Single Molecule Studies of Recombinational DNA Structure and Dynamics

重组 DNA 结构和动力学的单分子研究

• 批准号: 0615590
• 负责人: Yuri Lyubchenko
• 金额: --
• 依托单位: University of Nebraska Medical Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0615590&HistoricalAwards=false
• 关键词: Single; Molecule; Studies; Recombinational; DNA

The Holliday junction is an intermediate in homologous and site-specific DNA recombination, DNA replication, and DNA repair. Despite great efforts made in structural studies of Holliday junctions, the understanding of the branch migration process, as a central mechanism of DNA recombination, is rather poor. This work addresses this problem by focusing on structural studies of mobile Holliday junctions under conditions allowing branch migration. Single molecule techniques using Atomic Force Microscopy (AFM) and Fluorescence Resonance Energy Transfer (FRET) will be applied, and three specific aims will be addressed to test the models for branch migration and to shed new light on the molecular mechanisms of this crucial step in DNA recombination. 1) Structural dynamics of Holliday junctions will be characterized in real time to follow branch migration at the single base pair level; 2) Large-scale dynamics of mobile Holliday junctions and spontaneous branch migration will be explored; 3) The effect of DNA supercoiling will be systematically analyzed. This research is accompanied by a educational component that is designed to foster the development of single-molecule biophysics within the context of training at the University of Nebraska Medical Center and Creighton University. Particular emphasis of the training is an interdisciplinary course in bio-imaging and practical research experience in the university's nano-imagining facility.Funding for this award is provided through the Division of Physics in the Directorate for Mathematical and Physical Sciences and the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

霍利迪连接是同源和位点特异性DNA重组、DNA复制和DNA修复的中间体。尽管人们在霍利迪连接的结构研究方面做了大量的工作，但作为DNA重组的中心机制，对分支迁移过程的理解却相当贫乏。这项工作解决了这个问题，通过专注于结构研究的条件下，允许分支迁移的移动的霍利迪路口。使用原子力显微镜（AFM）和荧光共振能量转移（FRET）的单分子技术将被应用，并将解决三个具体的目标，以测试模型的分支迁移，并揭示新的光在DNA重组的这一关键步骤的分子机制。 1)霍利迪连接的结构动力学将在真实的时间表征，以在单碱基对水平上跟踪分支迁移; 2）移动的霍利迪连接和自发分支迁移的大规模动力学将被探索; 3）DNA超螺旋的影响将被系统地分析。 这项研究伴随着一个教育部分，旨在促进内布拉斯加大学医学中心和克雷顿大学培训背景下的单分子生物物理学的发展。 培训的重点是在大学的纳米成像设施中进行生物成像和实际研究经验的跨学科课程，该奖项的资金由数学和物理科学局物理学处和生物科学局分子和细胞生物科学处提供。