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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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Resolvase structures and activating mutations. (A) Crystal structure of a wild-type γδ resolvase dimer bound to site I [1GDT; (11)]. On the right, residues relevant to the experiments presented here are shown in spacefill on the orange resolvase subunit (in the same orientation); S10 in green; R2 and E56 in blue; G101, E102, M103 and K105 in cyan; A117, R121 and E124 in magenta. The hydroxyl group of S10 is the catalytic nucleophile in recombination. The sidechains of R2 and E56 contribute to the 2–3′ interface (see Introduction section). The other highlighted residues are the sites of activating mutations of Tn3 resolvase. The γδ resolvase residues E102 and K105 correspond to D102 and Q105, respectively, in Tn3 resolvase. (B) Crystal structure of a synaptic tetramer of γδ resolvase with two cleaved site Is [1ZR4; (16)], and on the right the orange resolvase subunit in the same orientation, showing residues in spacefill as in (A), except that the following residues are mutant; A2, K56, S101, Y102, I103, Q124. The images were created with PyMol.
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Figure 2: Resolvase structures and activating mutations. (A) Crystal structure of a wild-type γδ resolvase dimer bound to site I [1GDT; (11)]. On the right, residues relevant to the experiments presented here are shown in spacefill on the orange resolvase subunit (in the same orientation); S10 in green; R2 and E56 in blue; G101, E102, M103 and K105 in cyan; A117, R121 and E124 in magenta. The hydroxyl group of S10 is the catalytic nucleophile in recombination. The sidechains of R2 and E56 contribute to the 2–3′ interface (see Introduction section). The other highlighted residues are the sites of activating mutations of Tn3 resolvase. The γδ resolvase residues E102 and K105 correspond to D102 and Q105, respectively, in Tn3 resolvase. (B) Crystal structure of a synaptic tetramer of γδ resolvase with two cleaved site Is [1ZR4; (16)], and on the right the orange resolvase subunit in the same orientation, showing residues in spacefill as in (A), except that the following residues are mutant; A2, K56, S101, Y102, I103, Q124. The images were created with PyMol.

Mentions: Tn3 resolvase is a 185-amino acid protein consisting of two domains. The C-terminal domains (∼45 amino acids) of resolvase dimers make sequence-specific interactions with the DNA at each end of binding sites I, II and III in res. The N-terminal domain contains the active site for catalysis of strand exchange, and all known interactions between subunits involve residues of this domain (Figure 2). The structure of the N-terminal domain of γδ resolvase, solved by X-ray crystallography (6–9), revealed two important types of interaction between subunits. The 1–2 or dimer interface mediates dimerization of resolvase in solution and when bound to DNA. The 2–3′ interface connects dimers; it has essential functions for wild-type resolvase in assembly of the synapse of two res sites and in activation of recombination (5,7,10). The 1–2 dimer is present in a structure of γδ resolvase bound to site I DNA (11; Figure 2A), but the 2–3′ interaction has not yet been observed in X-ray structures of complexes containing DNA.Figure 2.


Synapsis and catalysis by activated Tn3 resolvase mutants.

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

Resolvase structures and activating mutations. (A) Crystal structure of a wild-type γδ resolvase dimer bound to site I [1GDT; (11)]. On the right, residues relevant to the experiments presented here are shown in spacefill on the orange resolvase subunit (in the same orientation); S10 in green; R2 and E56 in blue; G101, E102, M103 and K105 in cyan; A117, R121 and E124 in magenta. The hydroxyl group of S10 is the catalytic nucleophile in recombination. The sidechains of R2 and E56 contribute to the 2–3′ interface (see Introduction section). The other highlighted residues are the sites of activating mutations of Tn3 resolvase. The γδ resolvase residues E102 and K105 correspond to D102 and Q105, respectively, in Tn3 resolvase. (B) Crystal structure of a synaptic tetramer of γδ resolvase with two cleaved site Is [1ZR4; (16)], and on the right the orange resolvase subunit in the same orientation, showing residues in spacefill as in (A), except that the following residues are mutant; A2, K56, S101, Y102, I103, Q124. The images were created with PyMol.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Resolvase structures and activating mutations. (A) Crystal structure of a wild-type γδ resolvase dimer bound to site I [1GDT; (11)]. On the right, residues relevant to the experiments presented here are shown in spacefill on the orange resolvase subunit (in the same orientation); S10 in green; R2 and E56 in blue; G101, E102, M103 and K105 in cyan; A117, R121 and E124 in magenta. The hydroxyl group of S10 is the catalytic nucleophile in recombination. The sidechains of R2 and E56 contribute to the 2–3′ interface (see Introduction section). The other highlighted residues are the sites of activating mutations of Tn3 resolvase. The γδ resolvase residues E102 and K105 correspond to D102 and Q105, respectively, in Tn3 resolvase. (B) Crystal structure of a synaptic tetramer of γδ resolvase with two cleaved site Is [1ZR4; (16)], and on the right the orange resolvase subunit in the same orientation, showing residues in spacefill as in (A), except that the following residues are mutant; A2, K56, S101, Y102, I103, Q124. The images were created with PyMol.
Mentions: Tn3 resolvase is a 185-amino acid protein consisting of two domains. The C-terminal domains (∼45 amino acids) of resolvase dimers make sequence-specific interactions with the DNA at each end of binding sites I, II and III in res. The N-terminal domain contains the active site for catalysis of strand exchange, and all known interactions between subunits involve residues of this domain (Figure 2). The structure of the N-terminal domain of γδ resolvase, solved by X-ray crystallography (6–9), revealed two important types of interaction between subunits. The 1–2 or dimer interface mediates dimerization of resolvase in solution and when bound to DNA. The 2–3′ interface connects dimers; it has essential functions for wild-type resolvase in assembly of the synapse of two res sites and in activation of recombination (5,7,10). The 1–2 dimer is present in a structure of γδ resolvase bound to site I DNA (11; Figure 2A), but the 2–3′ interaction has not yet been observed in X-ray structures of complexes containing DNA.Figure 2.

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