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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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Half-sites strand transfer reactions catalyzed by XerA.A. Sequence of the natural dif site and half-site substrates. The predicted XerA binding sequence is in bold. The spacer sequence is in italics. The predicted cleavage sites are indicated by arrows. B. Covalent complex formation between XerA and half-site substrates was analyzed by 12% SDS-PAGE. Left, reactions on the left half site 5′-end labeled on the top strand. The 5′-end of the bottom strand was either a hydroxyl (LT*B) or a phosphate (LT*Bp). Center, representation of the covalent complexes formed and subsequent steps of the recombination reaction. Right, reactions on the right half site 5′-end labeled on the bottom strand. The 5′-end of the top strand was either a hydroxyl (RTB*) or a phosphate (RTpB*). C. Recombination products between half-site substrates were visualized on 15% polyacrylamide-urea gels. The sizes of the top strand exchange (FST*) and bottom strand exchange (FSB*) are indicated and correspond to the predicted product sizes (sequences are presented below the gel).
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pone-0063010-g006: Half-sites strand transfer reactions catalyzed by XerA.A. Sequence of the natural dif site and half-site substrates. The predicted XerA binding sequence is in bold. The spacer sequence is in italics. The predicted cleavage sites are indicated by arrows. B. Covalent complex formation between XerA and half-site substrates was analyzed by 12% SDS-PAGE. Left, reactions on the left half site 5′-end labeled on the top strand. The 5′-end of the bottom strand was either a hydroxyl (LT*B) or a phosphate (LT*Bp). Center, representation of the covalent complexes formed and subsequent steps of the recombination reaction. Right, reactions on the right half site 5′-end labeled on the bottom strand. The 5′-end of the top strand was either a hydroxyl (RTB*) or a phosphate (RTpB*). C. Recombination products between half-site substrates were visualized on 15% polyacrylamide-urea gels. The sizes of the top strand exchange (FST*) and bottom strand exchange (FSB*) are indicated and correspond to the predicted product sizes (sequences are presented below the gel).

Mentions: The reaction catalyzed by Tyr-recombinases involves a transient 3′-phosphotyrosine protein-DNA covalent complex. To identify this complex in XerA, we designed half-site suicide substrates (Figure 6A) similar to those previously designed for λ Int [47] and eukaryotic Flp recombinase [48]. Each half-site contains one of the two XerA binding sites present at the dif site and either the 6 nt spacer top strand (right half site) or bottom strand (left half site). Cleavage of these synthetic substrates traps the covalent complex, with the trinucleotide cleaved product diffusing out of the catalytic center (Figure 6A, B). XerA was incubated with either the left or right half-site substrates, and reaction products were analyzed by SDS-PAGE (Figure 6B). The appearance of radiolabeled, low mobility complexes revealed that XerA can cleave and generate 3′-phosphotyrosine covalent complexes with both strands of the dif site. This confirmed that the polarity of strand cleavage by XerA is the same as that of other Tyr-recombinases. For both substrates, phosphorylation of the 5′-end of the uncleaved strand resulted in a three-fold increase of covalent complex (Figure 6B). This result strongly suggests that the free 5′ hydroxyl of the uncleaved strand is able to attack the covalent complex either intra- or inter-molecularly as previously observed with the Flp recombinase [48]. Finally, no DNA-protein covalent complex was detected when using the Y261F active site mutant (data not shown), confirming that the catalytic Tyr is required for activity.


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)

Half-sites strand transfer reactions catalyzed by XerA.A. Sequence of the natural dif site and half-site substrates. The predicted XerA binding sequence is in bold. The spacer sequence is in italics. The predicted cleavage sites are indicated by arrows. B. Covalent complex formation between XerA and half-site substrates was analyzed by 12% SDS-PAGE. Left, reactions on the left half site 5′-end labeled on the top strand. The 5′-end of the bottom strand was either a hydroxyl (LT*B) or a phosphate (LT*Bp). Center, representation of the covalent complexes formed and subsequent steps of the recombination reaction. Right, reactions on the right half site 5′-end labeled on the bottom strand. The 5′-end of the top strand was either a hydroxyl (RTB*) or a phosphate (RTpB*). C. Recombination products between half-site substrates were visualized on 15% polyacrylamide-urea gels. The sizes of the top strand exchange (FST*) and bottom strand exchange (FSB*) are indicated and correspond to the predicted product sizes (sequences are presented below the gel).
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Related In: Results  -  Collection

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pone-0063010-g006: Half-sites strand transfer reactions catalyzed by XerA.A. Sequence of the natural dif site and half-site substrates. The predicted XerA binding sequence is in bold. The spacer sequence is in italics. The predicted cleavage sites are indicated by arrows. B. Covalent complex formation between XerA and half-site substrates was analyzed by 12% SDS-PAGE. Left, reactions on the left half site 5′-end labeled on the top strand. The 5′-end of the bottom strand was either a hydroxyl (LT*B) or a phosphate (LT*Bp). Center, representation of the covalent complexes formed and subsequent steps of the recombination reaction. Right, reactions on the right half site 5′-end labeled on the bottom strand. The 5′-end of the top strand was either a hydroxyl (RTB*) or a phosphate (RTpB*). C. Recombination products between half-site substrates were visualized on 15% polyacrylamide-urea gels. The sizes of the top strand exchange (FST*) and bottom strand exchange (FSB*) are indicated and correspond to the predicted product sizes (sequences are presented below the gel).
Mentions: The reaction catalyzed by Tyr-recombinases involves a transient 3′-phosphotyrosine protein-DNA covalent complex. To identify this complex in XerA, we designed half-site suicide substrates (Figure 6A) similar to those previously designed for λ Int [47] and eukaryotic Flp recombinase [48]. Each half-site contains one of the two XerA binding sites present at the dif site and either the 6 nt spacer top strand (right half site) or bottom strand (left half site). Cleavage of these synthetic substrates traps the covalent complex, with the trinucleotide cleaved product diffusing out of the catalytic center (Figure 6A, B). XerA was incubated with either the left or right half-site substrates, and reaction products were analyzed by SDS-PAGE (Figure 6B). The appearance of radiolabeled, low mobility complexes revealed that XerA can cleave and generate 3′-phosphotyrosine covalent complexes with both strands of the dif site. This confirmed that the polarity of strand cleavage by XerA is the same as that of other Tyr-recombinases. For both substrates, phosphorylation of the 5′-end of the uncleaved strand resulted in a three-fold increase of covalent complex (Figure 6B). This result strongly suggests that the free 5′ hydroxyl of the uncleaved strand is able to attack the covalent complex either intra- or inter-molecularly as previously observed with the Flp recombinase [48]. Finally, no DNA-protein covalent complex was detected when using the Y261F active site mutant (data not shown), confirming that the catalytic Tyr is required for activity.

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