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An important step in Tn5 transposition requires transposase-transposase homodimerization to form a synaptic complex competent for cleavage of transposon DNA free from the flanking sequence. We demonstrate that the C-terminal helix of Tn5 transposase (residues 458 -468 of 476 total amino acids) is required for synaptic complex formation during Tn5 transposition. Specifically, deletion of eight amino acids or more from the C terminus greatly reduces or abolishes synaptic complex formation in vitro. Due to this impaired synaptic complex formation, transposases lacking eight amino acids are also defective in the cleavage step of transposition. Interactions within the synaptic complex dimer interface were investigated by site-directed mutagenesis, and residues required for synaptic complex formation include amino acids comprising the dimer interface in the Tn5 inhibitor x-ray crystal structure dimer. Because the crystal structure dimer was hypothesized to be the inhibitory complex and not a synaptic complex, this result was surprising. Based on these data, models for both in vivo and in vitro synaptic complex formation are presented.Tn5 is a composite, prokaryotic transposon consisting of two IS50 elements (termed IS50R and IS50L) flanking a region of DNA encoding three antibiotic resistance genes. Tn5 is bracketed by 19-bp 1 inverted repeat transposase recognition sequences termed outside ends (OEs). Two OEs (or similar end sequences) are absolutely required for movement of the Tn5 transposon (1). IS50R encodes two proteins, a 476-amino acid (aa) cis-acting transposase (Tnp) protein, which is essential for transposition, and a 421-aa inhibitor protein (Inh), which inhibits transposition both in cis and in trans (2) by forming nonproductive multimers with Tnp (3). Because translation of Inh begins from a downstream, in-frame, independent start codon, Inh is identical to Tnp except that 55 aa on the N terminus are missing. IS50L encodes proteins P3 and P4 corresponding to Tnp and Inh of IS50R. These proteins are nonfunctional because of an ochre codon at residue Glu-451 (4, see Ref. 5 for review).Tn5 transposes by a cut and paste mechanism (Fig. 1) (6). First, a monomer of Tn5 Tnp binds each OE sequence (7). Interaction of these bound Tnp monomers through Tnp-Tnp dimerization forms a complex nucleoprotein structure termed a synapse (3,8). Nicking of one DNA strand then occurs via nucleophilic attack of a water molecule (activated by a Tnpcoordinated Mg 2ϩ ) on the phosphodiester backbone between the ϩ1 position of the OE and the Ϫ1 position of the flanking DNA (donor backbone). This released 3Ј-OH then attacks the opposite DNA strand creating a hairpin intermediate (8) and releasing the blunt-ended transposon from the donor backbone (dbb) DNA (6). A second activated water molecule then resolves the hairpin intermediate. Strand transfer then occurs via a transesterification reaction in which the 3Ј-OH groups of the transposon attack the phosphodiester backbone of the captured target DNA in a staggered fashion. Formatio...

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