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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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Cysteine substitutions in the helix-E region. (A) Hin dimer model with the locations of the cysteine substitutions denoted with red spheres. (B) Hin tetramer model (GM4-based) with the locations of the cysteine substitutions denoted with red spheres. (C) Hin tetramer model after a 90° clockwise rotation. Substituted residues have been converted to cysteine with Sγ atoms denoted as spheres. Red designates efficient crosslinking [residues 134, 133, 130, 126 and 122 listed from the C-terminal (left) end]; orange designates crosslinking (residues 131 and 129); green designates poor or no crosslinking (residues 136, 132, 128, 124, 121 and 118); yellow designates cysteines that exhibit poor or no crosslinking and are within the catalytic core and excluded from solvent (residues 115, 112, 109, 105) (Table 1 and Figure 6). The catalytic core is rendered as a transparent surface. (D) End-on view looking down the E helices from the C-terminal ends of the yellow and blue subunits after a 90° subunit rotation. To obtain this view, the image in C was rotated about the y-axis. The dotted line denotes the rotating interface. Coloring is the same as for C. Note that rotations of 80°–105° are required to optimally position individual cysteine pairs for crosslinking. See the Supplementary Data movie for an animated view of the rotations and crosslinking.
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Figure 5: Cysteine substitutions in the helix-E region. (A) Hin dimer model with the locations of the cysteine substitutions denoted with red spheres. (B) Hin tetramer model (GM4-based) with the locations of the cysteine substitutions denoted with red spheres. (C) Hin tetramer model after a 90° clockwise rotation. Substituted residues have been converted to cysteine with Sγ atoms denoted as spheres. Red designates efficient crosslinking [residues 134, 133, 130, 126 and 122 listed from the C-terminal (left) end]; orange designates crosslinking (residues 131 and 129); green designates poor or no crosslinking (residues 136, 132, 128, 124, 121 and 118); yellow designates cysteines that exhibit poor or no crosslinking and are within the catalytic core and excluded from solvent (residues 115, 112, 109, 105) (Table 1 and Figure 6). The catalytic core is rendered as a transparent surface. (D) End-on view looking down the E helices from the C-terminal ends of the yellow and blue subunits after a 90° subunit rotation. To obtain this view, the image in C was rotated about the y-axis. The dotted line denotes the rotating interface. Coloring is the same as for C. Note that rotations of 80°–105° are required to optimally position individual cysteine pairs for crosslinking. See the Supplementary Data movie for an animated view of the rotations and crosslinking.

Mentions: In order to investigate the structure of the putative helix E segment in Hin at the time of crosslinking and further test the subunit rotation model, cysteines were substituted at 21 positions throughout the region [denoted in Figure 5A (dimer model) and B (closed tetramer model)]. We reasoned that a helical pattern of crosslinking efficiency would provide evidence that the segment (extending to 134) was folded into an α-helix at the time of crosslinking and would provide further support for formation of a rotational conformer in which the E helices were aligned. The cysteines were introduced into the Fis-independent Hin–H107Y background to enable crosslinking using labeled oligonucleotide substrates as described above. Most of these cysteine mutants retained good activity in assembling cleaved DNA complexes on supercoiled DNA in both the presence and absence of Fis (Table 1). Thirteen formed sufficient amounts of synaptic complexes on oligonucleotide substrates to evaluate crosslinking efficiencies. One minute crosslinking reactions were performed on these mutants, and the complexes were purified on native gels followed by SDS–PAGE to evaluate crosslinking, as described for the helix D cysteine mutants (Figure 6A, Table 1). One minute crosslinking reactions were also performed on Fis/enhancer-activated reactions employing supercoiled plasmids. After crosslinking on plasmid substrates, the DNA was digested with EcoR1, which cleaves 50 bp on either side of both recombination sites, end-filled with radiolabeled nucleotides, and the products electrophoresed in SDS gels. The increased activity by Fis allowed us to obtain cysteine crosslinking data on six additional residues where cysteine mutants failed to form synaptic complexes in oligonucleotide reactions (Figure 6B, Table 1). Where data on both substrates were obtained, relative crosslinking efficiencies with different length crosslinkers were similar.Table 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)

Cysteine substitutions in the helix-E region. (A) Hin dimer model with the locations of the cysteine substitutions denoted with red spheres. (B) Hin tetramer model (GM4-based) with the locations of the cysteine substitutions denoted with red spheres. (C) Hin tetramer model after a 90° clockwise rotation. Substituted residues have been converted to cysteine with Sγ atoms denoted as spheres. Red designates efficient crosslinking [residues 134, 133, 130, 126 and 122 listed from the C-terminal (left) end]; orange designates crosslinking (residues 131 and 129); green designates poor or no crosslinking (residues 136, 132, 128, 124, 121 and 118); yellow designates cysteines that exhibit poor or no crosslinking and are within the catalytic core and excluded from solvent (residues 115, 112, 109, 105) (Table 1 and Figure 6). The catalytic core is rendered as a transparent surface. (D) End-on view looking down the E helices from the C-terminal ends of the yellow and blue subunits after a 90° subunit rotation. To obtain this view, the image in C was rotated about the y-axis. The dotted line denotes the rotating interface. Coloring is the same as for C. Note that rotations of 80°–105° are required to optimally position individual cysteine pairs for crosslinking. See the Supplementary Data movie for an animated view of the rotations and crosslinking.
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Figure 5: Cysteine substitutions in the helix-E region. (A) Hin dimer model with the locations of the cysteine substitutions denoted with red spheres. (B) Hin tetramer model (GM4-based) with the locations of the cysteine substitutions denoted with red spheres. (C) Hin tetramer model after a 90° clockwise rotation. Substituted residues have been converted to cysteine with Sγ atoms denoted as spheres. Red designates efficient crosslinking [residues 134, 133, 130, 126 and 122 listed from the C-terminal (left) end]; orange designates crosslinking (residues 131 and 129); green designates poor or no crosslinking (residues 136, 132, 128, 124, 121 and 118); yellow designates cysteines that exhibit poor or no crosslinking and are within the catalytic core and excluded from solvent (residues 115, 112, 109, 105) (Table 1 and Figure 6). The catalytic core is rendered as a transparent surface. (D) End-on view looking down the E helices from the C-terminal ends of the yellow and blue subunits after a 90° subunit rotation. To obtain this view, the image in C was rotated about the y-axis. The dotted line denotes the rotating interface. Coloring is the same as for C. Note that rotations of 80°–105° are required to optimally position individual cysteine pairs for crosslinking. See the Supplementary Data movie for an animated view of the rotations and crosslinking.
Mentions: In order to investigate the structure of the putative helix E segment in Hin at the time of crosslinking and further test the subunit rotation model, cysteines were substituted at 21 positions throughout the region [denoted in Figure 5A (dimer model) and B (closed tetramer model)]. We reasoned that a helical pattern of crosslinking efficiency would provide evidence that the segment (extending to 134) was folded into an α-helix at the time of crosslinking and would provide further support for formation of a rotational conformer in which the E helices were aligned. The cysteines were introduced into the Fis-independent Hin–H107Y background to enable crosslinking using labeled oligonucleotide substrates as described above. Most of these cysteine mutants retained good activity in assembling cleaved DNA complexes on supercoiled DNA in both the presence and absence of Fis (Table 1). Thirteen formed sufficient amounts of synaptic complexes on oligonucleotide substrates to evaluate crosslinking efficiencies. One minute crosslinking reactions were performed on these mutants, and the complexes were purified on native gels followed by SDS–PAGE to evaluate crosslinking, as described for the helix D cysteine mutants (Figure 6A, Table 1). One minute crosslinking reactions were also performed on Fis/enhancer-activated reactions employing supercoiled plasmids. After crosslinking on plasmid substrates, the DNA was digested with EcoR1, which cleaves 50 bp on either side of both recombination sites, end-filled with radiolabeled nucleotides, and the products electrophoresed in SDS gels. The increased activity by Fis allowed us to obtain cysteine crosslinking data on six additional residues where cysteine mutants failed to form synaptic complexes in oligonucleotide reactions (Figure 6B, Table 1). Where data on both substrates were obtained, relative crosslinking efficiencies with different length crosslinkers were similar.Table 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