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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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Activities and crosslinking of helix D mutants. (A) Inversion of DNA between the hix sites on pMS551 by Hin reorients the HindIII site to produce different sized fragments on an agarose gel when co-digested with Pst I (32). Time courses were performed with the indicated Hin mutant together with Fis and HU on supercoiled pMS551, and aliquots were taken at the times indicated. Hin–H107Y, H107Y/K72C and H107Y/A76C exhibit similar inversion kinetics. (B) Synaptic complex assembly on oligonucleotide substrates. Hin was incubated with 36 bp 3′ 32P-labeled DNA for 20 min and electrophoresed on a native polyacrylamide gel containing 10% glycerol to enhance synaptic complex formation. Each of the mutants forms large amounts of synaptic complexes. (C) Crosslinking of mutants with cysteines in helix D. Reactions were performed as in panel B and then subjected to crosslinking for 1 min. Synaptic complexes were excised from a native gel, eluted and crosslinked products were separated by SDS–PAGE. Crosslinkers were diamide (0 Å), BMOE (8 Å spacer) and BMH (16 Å spacer). The locations of non-crosslinked and crosslinked Hin–DNA(32P) covalent complexes are shown on the autoradiograph.
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Figure 3: Activities and crosslinking of helix D mutants. (A) Inversion of DNA between the hix sites on pMS551 by Hin reorients the HindIII site to produce different sized fragments on an agarose gel when co-digested with Pst I (32). Time courses were performed with the indicated Hin mutant together with Fis and HU on supercoiled pMS551, and aliquots were taken at the times indicated. Hin–H107Y, H107Y/K72C and H107Y/A76C exhibit similar inversion kinetics. (B) Synaptic complex assembly on oligonucleotide substrates. Hin was incubated with 36 bp 3′ 32P-labeled DNA for 20 min and electrophoresed on a native polyacrylamide gel containing 10% glycerol to enhance synaptic complex formation. Each of the mutants forms large amounts of synaptic complexes. (C) Crosslinking of mutants with cysteines in helix D. Reactions were performed as in panel B and then subjected to crosslinking for 1 min. Synaptic complexes were excised from a native gel, eluted and crosslinked products were separated by SDS–PAGE. Crosslinkers were diamide (0 Å), BMOE (8 Å spacer) and BMH (16 Å spacer). The locations of non-crosslinked and crosslinked Hin–DNA(32P) covalent complexes are shown on the autoradiograph.

Mentions: Cysteines were independently substituted for Lys72 and Ala76 in an otherwise wild-type and in a Hin–H107Y background. Hin–H107Y is a strong hyperactive mutant that can promote DNA exchange within stable, DNA-cleaved, synaptic complexes without the requirement for Fis, the enhancer, or DNA supercoiling (17). The cysteine mutants were purified and evaluated for their ability to promote DNA inversion in vitro. Hin–A76C exhibited Fis-activated inversion activity that was slightly elevated over wild-type Hin, whereas inversion by Hin–K72C was reduced to <10% wild-type rates (data not shown). Qualitative in vivo assays measuring transcription of a lacZ reporter gene as a function of Hin-catalyzed inversion (31) also showed that Hin–K72C was severely defective in promoting inversion and that Hin–A76C exhibited a slightly faster inversion rate. As shown in Figure 3A, combining K72C with the H107Y hyperactive mutation rescues the inversion defect; Hin–H107Y/A76C also exhibits similar Fis-activated inversion rates as Hin–H107Y.


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

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

Activities and crosslinking of helix D mutants. (A) Inversion of DNA between the hix sites on pMS551 by Hin reorients the HindIII site to produce different sized fragments on an agarose gel when co-digested with Pst I (32). Time courses were performed with the indicated Hin mutant together with Fis and HU on supercoiled pMS551, and aliquots were taken at the times indicated. Hin–H107Y, H107Y/K72C and H107Y/A76C exhibit similar inversion kinetics. (B) Synaptic complex assembly on oligonucleotide substrates. Hin was incubated with 36 bp 3′ 32P-labeled DNA for 20 min and electrophoresed on a native polyacrylamide gel containing 10% glycerol to enhance synaptic complex formation. Each of the mutants forms large amounts of synaptic complexes. (C) Crosslinking of mutants with cysteines in helix D. Reactions were performed as in panel B and then subjected to crosslinking for 1 min. Synaptic complexes were excised from a native gel, eluted and crosslinked products were separated by SDS–PAGE. Crosslinkers were diamide (0 Å), BMOE (8 Å spacer) and BMH (16 Å spacer). The locations of non-crosslinked and crosslinked Hin–DNA(32P) covalent complexes are shown on the autoradiograph.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2724282&req=5

Figure 3: Activities and crosslinking of helix D mutants. (A) Inversion of DNA between the hix sites on pMS551 by Hin reorients the HindIII site to produce different sized fragments on an agarose gel when co-digested with Pst I (32). Time courses were performed with the indicated Hin mutant together with Fis and HU on supercoiled pMS551, and aliquots were taken at the times indicated. Hin–H107Y, H107Y/K72C and H107Y/A76C exhibit similar inversion kinetics. (B) Synaptic complex assembly on oligonucleotide substrates. Hin was incubated with 36 bp 3′ 32P-labeled DNA for 20 min and electrophoresed on a native polyacrylamide gel containing 10% glycerol to enhance synaptic complex formation. Each of the mutants forms large amounts of synaptic complexes. (C) Crosslinking of mutants with cysteines in helix D. Reactions were performed as in panel B and then subjected to crosslinking for 1 min. Synaptic complexes were excised from a native gel, eluted and crosslinked products were separated by SDS–PAGE. Crosslinkers were diamide (0 Å), BMOE (8 Å spacer) and BMH (16 Å spacer). The locations of non-crosslinked and crosslinked Hin–DNA(32P) covalent complexes are shown on the autoradiograph.
Mentions: Cysteines were independently substituted for Lys72 and Ala76 in an otherwise wild-type and in a Hin–H107Y background. Hin–H107Y is a strong hyperactive mutant that can promote DNA exchange within stable, DNA-cleaved, synaptic complexes without the requirement for Fis, the enhancer, or DNA supercoiling (17). The cysteine mutants were purified and evaluated for their ability to promote DNA inversion in vitro. Hin–A76C exhibited Fis-activated inversion activity that was slightly elevated over wild-type Hin, whereas inversion by Hin–K72C was reduced to <10% wild-type rates (data not shown). Qualitative in vivo assays measuring transcription of a lacZ reporter gene as a function of Hin-catalyzed inversion (31) also showed that Hin–K72C was severely defective in promoting inversion and that Hin–A76C exhibited a slightly faster inversion rate. As shown in Figure 3A, combining K72C with the H107Y hyperactive mutation rescues the inversion defect; Hin–H107Y/A76C also exhibits similar Fis-activated inversion rates as Hin–H107Y.

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