically linked as a simple (two-noded) catenane (Fig. la). Resolvase is highly selective in forming this product rather than unlinked circles or more complex catenanes. It does not catalyse recombination between sites on separate supercoiled molecules, or between inverted sites in the same supercoiled molecule3–5. Tn3 resolution removes four negative supercoils from the substrate, an energetically favourable change which may drive the reaction6: in relaxed or nicked circular substrates, resolution is incomplete and slower. Resolvase can catalyse fusion of the circles of a nicked or relaxed catenane, giving a single unknotted circular product6,7. The fusion is the precise topological reversal of resolution, introducing four negative supercoils into a relaxed catenane substrate6, and should therefore not proceed if the catenane is already negatively supercoiled. Here we study recombination between res sites in non-supercoiled DNA circles linked into simple catenanes. We used (+2) and (−2) catenanes, which differ only in the direction in which one circle is threaded through the other (Fig. 2a). Although stereoselectivity is a feature of enzyme catalysis, it is not obvious how Resolvase can distinguish between these subtly different catenane diastereomers. A model for the intertwining of the res site DNA in the catalytically active complex4,7 predicts that only the (−2) catenane will recombine, giving unknotted and 4-noded knot circular products. We have confirmed this prediction for the Tn3 and Tn21 Resolvases.

W. Marshall Stark - One of the best experts on this subject based on the ideXlab platform.

  • Catalysis of site-specific recombination by Tn3 Resolvase.
    Biochemical Society transactions, 2010
    Co-Authors: Femi J. Olorunniji, W. Marshall Stark
    Abstract:

    The active-site interactions involved in the catalysis of DNA site-specific recombination by the serine recombinases are still incompletely understood. Recent crystal structures of synaptic gammadelta Resolvase-DNA intermediates and biochemical analysis of Tn3 Resolvase mutants have provided new insights into the structure of the Resolvase active site, and how interactions of the catalytic residues with the DNA substrate might promote the phosphoryl transfer reactions.

  • Synapsis and catalysis by activated Tn3 Resolvase mutants
    Nucleic acids research, 2008
    Co-Authors: Femi J. Olorunniji, Martin R. Boocock, Sandra V. C. T. Wenwieser, W. Marshall Stark
    Abstract:

    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.

  • Solution Structure of the Tn3 Resolvase-Crossover Site Synaptic Complex
    Molecular cell, 2004
    Co-Authors: Marcelo Nollmann, Olwyn Byron, W. Marshall Stark
    Abstract:

    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.

  • Activating mutations of Tn3 Resolvase marking interfaces important in recombination catalysis and its regulation
    Molecular microbiology, 2003
    Co-Authors: Mary E. Burke, Martin R. Boocock, Sandra V. C. T. Wenwieser, Patricia H. Arnold, Sally-j. Rowland, W. Marshall Stark
    Abstract:

    Summary 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.

  • A Model for the γδ Resolvase Synaptic Complex
    Molecular cell, 2001
    Co-Authors: Gary J. Sarkis, Martin R. Boocock, W. Marshall Stark, Laura Lea Murley, Andres E. Leschziner, Nigel D. F. Grindley
    Abstract:

    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.

Nigel D. F. Grindley - One of the best experts on this subject based on the ideXlab platform.

  • Single-molecule Study of Site-specific DNA Recombination by γδ Resolvase
    Biophysical Journal, 2009
    Co-Authors: Mingxuan Sun, Nigel D. F. Grindley, Hua Bai, John F. Marko
    Abstract:

    γδ Resolvase is a serine recombinase coded by γδ transposon, which catalyzes DNA recombination between two res sites (114 bp) on a negatively supercoiled circular DNA, resulting in two catenated DNA circles. Each res site contains 3 different Resolvase binding sites - site I, II and III, and each binds to a Resolvase dimer. We have developed a single-DNA based system whereby synapsis and recombination should lead to torsional relaxation of a single supercoiled DNA. DNA relaxation catalyzed by γδ Resolvase occurs at much higher rate on DNA substrate containing 2 res sites than those containing 1 or 0 res site. Furthermore, reactions on a 2-res-site substrate show a characteristic ∼200 nm relaxation consistent with the +4 ΔLk observed to be associated with the recombination reaction in bulk experiments. We also have observed topoisomerase activity of γδ Resolvase on the non-specific (0 res) DNA substrate.

  • Implications of structures of synaptic tetramers of γδ Resolvase for the mechanism of recombination
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Satwik Kamtekar, Martin R. Boocock, Sandra V. C. T. Wenwieser, Nigel D. F. Grindley, Melanie J. Cocco, Thomas A. Steitz
    Abstract:

    The structures of two mutants of the site-specific recombinase, γδ 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 γδ 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.

  • The Architecture of the γδ Resolvase Crossover Site Synaptic Complex Revealed by Using Constrained DNA Substrates
    Molecular cell, 2003
    Co-Authors: Andres E. Leschziner, Nigel D. F. Grindley
    Abstract:

    Activated mutants of the serine recombinase, γδ 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.

  • a model for the γδ Resolvase synaptic complex
    Molecular Cell, 2001
    Co-Authors: Gary J. Sarkis, Martin R. Boocock, Laura Lea Murley, Andres E. Leschziner, Marshall W Stark, Nigel D. F. Grindley
    Abstract:

    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.

  • A Model for the γδ Resolvase Synaptic Complex
    Molecular cell, 2001
    Co-Authors: Gary J. Sarkis, Martin R. Boocock, W. Marshall Stark, Laura Lea Murley, Andres E. Leschziner, Nigel D. F. Grindley
    Abstract:

    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.

Martin R. Boocock - One of the best experts on this subject based on the ideXlab platform.

  • Synapsis and catalysis by activated Tn3 Resolvase mutants
    Nucleic acids research, 2008
    Co-Authors: Femi J. Olorunniji, Martin R. Boocock, Sandra V. C. T. Wenwieser, W. Marshall Stark
    Abstract:

    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.

  • Implications of structures of synaptic tetramers of γδ Resolvase for the mechanism of recombination
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Satwik Kamtekar, Martin R. Boocock, Sandra V. C. T. Wenwieser, Nigel D. F. Grindley, Melanie J. Cocco, Thomas A. Steitz
    Abstract:

    The structures of two mutants of the site-specific recombinase, γδ 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 γδ 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.

  • Activating mutations of Tn3 Resolvase marking interfaces important in recombination catalysis and its regulation
    Molecular microbiology, 2003
    Co-Authors: Mary E. Burke, Martin R. Boocock, Sandra V. C. T. Wenwieser, Patricia H. Arnold, Sally-j. Rowland, W. Marshall Stark
    Abstract:

    Summary 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.

  • a model for the γδ Resolvase synaptic complex
    Molecular Cell, 2001
    Co-Authors: Gary J. Sarkis, Martin R. Boocock, Laura Lea Murley, Andres E. Leschziner, Marshall W Stark, Nigel D. F. Grindley
    Abstract:

    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.

  • A Model for the γδ Resolvase Synaptic Complex
    Molecular cell, 2001
    Co-Authors: Gary J. Sarkis, Martin R. Boocock, W. Marshall Stark, Laura Lea Murley, Andres E. Leschziner, Nigel D. F. Grindley
    Abstract:

    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.

Thomas A. Steitz - One of the best experts on this subject based on the ideXlab platform.

  • Implications of structures of synaptic tetramers of γδ Resolvase for the mechanism of recombination
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Satwik Kamtekar, Martin R. Boocock, Sandra V. C. T. Wenwieser, Nigel D. F. Grindley, Melanie J. Cocco, Thomas A. Steitz
    Abstract:

    The structures of two mutants of the site-specific recombinase, γδ 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 γδ 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.

  • crystal structure of the site specific recombinase γδ Resolvase complexed with a 34 by cleavage site
    Cell, 1995
    Co-Authors: Wei Yang, Thomas A. Steitz
    Abstract:

    Abstract The structure of γδ Resolvase complexed with a 34 bp substrate DNA has been determined at 3.0 A resolution. The DNA is sharply bent by 60° toward the major groove and away from the Resolvase catalytic domains at the recombination crossover point. The C-terminal on third of Resolvase, which was disordered in the absence of DNA, forms an arm and a 3-helix DNA-binding domain on the opposite side of the DNA from the N-terminal domain. The arms wrap around the minor groove of the central 16 bp, and the DNA-binding domains interact with the major grooves near the outer boundaries of the binding site. The Resolvase dimer is asymmetric, particularly in the arm region, implying a conformational adaptability that may be important for Resolvase binding to different DNA sites in the synaptosome. It also raises the possibility of a sequential single-strand cleavage mechanism.

  • Crystal structure of the site-specific recombinase gamma delta Resolvase complexed with a 34 bp cleavage site.
    Cell, 1995
    Co-Authors: Wei Yang, Thomas A. Steitz
    Abstract:

    The structure of gamma delta Resolvase complexed with a 34 bp substrate DNA has been determined at 3.0 A resolution. The DNA is sharply bent by 60 degrees toward the major groove and away from the Resolvase catalytic domains at the recombination crossover point. The C-terminal one third of Resolvase, which was disordered in the absence of DNA, forms an arm and a 3-helix DNA-binding domain on the opposite side of the DNA from the N-terminal domain. The arms wrap around the minor groove of the central 16 bp, and the DNA-binding domains interact with the major grooves near the outer boundaries of the binding site. The Resolvase dimer is asymmetric, particularly in the arm region, implying a conformational adaptability that may be important for Resolvase binding to different DNA sites in the synaptosome. It also raises the possibility of a sequential single-strand cleavage mechanism.

  • Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta Resolvase subunits.
    The EMBO journal, 1994
    Co-Authors: Phoebe A. Rice, Thomas A. Steitz
    Abstract:

    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.

  • Protein-protein interactions directing Resolvase site-specific recombination: a structure-function analysis.
    The EMBO journal, 1993
    Co-Authors: Robert E. Hughes, Phoebe A. Rice, Thomas A. Steitz, Nigel D. F. Grindley
    Abstract:

    Recombination catalyzed by the gamma delta Resolvase requires assembly of a nucleo-protein complex, the synaptosome, whose structure is determined by Resolvase-res and Resolvase-Resolvase interactions. In crystals of the Resolvase catalytic domain, monomers of Resolvase were closely associated with one another across three different dyad axes; one of these subunit contacts was shown to be an essential inter-dimer interaction. To investigate the relevance of the remaining two interfaces, we have made site-directed mutations at positions suggested by the structure. Cysteine substitutions were designed to link the interfaces covalently, mutations to arginine were used to disrupt intersubunit contacts, and mutations to tryptophan were used to study the hydrophobicity and solvent accessibility of potential interfaces by fluorescence quenching. Characterization of the mutant proteins has allowed us to identify the dimer interface of Resolvase and to assign a structural role to a second intersubunit contact. The data presented here, together with our previous results, suggest that all three of the dyad-related intersubunit interactions observed in the crystal play specific roles in synapsis and recombination.