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The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

Serre MC, El Arnaout T, Brooks MA, Durand D, Lisboa J, Lazar N, Raynal B, van Tilbeurgh H, Quevillon-Cheruel S - PLoS ONE (2013)

Bottom Line: Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates.Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis.We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France. marie-claude.serre@igmors.u-psud.fr

ABSTRACT
Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

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The αN helix of XerA is essential for recombination.A. Plasmid-based recombination reactions catalysed by XerA and XerA-ΔC mutant. Recombination products between pBend plasmids harbouring the dif site were visualised on 1.2% agarose gels. B. Covalent complex formation between XerA or XerA-ΔC mutant and half-site substrates. Reactions on the left half site 5′-end labeled on the top strand. Positions of dimers and tetramers of XerA resistant to thermal denaturation are indicated.
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pone-0063010-g008: The αN helix of XerA is essential for recombination.A. Plasmid-based recombination reactions catalysed by XerA and XerA-ΔC mutant. Recombination products between pBend plasmids harbouring the dif site were visualised on 1.2% agarose gels. B. Covalent complex formation between XerA or XerA-ΔC mutant and half-site substrates. Reactions on the left half site 5′-end labeled on the top strand. Positions of dimers and tetramers of XerA resistant to thermal denaturation are indicated.

Mentions: To investigate the role of the αN helix in XerA-mediated recombination, we generated a mutant (XerA-ΔC) deleted for the last 13 amino-acids corresponding to the αN helix alone. XerA-ΔC was expressed and purified following the procedure described for the wild-type enzyme, and its catalytic properties were analyzed. The site-specific recombination activity of XerA-ΔC was evaluated in the in vitro assay previously described [3] and compared to the wild-type (Figure 8A). When incubated with a plasmid containing the dif site, XerA generates plasmid dimers and multimers to a lesser extent. As protein concentration increases, plasmid dimers are used as substrates and resolved into plasmid monomers. Under the same conditions, XerA-ΔC was unable to recombine the dif-containing plasmid even with two-fold excess of protein (Figure 8A). A trivial explanation is that XerA-ΔC is unable to bind and/or cleave the DNA substrate. This hypothesis was tested by using half-site substrates to evaluate covalent complex formation. XerA-ΔC was able to cleave this unconstrained DNA substrate (Figure 8B). However, formation of two additional complexes of high molecular weight, that probably correspond to dimers and tetramers assemblies, was reduced four-fold for the XerA-ΔC mutant as compared to XerA. This observation suggests that deletion of the αN helix severely impairs higher order assemblies required for the recombination process to occur. The interactions between XerA monomers via their C-terminal αN helices are therefore crucial for complete catalytic activity, probably by triggering the catalytic cycle, stabilizing the synaptic complex and the Holliday junction intermediate.


The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

Serre MC, El Arnaout T, Brooks MA, Durand D, Lisboa J, Lazar N, Raynal B, van Tilbeurgh H, Quevillon-Cheruel S - PLoS ONE (2013)

The αN helix of XerA is essential for recombination.A. Plasmid-based recombination reactions catalysed by XerA and XerA-ΔC mutant. Recombination products between pBend plasmids harbouring the dif site were visualised on 1.2% agarose gels. B. Covalent complex formation between XerA or XerA-ΔC mutant and half-site substrates. Reactions on the left half site 5′-end labeled on the top strand. Positions of dimers and tetramers of XerA resistant to thermal denaturation are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646895&req=5

pone-0063010-g008: The αN helix of XerA is essential for recombination.A. Plasmid-based recombination reactions catalysed by XerA and XerA-ΔC mutant. Recombination products between pBend plasmids harbouring the dif site were visualised on 1.2% agarose gels. B. Covalent complex formation between XerA or XerA-ΔC mutant and half-site substrates. Reactions on the left half site 5′-end labeled on the top strand. Positions of dimers and tetramers of XerA resistant to thermal denaturation are indicated.
Mentions: To investigate the role of the αN helix in XerA-mediated recombination, we generated a mutant (XerA-ΔC) deleted for the last 13 amino-acids corresponding to the αN helix alone. XerA-ΔC was expressed and purified following the procedure described for the wild-type enzyme, and its catalytic properties were analyzed. The site-specific recombination activity of XerA-ΔC was evaluated in the in vitro assay previously described [3] and compared to the wild-type (Figure 8A). When incubated with a plasmid containing the dif site, XerA generates plasmid dimers and multimers to a lesser extent. As protein concentration increases, plasmid dimers are used as substrates and resolved into plasmid monomers. Under the same conditions, XerA-ΔC was unable to recombine the dif-containing plasmid even with two-fold excess of protein (Figure 8A). A trivial explanation is that XerA-ΔC is unable to bind and/or cleave the DNA substrate. This hypothesis was tested by using half-site substrates to evaluate covalent complex formation. XerA-ΔC was able to cleave this unconstrained DNA substrate (Figure 8B). However, formation of two additional complexes of high molecular weight, that probably correspond to dimers and tetramers assemblies, was reduced four-fold for the XerA-ΔC mutant as compared to XerA. This observation suggests that deletion of the αN helix severely impairs higher order assemblies required for the recombination process to occur. The interactions between XerA monomers via their C-terminal αN helices are therefore crucial for complete catalytic activity, probably by triggering the catalytic cycle, stabilizing the synaptic complex and the Holliday junction intermediate.

Bottom Line: Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates.Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis.We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France. marie-claude.serre@igmors.u-psud.fr

ABSTRACT
Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

Show MeSH
Related in: MedlinePlus