ELECTRON PARAMAGNETIC RESONANCE OF DNA DYNAMICS

DNA 动力学的电子顺磁共振

• 批准号: 5212101
• 负责人: Bruce H Robinson
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5212101
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; conformation; electron spin resonance spectroscopy; electronic spectra; genetic recombination; intermolecular interaction; molecular dynamics; nucleic acid chemical synthesis; nucleic acid probes; nucleic acid sequence; nucleic acid structure; stress; transcription factor

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.

该提议概述了对DNA动力学的研究 电子顺磁共振（EPR）和脉冲电子双重 共振（脉冲 - 冠军）。 通过存在使DNA使EPR活跃 旋转探针通过乙酰基因共价与改良的胸苷连接 系绳。 一个探针（t*）已经证明了非常敏感的 到双链DNA的整体翻滚和长度依赖性 由于弯曲而引起的内部动作。 该探针的高灵敏度 使其成为研究DNA的理想候选者。 改进的版本的 旋转探针更牢固地耦合到修改的底座，为 合成。 这两个探针都将用于研究双链DNA的动力学 线性（放松），或包含一个结，或者由 蛋白质结合或超螺旋。 DNA将研究我 溶液和固态（在Sephadex-Deae树脂上）。 所有DNA 将通过合成o o DNA合成器，并且自旋探针将 在序列中放置在预选的战略位置。 EPR 因此，主动旋转探针是在特定的 DNA分子中定义明确的位置。 DNA蛋白相互作用的模型系统（使用TF-IID） 转录起始蛋白将用于研究 蛋白质对DNA柔韧性的结合。 DNA重组的模型系统 将研究以测量基本对的链交换速率 等级。 作为这项研究的一部分，总体翻滚运动将是 测量（获取全球结构信息）以及本地基础 配对运动（获取有关内部速度和幅度的信息 运动）。 这种动力数据对于绘制完整的数据至关重要 DNA下的迁移率和构象灵活性的图片 不同类型的压力。