Fluorescence-detected DNA Looping: Experimental Measurements and Computational Modeling

Stephen D. Levene

University of Texas at Dallas



IPHT, Raum 225

Abstract: I will present a quantitative approach for characterizing the probability of DNA-loop formation in solution based on time-dependent ensemble Förster resonance energy transfer (FRET) measurements of intra- and intermolecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications, including conjugated fluorophores, at specific sites in covalently closed DNA. Moreover, Cre recombination does not require protein cofactors or highly restrictive solution conditions and is thus a flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo. Although this experimental approach is robust, there remains the considerable challenge of determining the structure of a nucleoprotein complex from loop-closure-kinetics data. Numerical and simulationbased
approaches to the statistical mechanics of DNA-loop formation and its associated FRET signature will be discussed.