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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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Structure comparison of apo-XerA and apo-HP1 integrase dimers.C-terminal domain dimers of apo-XerA (top) and apo-HP1 Integrase (bottom). Within dimers, the C-terminal αMN helices of the green monomer are highlighted in blue and the C-terminal αMN helices of the grey monomer are highlighted in orange. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan.
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pone-0063010-g004: Structure comparison of apo-XerA and apo-HP1 integrase dimers.C-terminal domain dimers of apo-XerA (top) and apo-HP1 Integrase (bottom). Within dimers, the C-terminal αMN helices of the green monomer are highlighted in blue and the C-terminal αMN helices of the grey monomer are highlighted in orange. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan.

Mentions: The C-terminal domain of XerA is mainly α-helical, except for a 3-stranded β-sheet (Figure 1A). The first short β-strand is not well defined in the structure, as in the case of XerD or HP1-Int. The conserved β2–β3 loop constitutes the edge of the C-shaped clamp of the protein. As observed for XerD, the catalytic residue K160 carried by this loop is removed from the active site (Figure 1A). The β2–β3 loop is fairly mobile and dominates the active site where a sulfate ion is bound both in the XerA and HP1-Int C-terminal domain structures (Figure 4) [17]. The conserved catalytic residues R135 and H226 of XerA are within hydrogen-bonding distance of the sulfate ion. The same coordination is observed in HP1-Int [17]. However, the sulfate ion coordinates the catalytic Tyr in the HP1-Int active site but not in XerA (Figure 4). Interestingly, Y261 is located at the end of the most flexible loop of XerA, at the N-terminus of the αM helix, and is extruded away from the active site with its side chain pointing towards the solvent (Figures 1A and 4). Thus XerA, unlike the HP1-Int catalytic domain [17], or type IB DNA topoisomerase from Deinococcus radiodurans[46], only partially preassembles its active site in the absence of the DNA substrate. However, the flexible nature of the loop upstream of the XerA αMN helices may facilitate their repositioning upon assembly of the synaptic complex. The conformational change of αMN helices would be sufficient to direct the catalytic Y261 into the catalytic pocket.


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)

Structure comparison of apo-XerA and apo-HP1 integrase dimers.C-terminal domain dimers of apo-XerA (top) and apo-HP1 Integrase (bottom). Within dimers, the C-terminal αMN helices of the green monomer are highlighted in blue and the C-terminal αMN helices of the grey monomer are highlighted in orange. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063010-g004: Structure comparison of apo-XerA and apo-HP1 integrase dimers.C-terminal domain dimers of apo-XerA (top) and apo-HP1 Integrase (bottom). Within dimers, the C-terminal αMN helices of the green monomer are highlighted in blue and the C-terminal αMN helices of the grey monomer are highlighted in orange. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan.
Mentions: The C-terminal domain of XerA is mainly α-helical, except for a 3-stranded β-sheet (Figure 1A). The first short β-strand is not well defined in the structure, as in the case of XerD or HP1-Int. The conserved β2–β3 loop constitutes the edge of the C-shaped clamp of the protein. As observed for XerD, the catalytic residue K160 carried by this loop is removed from the active site (Figure 1A). The β2–β3 loop is fairly mobile and dominates the active site where a sulfate ion is bound both in the XerA and HP1-Int C-terminal domain structures (Figure 4) [17]. The conserved catalytic residues R135 and H226 of XerA are within hydrogen-bonding distance of the sulfate ion. The same coordination is observed in HP1-Int [17]. However, the sulfate ion coordinates the catalytic Tyr in the HP1-Int active site but not in XerA (Figure 4). Interestingly, Y261 is located at the end of the most flexible loop of XerA, at the N-terminus of the αM helix, and is extruded away from the active site with its side chain pointing towards the solvent (Figures 1A and 4). Thus XerA, unlike the HP1-Int catalytic domain [17], or type IB DNA topoisomerase from Deinococcus radiodurans[46], only partially preassembles its active site in the absence of the DNA substrate. However, the flexible nature of the loop upstream of the XerA αMN helices may facilitate their repositioning upon assembly of the synaptic complex. The conformational change of αMN helices would be sufficient to direct the catalytic Y261 into the catalytic pocket.

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