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The Hin recombinase assembles a tetrameric protein swivel that exchanges DNA strands.

Dhar G, McLean MM, Heiss JK, Johnson RC - Nucleic Acids Res. (2009)

Bottom Line: Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements.Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase.Efficient crosslinking between residues corresponding to the 'D-helix' region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.

ABSTRACT
Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the 'D-helix' region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange.

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The Hin site-specific DNA inversion reaction from Salmonella sp. (A) Inversion of the chromosomal segment between the two hix recombination sites switches the orientation of an internal promoter. In one orientation, this promoter directs transcription of a flagellin gene and a negative regulator of an alternative flagellin gene located elsewhere in the chromosome. The recombinational enhancer with the two Fis binding sites (red) is depicted in its normal location within the N-terminal coding region of the hin gene. (B) Recombination intermediate (invertasome) formed during the native DNA inversion reaction. A tripartite complex containing a tetramer of Hin bound to the two hix sites, and two dimers of Fis (brown ellipses) bound to the enhancer assembles at the base of a supercoiled branch. Formation of this complex requires DNA supercoiling and is aided by HU (red half-sphere), which stabilizes short loops. (C) Recombination on linear DNA substrates by Fis-independent Hin mutants. Hin dimers bound to DNA fragments containing hix recombination sites assemble into a tetrameric synaptic complex. Each subunit of Hin cleaves a DNA strand at the center of hix to form a serine-phosphodiester bond with the 5′ end of the broken DNA. Data in this article and elsewhere (10,20) indicate that one set of synapsed subunits can rotate relative to the other to position the DNA strands in the recombinant configuration. Ligation of the DNA ends completes the reaction. Reactions performed in the presence of ethylene glycol and without Mg2+ generate stable cleaved synaptic complexes that support subunit rotation but not ligation.
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Figure 1: The Hin site-specific DNA inversion reaction from Salmonella sp. (A) Inversion of the chromosomal segment between the two hix recombination sites switches the orientation of an internal promoter. In one orientation, this promoter directs transcription of a flagellin gene and a negative regulator of an alternative flagellin gene located elsewhere in the chromosome. The recombinational enhancer with the two Fis binding sites (red) is depicted in its normal location within the N-terminal coding region of the hin gene. (B) Recombination intermediate (invertasome) formed during the native DNA inversion reaction. A tripartite complex containing a tetramer of Hin bound to the two hix sites, and two dimers of Fis (brown ellipses) bound to the enhancer assembles at the base of a supercoiled branch. Formation of this complex requires DNA supercoiling and is aided by HU (red half-sphere), which stabilizes short loops. (C) Recombination on linear DNA substrates by Fis-independent Hin mutants. Hin dimers bound to DNA fragments containing hix recombination sites assemble into a tetrameric synaptic complex. Each subunit of Hin cleaves a DNA strand at the center of hix to form a serine-phosphodiester bond with the 5′ end of the broken DNA. Data in this article and elsewhere (10,20) indicate that one set of synapsed subunits can rotate relative to the other to position the DNA strands in the recombinant configuration. Ligation of the DNA ends completes the reaction. Reactions performed in the presence of ethylene glycol and without Mg2+ generate stable cleaved synaptic complexes that support subunit rotation but not ligation.

Mentions: Hin is a member of the serine recombinase family (3), which is named because a conserved serine is the active site residue that catalyzes the DNA cleavage/joining reaction. Hin promotes inversion of a ∼1 kb DNA segment between two hix recombination sites within the chromosome of Salmonella sp (7). The flipping of the DNA segment reorients an internal promoter, which results in alternate expression of antigenically distinct flagellin protein genes (Figure 1A). Hin is a member of a subgroup of serine recombinases called DNA invertases that promote inversion reactions in different biological contexts. Members of another subgroup of serine recombinases, DNA resolvases, promote site-specific deletions of DNA (8). DNA invertases and resolvases are similar in size and share a similar ∼100 residue N-terminal catalytic domain linked to a ∼34 residue oligomerization helix (‘helix E’). The C-terminal DNA binding domains are more divergent at the sequence level, but crystal structures have shown that the 40–50 amino acid residue domains from γδ resolvase and Hin share a similar helix-turn-helix fold, which are positioned on opposite sides of the DNA duplex from the catalytic domain (9). Crystal structures of the catalytic domain of γδ resolvase have been determined in isolation, as a full-length dimer bound to a single recombination site and as tetrameric synaptic complexes (10–14). A goal of the present work is to establish the relationship between the resolvase tetrameric structures and the structure of Hin within active recombination complexes. A third class of serine recombinases promote phage integration/excision and DNA transposition reactions (3). The catalytic cores of these recombinases (15) are present within a much larger polypeptide chain.Figure 1.


The Hin recombinase assembles a tetrameric protein swivel that exchanges DNA strands.

Dhar G, McLean MM, Heiss JK, Johnson RC - Nucleic Acids Res. (2009)

The Hin site-specific DNA inversion reaction from Salmonella sp. (A) Inversion of the chromosomal segment between the two hix recombination sites switches the orientation of an internal promoter. In one orientation, this promoter directs transcription of a flagellin gene and a negative regulator of an alternative flagellin gene located elsewhere in the chromosome. The recombinational enhancer with the two Fis binding sites (red) is depicted in its normal location within the N-terminal coding region of the hin gene. (B) Recombination intermediate (invertasome) formed during the native DNA inversion reaction. A tripartite complex containing a tetramer of Hin bound to the two hix sites, and two dimers of Fis (brown ellipses) bound to the enhancer assembles at the base of a supercoiled branch. Formation of this complex requires DNA supercoiling and is aided by HU (red half-sphere), which stabilizes short loops. (C) Recombination on linear DNA substrates by Fis-independent Hin mutants. Hin dimers bound to DNA fragments containing hix recombination sites assemble into a tetrameric synaptic complex. Each subunit of Hin cleaves a DNA strand at the center of hix to form a serine-phosphodiester bond with the 5′ end of the broken DNA. Data in this article and elsewhere (10,20) indicate that one set of synapsed subunits can rotate relative to the other to position the DNA strands in the recombinant configuration. Ligation of the DNA ends completes the reaction. Reactions performed in the presence of ethylene glycol and without Mg2+ generate stable cleaved synaptic complexes that support subunit rotation but not ligation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: The Hin site-specific DNA inversion reaction from Salmonella sp. (A) Inversion of the chromosomal segment between the two hix recombination sites switches the orientation of an internal promoter. In one orientation, this promoter directs transcription of a flagellin gene and a negative regulator of an alternative flagellin gene located elsewhere in the chromosome. The recombinational enhancer with the two Fis binding sites (red) is depicted in its normal location within the N-terminal coding region of the hin gene. (B) Recombination intermediate (invertasome) formed during the native DNA inversion reaction. A tripartite complex containing a tetramer of Hin bound to the two hix sites, and two dimers of Fis (brown ellipses) bound to the enhancer assembles at the base of a supercoiled branch. Formation of this complex requires DNA supercoiling and is aided by HU (red half-sphere), which stabilizes short loops. (C) Recombination on linear DNA substrates by Fis-independent Hin mutants. Hin dimers bound to DNA fragments containing hix recombination sites assemble into a tetrameric synaptic complex. Each subunit of Hin cleaves a DNA strand at the center of hix to form a serine-phosphodiester bond with the 5′ end of the broken DNA. Data in this article and elsewhere (10,20) indicate that one set of synapsed subunits can rotate relative to the other to position the DNA strands in the recombinant configuration. Ligation of the DNA ends completes the reaction. Reactions performed in the presence of ethylene glycol and without Mg2+ generate stable cleaved synaptic complexes that support subunit rotation but not ligation.
Mentions: Hin is a member of the serine recombinase family (3), which is named because a conserved serine is the active site residue that catalyzes the DNA cleavage/joining reaction. Hin promotes inversion of a ∼1 kb DNA segment between two hix recombination sites within the chromosome of Salmonella sp (7). The flipping of the DNA segment reorients an internal promoter, which results in alternate expression of antigenically distinct flagellin protein genes (Figure 1A). Hin is a member of a subgroup of serine recombinases called DNA invertases that promote inversion reactions in different biological contexts. Members of another subgroup of serine recombinases, DNA resolvases, promote site-specific deletions of DNA (8). DNA invertases and resolvases are similar in size and share a similar ∼100 residue N-terminal catalytic domain linked to a ∼34 residue oligomerization helix (‘helix E’). The C-terminal DNA binding domains are more divergent at the sequence level, but crystal structures have shown that the 40–50 amino acid residue domains from γδ resolvase and Hin share a similar helix-turn-helix fold, which are positioned on opposite sides of the DNA duplex from the catalytic domain (9). Crystal structures of the catalytic domain of γδ resolvase have been determined in isolation, as a full-length dimer bound to a single recombination site and as tetrameric synaptic complexes (10–14). A goal of the present work is to establish the relationship between the resolvase tetrameric structures and the structure of Hin within active recombination complexes. A third class of serine recombinases promote phage integration/excision and DNA transposition reactions (3). The catalytic cores of these recombinases (15) are present within a much larger polypeptide chain.Figure 1.

Bottom Line: Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements.Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase.Efficient crosslinking between residues corresponding to the 'D-helix' region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.

ABSTRACT
Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the 'D-helix' region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange.

Show MeSH
Related in: MedlinePlus