FACILITATED DISTORTION OF THE DNA SITE ENHANCES ECORI ENDONUCLEASE DNA RECOGNITION

We have measured the binding of EcoRI endonuclease to a complete set o f purine-base analogue sites, each of which deletes one functional gro up that forms a hydrogen bond with the endonuclease in the canonical G AATTC complex. For five of six functional group deletions, the observe d 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-b ase hydrogen bond is removed without deleting an interstrand Watson-Cr ick hydrogen bond or causing structural ''adaptation'' in the complex. This observation establishes that the incremental energetic contribut ion of one protein-base hydrogen bond is about -1.5 kcal/mol. By contr ast, deletion of the N6-amino group of the inner adenine in the site i mproves binding by -1.0 kcal/mol because the penalty for deleting a pr otein-base hydrogen bond is outweighed by facilitation of the required DNA distortion (''kinking'') in the complex. This result provides dir ect evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.