Single Molecule Studies of Recombinational DNA Structure and Dynamics

点击翻译按钮获取中文摘要

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.