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，例如重组或修复。多年以来，我们一直有不寻常的DNA图案，这些基序被用来表征霍利迪连接的物理化学。 Holliday结的概括是一种称为PX DNA的结构。这是两个双螺旋的包裹间结构。这种结构的一个预测是，超编层质粒中同源性的存在应导致超涂层的放松。在当前的项目期间，我们通过2D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结构一致的同源性较低程度的PX-词素产生相同的效果。在此应用中，我们提出了实验以进一步表征这种效果，并确定它是否确实是由于PX结构所致。涉及这种现象的具体目的是：（i）建立影响放松的参数； （ii.1）完成有关PX同源性的数据； （ii.2）扩展该结构是否需要包裹两个同源双螺旋； （ii.3）获得建立同源性抗性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作用是否发生在体内； （iii.2）寻求与结构相互作用的蛋白质；并确定PX同源性是否可以充当重组中的常规同源性。另一个特定的目的涉及在当前项目期间开发的DNA设备，该设备测量了DNA延伸蛋白与DNA结合时可以起作用的负载。我们将使用MUTS DNA修复蛋白识别的各种差异位点进行这些实验。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，这些结构将作为人和酵母拓扑异构酶II的可能底物进行测试。