ELECTRON PARAMAGNETIC RESONANCE OF DNA DYNAMICS

该建议概述了对DNA动力学的研究，该研究由 电子顺磁共振(EPR)和脉冲电子倍增 共振(脉冲Eldor)。DNA通过存在于EPR中而激活 一种通过乙炔共价连接到修饰的胸苷的自旋探针 系绳。一个探测器(T*)已经证明了它是非常敏感的 对双链DNA的整体翻滚和对长度的依赖 由于弯曲而产生的内部运动。这种探头的高灵敏度 这使它成为研究DNA的理想候选者。改进版本的 自旋探测器更坚固地连接到改进的基座上， 正在被合成。 这两个探针都将用于研究双链DNA的动力学 线性的(和松弛的)，或包含连接点，或由 蛋白质结合或超级卷曲。将对DNA进行研究，包括 溶液和固态(在Sephadex-DEAE树脂上)。所有DNA 将在DNA合成器上合成，自旋探测器将 被安排在序列中预先选定的战略位置。《环境政策报告》 因此，主动自旋探测器报告了特定位置的局部运动， DNA分子中明确的位置。 DNA蛋白质相互作用的模型系统(使用Tf-IID) 转录起始蛋白将被用来研究 蛋白质结合对DNA柔韧性的影响。一种DNA重组模型系统 将被研究以测量碱基对的链交换速率 水平。作为这项研究的一部分，整体翻滚运动将是 测量(以获得全球结构信息)以及局部基础- 配对运动(以获取有关内部运动的速率和幅度的信息 动议)。这样的动态数据对于绘制完整的 DNA的迁移性和构象灵活性 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.