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Keeping You in the Loop: The mechanics of DNA shape and flexibility in site-specific recombination.Mathematical Biology
Many methods have been used to directly observe DNA looping or knotting in vitro, such as scanning-probe and electron microscopy. However, the number of techniques available for quantifying DNA looping in solution is limited. Characterization of the rates at which proteins bind to, and bend, DNA provides insights into the reaction intermediates; hence it is a natural way to probe the mechanism of these interactions. Our approach characterizes the rate-determining steps for inter- and intramolecular synapsis in a Cre site-specific recombination reaction to answer the following questions: does Cre induce a bend in its DNA substrate, and can the Cre-loxP system report on DNA flexibility? This methodology, which can be extended to future in-vivo studies, uses time-dependent FRET in conjunction with numerical modeling of the recombination pathway to monitor target-site synapsis and the Cre-recombination products. In this talk, I will show that the quotient of apparent equilibrium constants for the intramolecular and equivalent intermolecular Cre reaction yields several parameters that determine the free energy of the looped intermediate including DNA helical repeat, DNA bending and torsional flexibilities, and most significantly, the geometry of the nucleoprotein complex.
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