DYNAMICS, RELAXATION, AND LOCAL MOTION IN DNA

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It is proposed to investigate certain aspects of DNA dynamics, structure, and hydration that may have significant roles in various biological processes. The amplitudes of local angular motion of selected groups in DNA provide information about its local deformability, which may be a significant factor in site-specific recognition and/or action of various proteins that interact with DNA. Whether the amplitudes of local angular motions in less stable non-standard structures, such as a hairpin loop, the third strand of a triplex, and a 4-arm Holiday junction, significantly exceed those in a normal duplex DNA is another question addressed here. Such amplitudes of local angular motion are determined by NMR relaxation measurements of selectively labelled synthetic DNAs in solution in conjunction with time-resolved fluorescence polarization anisotropy (FPA) measurements of intercalated ethidium (to characterize the global rotations) and recently developed theory. That same theory will also be used to estimate the errors incurred when the so-called model-free formulas for monodisperse spherical diffusors are applied to determine the amplitudes local angular motion of non-spherical molecules and molecules that dimerize appreciably. Possible long range effects of the SP1 transcriptional activator recognition sequence on the secondary structure of its flanking DNA are investigated by a variety of physical and biochemical methods, including dynamic light scattering (DLS), FPA, circular dichroism (CD) spectroscopy, optical melting and susceptibility to S1 nuclease. Any modulation of such long range effects upon binding the Sp1 protein will be similarly examined. Possible long range effects due to a particular mutation in the 434 repressor recognition sequence are also investigated by physical and biochemical techniques. The intrinsic twist and twist energy parameter are determined from measured topoisomer ratios after circularizing in the presence of various amounts of ethidium, again using recently developed theory. The effects of particular cations, anions, temperature, and other agents on the hydration of small DNA duplexes are examined via their effects on the hydrodynamic radius, which is measured by FPA of intercalated ethidium.