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Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens

Alain Raynal1, Annick Friedmann1, Karine Tuphile1

  • 1Laboratoire de Biologie et Génétique Moléculaire, Institut de Génétique et Microbiologie, UMR CNRS 8621, Bât. 400, Université Paris-Sud, F-91405 Orsay Cedex, France1.

Microbiology (Reading, England)
|January 10, 2002
PubMed

Insights

Site-specific recombination in Streptomyces ambofaciens involves the pSAM2 integrase protein. Studies in E. coli identified essential binding sites (P1/P2) within the attP region required for this integration process.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • pSAM2 integrates into the Streptomyces ambofaciens chromosome via site-specific recombination.
  • This process relies on the integrase protein encoded by pSAM2, which catalyzes recombination between attP and attB sites.

Purpose of the Study:

  • To investigate the mechanisms of pSAM2 site-specific recombination.
  • To identify the DNA regions and protein interactions critical for integration.

Main Methods:

  • Development of tools for studying site-specific recombination in Escherichia coli.
  • In vivo recombination assays to determine essential DNA fragments.
  • Protein overexpression and purification of pSAM2 integrase.
  • DNaseI footprinting to map integrase binding sites on attP.

Main Results:

  • A 360 bp fragment of attP is necessary for efficient recombination.
  • The integrase (int) can be supplied in trans.
  • DNaseI footprinting identified two strong integrase binding sites (P1/P2) within attP.
  • These sites contain a conserved 17 bp motif and are essential for recombination.

Conclusions:

  • pSAM2 integrase binds specifically to the P1 and P2 sites within the attP region.
  • These P1/P2 binding sites are crucial for mediating site-specific integration of pSAM2 into the Streptomyces chromosome.

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