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EcoRII: a restriction enzyme evolving recombination functions?

Merlind Mücke1, Gerlinde Grelle, Joachim Behlke

  • 1Institut für Virologie, Medizinische Fakultät (Charité) der Humboldt-Universität zu Berlin, D-10098 Berlin, Germany.

The EMBO Journal
|October 3, 2002
PubMed
Summary
This summary is machine-generated.

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The restriction enzyme EcoRII uses two DNA sites for cleavage. Its C-terminal domain, EcoRII-C, cleaves single sites, while the N-terminal domain regulates activity, suggesting an evolutionary role.

Area of Science:

  • Molecular Biology
  • Enzymology
  • Genetics

Background:

  • The restriction endonuclease EcoRII recognizes and cleaves DNA at specific sequences, requiring cooperative binding to two 5'CCWGG sites.
  • Understanding the structural and functional basis of this cooperative DNA binding is crucial for elucidating its mechanism and evolutionary significance.

Purpose of the Study:

  • To investigate the domain structure of EcoRII and its role in DNA cleavage and recognition.
  • To characterize a novel C-terminal restriction endonuclease domain (EcoRII-C) and compare its activity to the wild-type enzyme.
  • To propose an evolutionary model for EcoRII based on its domain organization and function.

Main Methods:

  • Limited proteolysis to determine the domain structure of EcoRII.
  • Biochemical assays to assess DNA cleavage activity of wild-type EcoRII and the isolated EcoRII-C domain.

Related Experiment Videos

  • Analysis of DNA substrates with single and multiple 5'CCWGG sites.

    • Main Results:

    • EcoRII possesses a two-domain structure, comprising an N-terminal DNA-binding domain and a C-terminal catalytic domain (EcoRII-C).
    • EcoRII-C functions as a restriction endonuclease that cleaves DNA at single 5'CCWGG sites.
    • EcoRII-C exhibits significantly higher cleavage efficiency on substrates with multiple cooperative 5'CCWGG sites compared to wild-type EcoRII.
    • The N-terminal domain specifically binds DNA and attenuates EcoRII activity, enforcing dependence on a second 5'CCWGG site.

    Conclusions:

    • The two-domain structure of EcoRII facilitates cooperative binding to two 5'CCWGG sites, essential for its wild-type activity.
    • EcoRII-C represents a novel, simplified restriction endonuclease capable of cleaving single recognition sites.
    • EcoRII's domain structure suggests it is an evolutionary intermediate, potentially acquiring a DNA-binding domain to achieve two-site recognition, bridging simple endonucleases and proteins like recombinases and transposases.