Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition.

AUTOR(ES)

Lesser, D R

RESUMO

We have measured the binding of EcoRI endonuclease to a complete set of purine-base analogue sites, each of which deletes one functional group that forms a hydrogen bond with the endonuclease in the canonical GAATTC complex. For five of six functional group deletions, the observed penalty in binding free energy is +1.3 to +1.7 kcal/mol. For two of these cases (replacement of adenine N7 with carbon) a single protein-base hydrogen bond is removed without deleting an interstrand Watson-Crick hydrogen bond or causing structural "adaptation" in the complex. This observation establishes that the incremental energetic contribution of one protein-base hydrogen bond is about -1.5 kcal/mol. By contrast, deletion of the N6-amino group of the inner adenine in the site improves binding by -1.0 kcal/mol because the penalty for deleting a protein-base hydrogen bond is outweighed by facilitation of the required DNA distortion ("kinking") in the complex. This result provides direct evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.

Documentos Relacionados

• "Ultraviolet footprinting" accurately maps sequence-specific contacts and DNA kinking in the EcoRI endonuclease-DNA complex.
• Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.
• Does the specific recognition of DNA by the restriction endonuclease EcoRI involve a linear diffusion step? Investigation of the processivity of the EcoRI endonuclease.
• Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites.
• Specificity of substrate recognition by the EcoRI restriction endonuclease.