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Related Experiment Videos

Protein-protein interactions directing resolvase site-specific recombination: a structure-function analysis

R E Hughes1, P A Rice, T A Steitz

  • 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.

The EMBO Journal
|April 1, 1993
PubMed
Summary
This summary is machine-generated.

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Gamma delta resolvase (GDR) recombination requires a synaptosome complex. This study identified key resolvase-resolvase interactions, revealing how three specific subunit contacts are crucial for DNA synapsis and recombination.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • Recombination catalyzed by gamma delta resolvase (GDR) involves the formation of a nucleoprotein complex called the synaptosome.
  • The structure of the synaptosome is dictated by interactions between resolvase subunits and with DNA (res).
  • Previous studies identified three distinct dyad axes of resolvase association in crystal structures of the catalytic domain.

Purpose of the Study:

  • To investigate the functional significance of two previously uncharacterized inter-subunit interfaces in gamma delta resolvase.
  • To elucidate the specific roles of resolvase-resolvase interactions in the assembly of the synaptosome and the catalysis of recombination.
  • To determine the precise dimer interface and other inter-subunit contacts essential for GDR function.

Main Methods:

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  • Site-directed mutagenesis was employed to alter specific amino acid positions within the resolvase catalytic domain.
  • Cysteine substitutions were introduced to create covalent cross-links at potential interfaces.
  • Arginine and tryptophan mutations were used to disrupt inter-subunit contacts and assess hydrophobicity/solvent accessibility via fluorescence quenching.

Main Results:

  • Characterization of mutant proteins identified the primary dimer interface of gamma delta resolvase.
  • A second inter-subunit contact was assigned a specific structural role in synaptosome assembly.
  • Mutational analysis confirmed the functional importance of the identified interfaces in recombination.

Conclusions:

  • All three dyad-related inter-subunit interactions observed in the crystal structure of gamma delta resolvase are functionally significant.
  • These interactions play specific roles in the process of DNA synapsis and subsequent recombination.
  • The findings provide a deeper understanding of the structural basis for gamma delta resolvase-mediated recombination.