The goal of this proposal is to contribute to the understanding of sequence directed thermodynamic behavior of DNA. A novel approach will be employed to evaluate thermodynamic parameters of base pair stability, intramolecular hairpin loop formation and internal loop formation in DNA as a function of solvent ionic strength. Automated DNA synthesis will be employed to construct a series of self-complementary DNA loops (dumbbells). Absorbance versus temperature melting profiles of these molecules over a wide range of solvent ionic strengths will be measured and analyzed in terms of an exact equilibrium statistical thermodynamic model that has been formulated for predicting helix-coil transitions in DNA dumbbells. Dumbbells have several advantages over linear fragments in physical studies investigating thermostability of DNA. Because """"""""end-fraying"""""""", strand dissociation and the associated concentration dependence of the melting process are eliminated, only the molecular internal degrees of freedom contribute to the melting process of DNA dumbbells. Sequence dependent base pair stability, internal loop melting, melting cooperativity and intramolecular loop formation all constitute contributions from the internal degrees of freedom. Thermodynamic parameters representative of these internal molecular degrees of freedom as a function of solvent ionic strength will be empirically evaluated by adjusting them in the theoretical calculation to fit experimental transitions. Heterogeneous sequence DNA restriction fragments with hairpin loop attached to the ends of the duplex will also be constructed. NMR and dynamic light scattering will be employed to investigate the solution dynamics of these """"""""long"""""""" DNA dumbbells. The interactions of the catabolite activator protein and EcoRI endonuclease with their specific recognition sequence on a DNA dumbbell will also be investigated.
