We propose to use the highly specific interaction between EcoRI endonuclease and oligonucleotides containing the normal (GAATTC) or variant recognition sites as a model system for the study of sequence-specific DNA-protein interactions. We have demonstrated an unexpected role for electrostatic interactions at GAATpTC in DNA cleavage. We have used data on equilibrium binding and cleavage kinetics to formulate a refined model which correctly orders the effects of base substitutions in the recognition site. We have also demonstrated that contacts between the protein and particular DNA phosphates are altered when the enzyme binds to recognition sites containing a single base substitution, and that the phosphate contact at pXGAATTC is dependent upon flanking sequence. Here we propose to build upon this foundation by: a) Determining whether and how hydrogen bonding interactions to bases in the recognition site depend upon each other; b) Assessing the functional roles and interdependences of interactions between the protein and individual DNA phosphates; c) Systematically investigating the basis for the influence of flanking base sequence on phosphate interactions; and d) Identifying a carboxyl residue whose chemical modification inactivates the endonuclease.
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