Redesign of high-affinity nonspecific nucleases with altered sequence preference

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• Understanding protein-DNA interactions is key for molecular biology and biotechnology.
• The nuclease domain of Colicin E7 (nColE7) is a model system for studying DNA binding.
• Wild-type nColE7 has a preference for cleaving DNA after thymine and adenine.

Purpose of the Study:

• To generate redesigned nucleases with enhanced DNA-binding affinity and altered sequence specificity.
• To investigate the structural and energetic basis for improved protein-DNA interactions.

Main Methods:

• Systematic computational screening to predict high-affinity nColE7 mutants.
• Site-directed mutagenesis, protein purification, and fluorescence kinetic assays for DNA binding.
• DNA footprinting assays to determine sequence cleavage preferences.
• X-ray crystallography and free energy decomposition analysis for structural insights.

Main Results:

• Four out of five engineered nColE7 mutants exhibited 3- to 5-fold higher DNA binding affinity.
• Three mutants (D493N, D493Q, D493R) showed an increased preference for cleaving DNA at guanine residues.
• Structural analysis revealed that replacing D493 resolved unfavorable electrostatic repulsion, improving protein-DNA interactions.

Conclusions:

• Computational screening is effective for designing nucleases with improved DNA binding.
• Mutations can alter both DNA binding affinity and sequence cleavage specificity.
• This approach provides a powerful strategy for engineering custom DNA-binding proteins.