Recent experiments have suggested that, in concentrated solutions, DNA dynamics is profoundly affected by intermolecular interactions. This interpretation is especially intriguing because DNA performs is cellular functions in a concentrated environment. The experiments proposed herein are designed to explore, in greater depth, the nature of DNA dynamics at concentrations that are representative of the physiological state. Two fluorescence techniques--Fluorescence Photobleaching Recovery (FPR) and Fluorescence Correlation Spectroscopy (FCS)-- and a pulsed Electron Electron Double Resonance (ELDOR) technique will be used. Rotational and translational diffusion of DNA in concentrated solutions and in gels will be measured. Our newly introduced polarized FPR technique and pulsed ELDOR will be used to measure the effects that concentration, salt, DNA size and form and the binding of important enzymes such as RNA polymerase have on reorientational relaxation. Polarized FPR has recently been shown to be well suited to the task of monitoring slow (longer than 1 mu sec) reorientational relaxation. The pulsed ELDOR technique is expected to yield excellent angular resolution approximately 10 degrees, and to be capable of measuring rotational correlation times from about 200 nanoseconds to one millisecond; the interpretation of ELDOR is also conceptually quite straightforward. For use in the ELDOR studies, three new Electron Paramagnetic Resonance (EPR) spin label probes--a nitroxide-psoralen compound, and nitroxide-labeled adenosine and thymidine bases--have been synthesized by us and our collaborators. The latter molecule has been shown to be nonperturbing to the DNA and to be a faithful reporter of DNA motion. Translational FPR will be used to determine the interaction dependence of the self-diffusion coefficient of DNA in concentrated samples; complementary FCS experiments will allow us to measure mutual diffusion. The reorientation and translation studies proposed here are expected to reveal novel information about the dynamics that underlies important biological processes such as transcription, DNA replication and DNA condensation.
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