Our long-term objective is to use restriction endonucleases as models to understand the structural and energetic factors that determine specificity in DNA-protein interactions. Our previous work with EcoRl endonuclease has shown that both thermodynamic and kinetic factors govern selectivity between DNA sites. We have demonstrated and quantified the importance not only of direct protein-base contacts, but also of sequence-specific formation of particular phosphate contacts and the energy required to distort the DNA into a particular conformation in the complex. In the next project period, we propose thermodynamic and kinetic studies: 1. To identify structural features that may govern the energetic contribution of DNA distortability to the EcoRI endonuclease-DNA interaction, by systematically varying major- and minor-groove functional groups with base analogue replacements. 2. To delineate the functional roles of interactions with DNA phosphates at pNGApATTC in both the enzyme-substrate complex and the transition state complex, by stereospecific substitutions with chiral phosphorothioates and methylphosphonates. 3. To use existing """"""""promiscuous"""""""" mutants of EcoRI endonuclease to determine how the endonuclease-DNA interface and the energetics of the interaction may be modified by the introduction of new favorable interactions or the elimination of unfavorable interactions. 4. To test the generality of the principles deduced from Eco RI endonuclease, by comparing particular properties of BamHI and EcoRV endonucleases. For BamHI endonuclease, we will determine by base analogue studies the relative contributions to the interaction made by direct protein-base contacts and DNA conformational properties. For EcoRV endonuclease, we will determine (using novel noncleavable phosphorothiolate oligonucleotides) if binding discrimination or protein-phosphate contacts are affected by Mg++ and whether flanking sequence has unusually strong effects on binding discrimination.
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