Physical Chemistry of Recombinational Intermediates

描述（由申请人提供）：本研究项目涉及DNA的异常结构及其在生物过程中的作用，如重组或修复。多年来，我们构建了不同寻常的DNA基序，这些基序被用来表征霍利迪结的物理化学性质。霍利迪结的概括是一种被称为PX DNA的结构。这是一个由两个双螺旋相互缠绕的结构。对这种结构的一个预测是，在超卷曲质粒中同源性的存在会导致超卷曲的松弛。在目前的项目期间，我们已经通过二维凝胶电泳和原子力显微镜验证了这一预测。我们还表明，与PX结构一致的较低程度的同源性PX-homology也会产生相同的效果。在本应用中，我们提出实验来进一步表征这种效应，并确定它是否确实是由于PX结构。涉及这一现象的具体目的是：(i)建立影响松弛的参数；（ii.1）完成PX同源性数据；（ii.2）确定该结构是否需要两个同源双螺旋相互缠绕；（ii.3）获得独立的结构数据，确定同源结构的性质；（iii.1）在细菌细胞内进行交联实验，确定这种效应在体内是否发生；（iii.2）寻找与该结构相互作用的蛋白质；确定PX同源性在重组中是否可以作为规则同源性。另一个具体目标是使用在当前项目期间开发的DNA设备，测量DNA扭曲蛋白与DNA结合时所能承受的负荷。我们将使用MutS DNA修复蛋白识别的各种错配对位点进行这些实验。在第三个目标中，我们将从基于DNA平行四边形的结构中开发一系列不同的潜在底物，这些底物将作为人类和酵母拓扑异构酶II的可能底物进行测试。

DESCRIPTION (provided by applicant): This research program is concerned with unusual structures of DNA and their role in biological processes, such as recombination or repair. For many years, we have bulit unusual DNA motifs that have been used to characterize the physical chemistry of Holliday junctions. A generalization of the Holliday junction is a structure known as PX DNA. This is an inter-wrapped structure of two double helices. A prediction of this structure, is that the presence of homology in a supercoiled plasmid should lead to the relaxation of the supercoiling. During the current project period, we have verified this prediction, both by 2D gel electrophoresis and by atomic force microscopy. We have also shown that PX-homology, a lower extent of homology consistent with the PX structure, produces the same effect. In this application, we propose experiments to characterize this effect further, and to establish whether it is indeed due to a PX structure. The specific aims involving this phenomenon are: to (i) establish the parameters that affect the relaxation; (ii.1) to complete the data on the PX homology; (ii.2) to extablish whether the structure entails a wrapping of the two homologous double helices around each other; (ii.3) to obtain independent structural data that establish the nature of the homology strutcure; (iii.1) to perform crosslinking experiments within bacterial cells to determine whether this effect occurs in vivo; (iii.2) to seek proteins that interact with the structure; and to determine whether PX homology can function as regular homology in recombination. Another specific aim involves using a DNA device, developed during the current project period, that measures the load against which a DNA-distorting protein can work when it binds to DNA. We will perform these experiments with the various mis-paired sites recognized by the MutS DNA repair protein. In a third aim, we will develop a series of different potential substrates from DNA parallelogram-based structures, that will be tested as possible substrates for human and yeast topoisomerase II.