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Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
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Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several

Karol H Wyszomirski1, Ute Curth, Jürgen Alves

  • 1Institute of Medical Virology, Helmut-Ruska-Haus, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin,Germany.

Nucleic Acids Research
|December 27, 2011
PubMed

Insights

The restriction enzyme EcoP15I uses its single restriction subunit to bind DNA. Multiple DNA-binding regions on the restriction subunit

Area of Science:

  • Molecular Biology
  • Enzymology

Background:

  • Type III restriction endonucleases like EcoP15I are crucial for DNA cleavage.
  • EcoP15I functions as a multifunctional enzyme complex with methylation (Mod) and restriction (Res) subunits.

Purpose of the Study:

  • To determine the stoichiometry and structure of the EcoP15I enzyme complex.
  • To characterize the DNA-binding properties of the restriction (Res) subunit and its domains.

Main Methods:

  • Analytical ultracentrifugation and other biochemical methods to determine stoichiometry.
  • Expression and purification of the isolated translocase (Tr) domain.
  • ATP-hydrolysis assays and DNA-binding experiments.
  • Peptide array screening and computational modeling.

Main Results:

  • EcoP15I was found to have a Mod(2)Res stoichiometry, with a single Res subunit.
  • The isolated Tr domain exhibits ATP-hydrolyzing activity and binds DNA non-specifically.
  • Four DNA-binding regions were identified in the Tr domain and two in the endonuclease domain of the Res subunit.
  • These DNA-binding regions are surface-exposed and conserved in other Type III enzymes.

Conclusions:

  • The EcoP15I restriction subunit possesses multiple DNA-binding sites critical for its function.
  • The identified DNA-binding regions are conserved, suggesting a common mechanism for Type III restriction enzymes.

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