Physical Chemistry of Recombinational Intermediates

重组中间体的物理化学

• 批准号: 8312528
• 负责人: NADRIAN C. SEEMAN
• 金额: $ 32.76万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312528
• 关键词: Biological Process; Cells; DNA; DNA Repair; DNA Structure; Electrons; Genetic Recombination; Instruction; Label; Lead; Meiosis; Methods; Microscopic; Physical Chemistry; Plasmids; Process; Resolution; Scheme; Structure; Superhelical DNA; Techniques; base; crosslink; design; ds-DNA; improved; nanoparticle; recombinational repair; research study; response; three dimensional structure

This project entails the characterization of DNA molecules involved in the processes of DNA recombination. These are typically branched molecules, whose 3D structures are only partially known. A key issue is the recognition of homology by double stranded DNA molecules. We have established that there is an unusual DNA structure that is formed by homologous molecules when they are in a supercoiled plasmid. Specific Aim 1. The structure that is formed is a dumbbell-like, where the shaft contains the homologous DNA. We plan to ascertain the structure of the shaft of the homology structure. To see if (as hypothesized) the structure is indeed PX-DNA, we will proceed v^ath chemically-based experiments, including crosslinking the strands together andpadlocking the circle. We would like to have the best physical evidence that we can get on the shaft. The next level of characterization will entail electron microscopic examination of dumbbells that have been derivatized by metallic nanoparticles that will be visible. We will use this labeling scheme to try to make the relevant portions of the dumbbell molecules visible to the cryo-EM, so that those methods can be applied to this key structure. Specific Aim 2. We have produced crystals of a variety of branched and otherwise unusual DNA species relevant to recombination. These include designed rhombohedral lattices that self-assemble to form macroscopic crystals. We plan to determine the structures of these lattices. We also have crystals of the inside of a double crossover molecule (related to meiotic intermediates) that diffract to atomic resolution, and a third crystal that contains an unusual linkage. We will determine the structures ofthese molecules and will establish their 3D structures. In addition, we will try to improve the resolution of some of the larger self-assembling lattices by a lashing technique that will, if successful, lead to the ability to insert guests into the lattice.

该项目需要表征参与DNA重组过程的DNA分子。 这些通常是分支分子，其3D结构仅部分知道。一个关键问题是 双链DNA分子对同源性的识别。我们已经确定有一个不寻常的DNA 由同源分子形成的结构，当它们在超螺旋质粒中时。 特定的目标1。形成的结构是类似哑铃的样结构，其中轴包含同源DNA。 我们计划确定同源结构轴的结构。查看（如假设的）是否 结构确实是PX-DNA，我们将进行基于化学的实验，包括交联的实验 将链条一起并锁定圆圈。我们想拥有最好的物理证据 轴。下一个表征将需要对具有的哑铃进行电子显微镜检查 被将可见的金属纳米颗粒衍生。我们将使用此标签方案尝试制作 哑铃分子的相关部分可见，以便将这些方法应用于 这个关键结构。 具体目标2。我们生产了各种分支和其他不寻常DNA物种相关的晶体 重组。其中包括设计自组装以形成宏观的菱形晶格 晶体。我们计划确定这些晶格的结构。我们也有双层的晶体 衍射与原子分辨率的跨界分子（与减数分裂中间体有关），以及第三个晶体 包含不寻常的联系。我们将确定这些分子的结构，并建立其3D 结构。此外，我们将尝试通过一个 如果成功的话，绑扎技术将使宾客插入晶格。