Coordinate ion pair formation between EcoRI endonuclease and DNA

Area of Science:

• Molecular biology
• Biochemistry
• Structural biology

Background:

• EcoRI restriction endonuclease binds to a specific DNA sequence.
• The binding involves electrostatic and nonelectrostatic forces.
• A mutation S187 affects the electrostatic contribution to binding.

Purpose of the Study:

• To dissect the electrostatic and nonelectrostatic contributions to EcoRI-DNA binding.
• To investigate the role of arginine 187 in binding.
• To understand the functional separation of electrostatic interactions from DNA recognition and cleavage.

Main Methods:

• Measuring equilibrium binding constants at varying salt concentrations.
• Applying thermodynamic analyses (Record et al.).
• Utilizing endonuclease mutation S187.

Main Results:

• The S187 mutation primarily affects the electrostatic component of binding.
• Wild type EcoRI forms eight ionic interactions, while S187 forms six at pH 6.0 and two at pH 7.4.
• Electrostatic interactions are separable from sequence recognition and phosphodiester bond hydrolysis.

Conclusions:

• Arginine 187 is crucial for the electrostatic function of EcoRI.
• Electrostatic interactions are functionally distinct from DNA sequence recognition and cleavage.
• A model suggests mobile ionic groups on the protein mediate DNA interaction.