Towards our goal of understanding DNA transposition by the IS608 transposase, TnpA, we have recently reconstituted the entire recombination reaction in vitro using single-stranded DNA, and have solved the structures of six different complexes of TnpA with various oligonucleotides representing sequences located at the transposon ends. These allow us detailed views of sequential steps along the recombination pathway including (i) the pairing of transposon ends caused by TnpA binding to DNA hairpin structures located close to the transposon ends; (ii) the large-scale conformational change induced by hairpin binding that causes the transition from an inactive enzyme to one with a correctly assembled active site; (iii) the orientation of the transposon ends in the active site; and (iv) the mode of target site recognition. This last observation is particularly important as it immediately suggests ways to modify the system so that alternate targets might be used for transposition. In particular, we hope to devise methods to allow targets longer than a tetranucleotide to be used. If we can do this, this might allow the precise introduction of exogenous genes into benign locations in chromosomes or places where gene expression can be appropriately controlled in a cell- and development-specific manner.? ? ? Curcio, M.J. and Derbyshire, K.M. (2003) Nat. Rev. Mol. Cell. Biol. 4, 865-877. ? Debets-Ossenkopp, Y.J., Pot, R.G.J., van Westerloo, D.J., Goodwin, A., Vandenbroucke-Grauls, C.M.J.E., Berg, D.E., Hoffman, P.S., and Kusters, J.G. (1999) Antimicrob. Agents Chemother. 43, 2657-2662.? Kersulyte, D., Velapatino, B., Dailide, G., Mukhopadhyay, A.K., Ito, Y., Cahuayme, L., Parkinson, A.J., Gilman, R.H., and Berg, D.E. (2002) J. Bacteriol. 184, 992-1002. ? Sebaihia, M. et al. (2006) Nature Genet. 38, 779-786.
