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Synapsis and catalysis by activated Tn3 resolvase mutants.

Olorunniji FJ, He J, Wenwieser SV, Boocock MR, Stark WM - Nucleic Acids Res. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, Scotland, UK.

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 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.

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Synapsis and recombination by Tn3 resolvase. (A) Cartoon illustrating synapsis and strand exchange of a supercoiled substrate by wild-type and activated resolvase mutants. The two sites (res or site I) are indicated by arrowheads. Synapsis of two res sites in head-to-tail orientation by wild-type resolvase traps three topological nodes (shaded oval), and strand exchange results in a 2-noded catenane resolution product. Activated mutant resolvases can synapse sites by random collision, giving rise to products of variable topologies; in the example shown, a 5-noded knot inversion product. (B) The Tn3 recombination site res. The boxes represent binding sites for dimers of resolvase, with binding motifs represented by the wedges at the ends of each box. The lengths of the DNA segments are indicated (base pairs). The point within site I at which resolvase breaks and rejoins the DNA is marked by a staggered line. (C) Cartoon showing the path of the DNA in the res × res synapse, and the relationship of the catalytic and regulatory parts. The four resolvase subunits forming the tetramer in the site I synapse component of this complex are shown in red, and the grey shaded oval represents the eight subunits presumed to be bound to the accessory sites II and III.
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Figure 1: Synapsis and recombination by Tn3 resolvase. (A) Cartoon illustrating synapsis and strand exchange of a supercoiled substrate by wild-type and activated resolvase mutants. The two sites (res or site I) are indicated by arrowheads. Synapsis of two res sites in head-to-tail orientation by wild-type resolvase traps three topological nodes (shaded oval), and strand exchange results in a 2-noded catenane resolution product. Activated mutant resolvases can synapse sites by random collision, giving rise to products of variable topologies; in the example shown, a 5-noded knot inversion product. (B) The Tn3 recombination site res. The boxes represent binding sites for dimers of resolvase, with binding motifs represented by the wedges at the ends of each box. The lengths of the DNA segments are indicated (base pairs). The point within site I at which resolvase breaks and rejoins the DNA is marked by a staggered line. (C) Cartoon showing the path of the DNA in the res × res synapse, and the relationship of the catalytic and regulatory parts. The four resolvase subunits forming the tetramer in the site I synapse component of this complex are shown in red, and the grey shaded oval represents the eight subunits presumed to be bound to the accessory sites II and III.

Mentions: Much of our understanding of the mechanisms of serine recombinases has come from studies on the closely related cointegrate resolvases from the bacterial transposons Tn3 and γδ (4). Tn3 (or γδ) resolvase catalyses recombination between two 114 bp res sites (Figure 1A and B). Each res site contains binding sites for three resolvase dimers. The DNA strands are broken and rejoined at specific bonds near the centre of the 28 bp binding site I; catalysis of these reactions is by the subunits bound to site I. The ‘accessory’ binding sites II (34 bp) and III (25 bp) are also required for recombination. Resolvase dimers bound to sites II and III do not participate in the catalysis of strand cleavage and rejoining, but have an essential role in assembly of the synaptic complex (‘synapse’) of two res sites that is a prerequisite for strand exchange (Figure 1A and C). The synapse has regulatory functions which include restriction of recombination to pairs of res sites that are in the same orientation in a supercoiled DNA molecule, specification of a single round of recombination and specification of the 2-noded catenane recombinant product (5).Figure 1.


Synapsis and catalysis by activated Tn3 resolvase mutants.

Olorunniji FJ, He J, Wenwieser SV, Boocock MR, Stark WM - Nucleic Acids Res. (2008)

Synapsis and recombination by Tn3 resolvase. (A) Cartoon illustrating synapsis and strand exchange of a supercoiled substrate by wild-type and activated resolvase mutants. The two sites (res or site I) are indicated by arrowheads. Synapsis of two res sites in head-to-tail orientation by wild-type resolvase traps three topological nodes (shaded oval), and strand exchange results in a 2-noded catenane resolution product. Activated mutant resolvases can synapse sites by random collision, giving rise to products of variable topologies; in the example shown, a 5-noded knot inversion product. (B) The Tn3 recombination site res. The boxes represent binding sites for dimers of resolvase, with binding motifs represented by the wedges at the ends of each box. The lengths of the DNA segments are indicated (base pairs). The point within site I at which resolvase breaks and rejoins the DNA is marked by a staggered line. (C) Cartoon showing the path of the DNA in the res × res synapse, and the relationship of the catalytic and regulatory parts. The four resolvase subunits forming the tetramer in the site I synapse component of this complex are shown in red, and the grey shaded oval represents the eight subunits presumed to be bound to the accessory sites II and III.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2602789&req=5

Figure 1: Synapsis and recombination by Tn3 resolvase. (A) Cartoon illustrating synapsis and strand exchange of a supercoiled substrate by wild-type and activated resolvase mutants. The two sites (res or site I) are indicated by arrowheads. Synapsis of two res sites in head-to-tail orientation by wild-type resolvase traps three topological nodes (shaded oval), and strand exchange results in a 2-noded catenane resolution product. Activated mutant resolvases can synapse sites by random collision, giving rise to products of variable topologies; in the example shown, a 5-noded knot inversion product. (B) The Tn3 recombination site res. The boxes represent binding sites for dimers of resolvase, with binding motifs represented by the wedges at the ends of each box. The lengths of the DNA segments are indicated (base pairs). The point within site I at which resolvase breaks and rejoins the DNA is marked by a staggered line. (C) Cartoon showing the path of the DNA in the res × res synapse, and the relationship of the catalytic and regulatory parts. The four resolvase subunits forming the tetramer in the site I synapse component of this complex are shown in red, and the grey shaded oval represents the eight subunits presumed to be bound to the accessory sites II and III.
Mentions: Much of our understanding of the mechanisms of serine recombinases has come from studies on the closely related cointegrate resolvases from the bacterial transposons Tn3 and γδ (4). Tn3 (or γδ) resolvase catalyses recombination between two 114 bp res sites (Figure 1A and B). Each res site contains binding sites for three resolvase dimers. The DNA strands are broken and rejoined at specific bonds near the centre of the 28 bp binding site I; catalysis of these reactions is by the subunits bound to site I. The ‘accessory’ binding sites II (34 bp) and III (25 bp) are also required for recombination. Resolvase dimers bound to sites II and III do not participate in the catalysis of strand cleavage and rejoining, but have an essential role in assembly of the synaptic complex (‘synapse’) of two res sites that is a prerequisite for strand exchange (Figure 1A and C). The synapse has regulatory functions which include restriction of recombination to pairs of res sites that are in the same orientation in a supercoiled DNA molecule, specification of a single round of recombination and specification of the 2-noded catenane recombinant product (5).Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, Scotland, UK.

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 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.

Show MeSH
Related in: MedlinePlus