Crucial role for DNA supercoiling in Mu transposition: a kinetic study

Abstract

DNA supercoiling plays an indispensable role in an early step of bacteriophage Mu transposition. This step involves formation of a nucleoprotein complex in which the Mu ends synapse and undergo two concerted single-strand cleavages. We describe a kinetic analysis of the role of supercoiling in the Mu-end synapsis reaction as measured by the cleavage assay. We observe a dependence of the reaction rate on superhelical density as well as on the length of Mu donor plasmid DNA. The reaction has a high activation enthalpy (approximately 67 kcal/mol). These results imply that the free energy of supercoiling is used directly to lower the activation barrier of the rate-limiting step of the reaction. Only the free energy of supercoiling associated with DNA outside the Mu ends appears to be utilized, implying that the Mu ends come together before the supercoiling energy is used. Our results suggest an essential function for the bacterial sequences attached to the ends of Mu virion DNA.