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 EcoRI, BamHI and EcoRV endonucleases has established a rigorous thermodynamic and kinetic basis for a quantitative understanding of the free energy changes in protein-DNA interactions, including the contributions of direct protein- base and protein-phosphate contacts and the energy required to distort the DNA. For the next project period, we propose the following interrelated aims: 1. To determine and dissect the entropic and enthalpic contributions to the formation of """"""""canonical"""""""" protein-DNA complexes, using titration- calorimetric, van't Hoff and osmotic-stress methods. To combine this analysis with structure-perturbation methods to determine how flanking- sequence or intra-site conformational determinants affect the conformational and vibrational entropy changes (DeltaDeltaS(o) conf and DeltaDelta(o) vib) and to determine the thermodynamic characteristics of the """"""""adaptive"""""""" complexes formed with sites differing by one base-pair (""""""""star"""""""" sites) and with sites containing base-analogs that perturb base- phosphate networks. 2. To determine how particular sequence contexts (i.e., outside a recognition site) influence site-specific interactions of three restriction endonucleases (EcoRI, BamHI, EcoRV) that distort their DNA sites in different ways and have different patterns of phosphate contacts immediately flanking the recognition site. 3. To complete our systematic study of the structural features within the DNA recognition site that govern the energetic contribution of DNA distortability to the EcoRI endonuclease-DNA interaction, and to extend this analysis to BamHI endonuclease. 4. 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.
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