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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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Helices αM and αN pack in cis.A. Superimposition of the two models constructed from the XerA monomer structure. Model 1 (yellow): cis positioning of αMN helices. Model 2 (grey): trans positioning of αMN helices. The two possible positions for αMN helices according to each model are in orange and blue respectively. B. Distance distribution functions P(r). Black line: experimental curve. Orange dotted curve: XerA monomer with αMN helices packed in cis. Blue dotted curve: XerA monomer with αMN helices packed in trans.
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pone-0063010-g005: Helices αM and αN pack in cis.A. Superimposition of the two models constructed from the XerA monomer structure. Model 1 (yellow): cis positioning of αMN helices. Model 2 (grey): trans positioning of αMN helices. The two possible positions for αMN helices according to each model are in orange and blue respectively. B. Distance distribution functions P(r). Black line: experimental curve. Orange dotted curve: XerA monomer with αMN helices packed in cis. Blue dotted curve: XerA monomer with αMN helices packed in trans.

Mentions: The weak electron density between residues H252 and T258 in the XerA crystal structure (Figure 1) rendered uncertain the assignment of αMN helices to their parent monomers. SAXS analysis was used to determine the location of the αMN helices in solution. Two models were constructed from the XerA monomer crystal structure (Figure 5A) and addition of the missing residues in the N and C-terminal domains. The first model corresponded to a cis positioning of αMN helices and the second model corresponded to trans positioning of these helices (i.e. helix swapping between monomers). SAXS curves were calculated for both models using CRYSOL [38] and adjusted to the experimental curve. The χ parameters characterizing the goodness of the fit were then compared. The best fit was obtained for the cis model (χ value of 0.80) compared to the trans model (χ value of 1.25). Moreover, comparison of the distance distribution functions clearly shows that the experimental curve fits better to the curve calculated with the cis conformation (Figure 5B). Finally, using SASREF [39] we reproduced the experimental curve by means of a curve calculated from a model based on the crystal structure where the αMN helices are free to move. Strikingly several independent runs starting from either the cis conformation or the trans conformation converged to similar models where the αMN helices are very close to the cis positioning. Therefore SAXS data are compatible with a cis positioning of the αMN helices in solution. However, we cannot exclude that it was the compact configuration of the monomer without helix swapping that crystallized.


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)

Helices αM and αN pack in cis.A. Superimposition of the two models constructed from the XerA monomer structure. Model 1 (yellow): cis positioning of αMN helices. Model 2 (grey): trans positioning of αMN helices. The two possible positions for αMN helices according to each model are in orange and blue respectively. B. Distance distribution functions P(r). Black line: experimental curve. Orange dotted curve: XerA monomer with αMN helices packed in cis. Blue dotted curve: XerA monomer with αMN helices packed in trans.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063010-g005: Helices αM and αN pack in cis.A. Superimposition of the two models constructed from the XerA monomer structure. Model 1 (yellow): cis positioning of αMN helices. Model 2 (grey): trans positioning of αMN helices. The two possible positions for αMN helices according to each model are in orange and blue respectively. B. Distance distribution functions P(r). Black line: experimental curve. Orange dotted curve: XerA monomer with αMN helices packed in cis. Blue dotted curve: XerA monomer with αMN helices packed in trans.
Mentions: The weak electron density between residues H252 and T258 in the XerA crystal structure (Figure 1) rendered uncertain the assignment of αMN helices to their parent monomers. SAXS analysis was used to determine the location of the αMN helices in solution. Two models were constructed from the XerA monomer crystal structure (Figure 5A) and addition of the missing residues in the N and C-terminal domains. The first model corresponded to a cis positioning of αMN helices and the second model corresponded to trans positioning of these helices (i.e. helix swapping between monomers). SAXS curves were calculated for both models using CRYSOL [38] and adjusted to the experimental curve. The χ parameters characterizing the goodness of the fit were then compared. The best fit was obtained for the cis model (χ value of 0.80) compared to the trans model (χ value of 1.25). Moreover, comparison of the distance distribution functions clearly shows that the experimental curve fits better to the curve calculated with the cis conformation (Figure 5B). Finally, using SASREF [39] we reproduced the experimental curve by means of a curve calculated from a model based on the crystal structure where the αMN helices are free to move. Strikingly several independent runs starting from either the cis conformation or the trans conformation converged to similar models where the αMN helices are very close to the cis positioning. Therefore SAXS data are compatible with a cis positioning of the αMN helices in solution. However, we cannot exclude that it was the compact configuration of the monomer without helix swapping that crystallized.

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