Characterization of DNA/Protein Interactions at the Single Molecule Level

Arizona State University, Scottsdale AZ

Investigators

Abstract

The goal of this work is to explore, at the single molecule level, three different types of DNA/protein interactions: 1) Binding interactions between proteins and DNA with a particular emphasis on the role of one-dimensional diffusion, 2) Generation of loops in DNA by protein complexes that bind at two or more nonadjacent sites, 3) Protein interactions which disrupt base stacking by pulling a base out of the stacking configuration with neighboring bases. Each of these processes will be explored at both the level of population distributions (relative populations of different geometries or conformations of the DNA/protein complex at equilibrium) and in terms of dynamics (times required for interconversion between conformations or geometries, again primarily at equilibrium). Single molecule fluorescence spectroscopy is well suited for such measurements. The primary emphasis will be placed on three DNA/Protein systems. These are the interaction of intercalating dyes with DNA as a model system for protein/DNA interaction and the study of various DNA binding domains with DNA with an emphasis on searching for binding sites and protein/DNA interactions which result in DNA looping. Some work will be performed on a final and ambitious project, monitoring the action of DNA polymerase at the single molecule level. This work will be done in collaboration with Dr. Reha-Krantz at the University of Alberta, who will provide some of the DNA templates and constructs required for the experiments. The project will be closely connected to an existing IGERT program at the university, and undergraduate students will be heavily involved in the research. This project is jointly funded by the Physics and Chemistry Divisions in the Directorate for Mathematical and Physical Sciences and the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.