Physical Chemistry of Recombinational Intermediates

这个项目需要对DNA重组过程中涉及的DNA分子进行表征。 这些通常是支链分子，其3D结构只有部分已知。一个关键问题是 双链DNA分子对同源性的识别。我们已经确定有一种不同寻常的DNA 同源分子在超卷绕质粒中时由同源分子形成的结构。 具体目的1.形成的结构是哑铃状，其中轴包含同源DNA。 我们计划确定轴的结构的同源结构。以查看(如假设的) 结构确实是px-DNA，我们将继续进行基于化学的实验，包括交联剂 缠绕在一起，锁住了圆圈。我们希望有最好的物证证明我们能继续下去 竖井。下一阶段的特征将需要对哑铃进行电子显微镜检查 由可见的金属纳米颗粒衍生而成。我们将使用这个标签方案来尝试使 冷冻机可以看到哑铃分子的相关部分，这样这些方法就可以应用于 这个关键结构。 具体目标2.我们已经生产出各种分枝的和其他不寻常的DNA物种的晶体 为了重组。这些包括设计的菱面体晶格，这些晶格自组装形成宏观 水晶。我们计划确定这些晶格的结构。我们也有双人床内部的水晶 衍射到原子分辨率的交叉分子(与减数分裂中间产物有关)和第三个晶体 包含一种不同寻常的联系。我们将确定这些分子的结构，并建立它们的3D 结构。此外，我们将尝试通过提高一些较大的自组装晶格的分辨率 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.