| The interior of a cell is a crowded and fluctuating environment where DNA and other biomolecules are both highly constrained and subject to many mechanical forces. The extensive compaction of DNA in living cells is a challenge to many critical biological functions. An evolutionary solution to this challenge may be the juxtaposition of cis-acting elements such that multimeric protein complexes simultaneously interact with two or more protein-binding sites. This mode of biological activity involves the formation of looped DNA structures, which, by themselves, are thermodynamically unfavorable. Our knowledge about the roles of DNA bending, twist, and their respective energetics in DNA looping has come mainly from analyses of ligase-dependent DNA-cyclization experiments, which are quantitatively described by the Jacobson-Stockmayer, or J factor. This dissertation focuses on the development of a quantitative approach for measuring the probability of DNA-loop formation in solution using ensemble Förster resonance energy transfer (FRET) measurements of intra- and inter-molecular Cre-recombination kinetics. In my dissertation, I describe a novel analysis method to characterize synaptic-complex formation based on the kinetics of the Cre recombination reaction, which yields a quantitative measure of the probability of DNA-loop formation, J. This approach uses time-dependent FRET to obtain the rates of both inter- and intramolecular recombination-site synapsis. In addition to providing information about the probability of Cre-mediated loop formation in vitro, our method is potentially applicable to studies of DNA-loop formation in living cells. |
