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Coiled-coil interactions mediate serine integrase directionality.

Kushol Gupta1, Robert Sharp1, Jimmy B Yuan1

  • 1Department of Biochemistry & Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 10104, USA.

Nucleic Acids Research
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This summary is machine-generated.

Serine integrases, crucial for viral DNA integration, exhibit directionality. This study reveals how coiled-coil domains in Listeria innocua integrase promote DNA integration while inhibiting excision.

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Area of Science:

  • Molecular Biology
  • Virology
  • Structural Biology

Background:

  • Serine integrases are essential bacteriophage enzymes mediating site-specific DNA integration and excision.
  • The integration process, involving phage (attP) and host (attB) attachment sites, forms hybrid sites (attL and attR) and is typically irreversible.
  • A proposed model suggests that coiled-coil (CC) domains in integrase subunits dictate reaction directionality by favoring integration over excision.

Purpose of the Study:

  • To investigate the role of coiled-coil (CC) domains in the directionality of serine integrase-mediated recombination.
  • To elucidate the structural basis of CC domain self-interaction and its functional implications in the Listeria innocua integrase (LI Int) system.

Main Methods:

  • Co-immunoprecipitation assays to assess Int self-interaction.
  • X-ray crystallography to determine the structure of the CC domain.
  • Site-directed mutagenesis (alanine scanning) to probe the functional significance of the CC dimer interface.

Main Results:

  • The CC domain of LI Int promotes self-interaction in both isolated and DNA-bound states.
  • Three crystal structures reveal the molecular details of the CC dimer interface.
  • Mutational analysis confirms that CC domain self-interaction is critical for both promoting integration and inhibiting excision.

Conclusions:

  • The CC domain's self-interaction is a key determinant of serine integrase directionality.
  • Structural and functional data provide a molecular basis for understanding how LI Int favors integration over excision.
  • This work offers insights into the stability of integrase-att site complexes.