Physical Chemistry of Recombinational Intermediates

重组中间体的物理化学

• 批准号: 7177131
• 负责人: NADRIAN C. SEEMAN
• 金额: $ 3.48万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7177131
• 关键词: DNA binding protein; DNA gyrase; DNA repair; X ray crystallography; atomic force microscopy; bacterial DNA; bacterial genetics; biophysics; chemical models; crosslink; fluorescence resonance energy transfer; genetic models; genetic recombination; intermolecular interaction; nucleic acid structure; plasmids; structural biology

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.

描述（由申请人提供）：该研究计划涉及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，我们已经建立了不寻常的DNA基序，已被用来表征霍利迪结的物理化学。霍利迪连接的一种概括是称为PX DNA的结构。这是两个双螺旋的相互缠绕结构。对这种结构的预测是，超螺旋质粒中同源性的存在应导致超螺旋的松弛。在目前的项目期间，我们已经验证了这一预测，无论是通过二维凝胶电泳和原子力显微镜。我们还表明，PX同源性，一个较低程度的同源性与PX结构一致，产生同样的效果。在这个应用程序中，我们提出的实验进一步表征这种效果，并确定它是否确实是由于PX结构。涉及这一现象的具体目标是：（i）建立影响弛豫的参数;（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）获得确定同源结构性质的独立结构数据;（iii.1）在细菌细胞内进行交联实验，以确定这种效应是否在体内发生;（iii.2）寻找与结构相互作用的蛋白质;并确定PX同源性是否可以在重组中起常规同源性的作用。另一个具体目标涉及使用在当前项目期间开发的DNA设备，该设备测量DNA扭曲蛋白质与DNA结合时可以工作的负荷。我们将用MutS DNA修复蛋白识别的各种错配位点进行这些实验。在第三个目标中，我们将开发一系列不同的潜在底物的DNA平行四边形为基础的结构，这将被测试为人类和酵母拓扑异构酶II的可能的基板。