ELECTRON PARAMAGNETIC RESONANCE OF DNA DYNAMICS

该提案概述了对DNA动力学的研究， 电子顺磁共振（EPR）和脉冲电子双 共振（脉冲ELDOR）。 DNA被EPR激活是由于 通过炔键与修饰的胸苷共价连接的自旋探针， 拴系。 一个探针（T*）已经证明是非常敏感的 双链体DNA的整体翻转和长度依赖性 由于弯曲引起的内部运动。 这个探测器的高灵敏度 使其成为DNA研究的理想候选者。 的改进版本 更刚性地耦合到改进的基座的自旋探针， 正在合成。 这两种探针将用于研究双链DNA的动力学， 或者是线性的（和松弛的），或者包含一个连接，或者是由 蛋白质结合或超螺旋。 DNA将被研究， 溶液和固态（在Sephadex-DEAE树脂上）。 所有DNA 将在DNA合成仪上合成，自旋探针将 被放置在序列中预先选择的战略位置。 的EPR 主动自旋探针因此报告在特定， 在DNA分子中定义明确的位置。 DNA蛋白质相互作用的模型系统（使用TF-IID） 转录起始蛋白将用于研究 蛋白质结合DNA的灵活性。 DNA重组的模型系统 将被研究来测量碱基对上链交换的速率 水平 作为这项研究的一部分，整体翻滚运动将是 测量（以获得全局结构信息）以及本地基础- 对运动（以获得关于内部运动的速率和幅度的信息） 运动）。 这些动态数据对于绘制完整的 DNA的迁移率和构象灵活性的图片， 不同类型的压力。

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.