ELECTRON PARAMAGNETIC RESONANCE OF DNA DYNAMICS

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This proposal outlines research on the dynamics of DNA as studied by Electron Paramagnetic Resonance (EPR), and Pulsed Electron Double Resonance (pulsed-ELDOR). The DNA is made EPR active by the presence of a spin probe covalently linked to a modified thymidine via an acetylenic tether. One probe (T*) has already demonstrated that is very sensitive to the overall tumbling of duplex DNA and to the length dependent internal motions due to flexure. The high sensitivity of this probe makes it an ideal candidate for studies of DNA. An improved version of the spin probe, which is more rigidly coupled to a modified base, is being synthesized. Both probes will be used to study the dynamics of duplex DNA that is either linear (and relaxed), or contains a junction, or is stressed by either protein binding or supercoiling. The DNA will be studied both i solution and with the solid state (on a Sephadex-DEAE resin). All DNAs will be made synthetically o a DNA synthesizer and the spin probe will be placed at pre-selected strategic positions in the sequence. The EPR active spin probe is therefore reporting the local motion at specific, well defined positions in the DNA molecule. A model system for DNA protein interaction (using the TF-IID) transcription initiation protein will be used to study the effects of protein binding on DNA flexibility. A model system for DNA recombination will be studied to measure the rate of strand exchange at the base-pair level. As part of this study, the overall tumbling motion will be measured (to obtain global structural information) as well as local base- pair motion (to obtain information on the rate and amplitude of internal motion). Such dynamical data are essential for drawing a complete picture of the mobility and conformation flexibility of DNA under different types of stress.