Characterization of a Tn5 pre-cleavage synaptic complex

Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures. In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis. We cha racterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described to date. We identified this complex by gel retardation a ssay using short, linear fragments and have shown that it contains a dimer of transposase, two DNA molecules, and is competent for DNA cleavage in the presence of Mg2+. We also used hydroxyl radical footprinting and interfere nce techniques to delineate the protein-DNA contacts made in the Tn5 synapt ic and monomer complexes. AU positions (except position 1) of the end seque nce are contacted by transposase in the synaptic complex. We have determine d that positions 2-5 of the end sequence are specifically required for syna ptic complex formation as they are not required for monomer complex formati on. In addition, in the synaptic complex, there is a strong, local distorti on centered around position 1 which Likely facilitates cleavage. (C) 2000 A cademic Press.