X-ray co-crystal structures of Tn5 transposase (Tnp) bound to its 19 base pair (bp) recognition end sequence (ES) reveal contacts between a β-loop (amino acids 240-260) and positions 3, 4, 5 and 6 of the ES. Here we show that mutations of residues in this loop affect both in vivo and in vitro transposition. Most mutations are detrimental, while some mutations at position 242 cause hyperactivity. More specifically, mutations to the β-loop affect every individual step of transposition tested. Mutants performing in vivo and in vitro transposition less efficiently also form fewer synaptic complexes, while hyperactive Tnps form more synaptic complexes. Surprisingly, two hypoactive mutations, K244R and R253L, also affect the cleavage steps of transposition with a much more dramatic effect on the second double end break (DEB) complex formation step, indicating that the β-loop likely plays an important roll in positioning the substrate DNA within the catalytic site. Finally, all mutants tested decrease efficiency of the final transposition step, strand transfer. A disparity in in vitro cleavage rate constants for mutants with changes to the proline at position 242 on transposons flanked by ESes differing in the orientation of the A-T base pair at position 4 allows us to postulate that P242 contacts the position 4 nucleotide pair. Based on these data, we propose a sequential model for end cleavage in Tn5 transposition in which the uncleaved PEC is not symmetrical and conformational changes are necessary between the first and second cleavage events and also for the final strand transfer step of transposition.Transposition is the process of moving DNA from one location to another. Tn5 is a well-studied composite, prokaryotic transposon consisting of two insertion sequences, IS50R and IS50L flanked by 19 base pair (bp) inverted repeats termed outside ends (OEs) (1). IS50R encodes the transposase (Tnp), the only protein required for transposition in this system (2). Tn5 is mobilized using a cut-and-paste mechanism in which the transposon is completely removed from its original location before being inserted in a new DNA site (3).Following translation, Tnp initially interacts with DNA non-specifically, but then localizes the OE by both inter and intramolecular transfer, possibly via a looping mechanism ( Figure 1A, C. D. Adams, personal communication and (4)). The OE bound Tnps then form a dimeric synaptic complex, the nucleoprotein complex required for catalysis (5-7). Following synapsis, cleavage occurs at one OE/flanking donor backbone (dbb) DNA junction (+1) followed by the other. First, a water molecule activated by Mg 2+ attacks the phosphodiester backbone of one DNA strand at the +1 location resulting in the generation of a 3′ hydroxyl group ( Figure 1B, column 1). This 3′ hydroxyl group then attacks the opposite DNA strand creating a hairpin intermediate and releasing the dbb DNA (8). The synaptic complex containing one cleaved * To whom correspondence should be addressed: William S. Reznikoff, Tel: (608) F...

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