Physical Chemistry of Recombinational Intermediates

该项目需要对参与DNA重组过程的DNA分子进行表征。 这些通常是分支分子，其3D结构仅部分已知。一个关键问题是 通过双链DNA分子的同源性识别。我们已经证实了在他的尸体上 同源分子在超螺旋质粒中形成的结构。 具体目标1。形成的结构是哑铃状的，其中轴包含同源DNA。 我们计划确定轴的结构的同源性结构。看一看，是不是真的。 结构确实是PX-DNA，我们将进行基于化学的实验，包括交联 把绳子绑在一起锁上圆圈。我们希望能找到最好的物证 轴。下一个层次的特征将需要电子显微镜检查哑铃， 被可见的金属纳米颗粒衍生化。我们将使用这个标签方案， 哑铃状分子的相关部分对cryo-EM可见，因此这些方法可以应用于 这个关键结构。 具体目标2。我们已经生产出了各种分支的晶体，以及其他不寻常的DNA物种， 到重组。这些包括设计的菱形晶格，自组装形成宏观 晶体我们计划确定这些晶格的结构。我们还有一个替身的内部晶体 交叉分子（与减数分裂中间体有关），其对原子分辨率起交叉作用，第三种晶体， 包含着一种不寻常的联系我们将确定这些分子的结构并建立它们的3D模型 结构.此外，我们将尝试通过一个更大的自组装晶格来提高一些较大自组装晶格的分辨率。 一种捆绑技术，如果成功，将导致能够将客人插入到晶格中。

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.