Purification and biochemical analyses of a monomeric form of Tn5 transposase.

Abstract

The binding of transposase (Tnp) to the specific Tn5 end sequences is the first dedicated reaction during transposition. In this study, comparative DNA-binding analyses were performed using purified full-length Tnp and a C-terminal deletion variant (delta369) that lacks the putative dimerization domain. The shape of the binding curve of full-length Tnp is sigmoidal in contrast to the hyperbolic-shaped binding curve of delta369. This observation is consistent with previous observations as well as a rate of binding study presented here, which suggest that the full-length Tnp-end interaction, unlike that of the truncated protein, is a complex time-dependent reaction possibly involving a subunit exchange. Circular permutation assay results indicate that both proteins are capable of distorting the Tn5end sequences upon binding. Molecular weight determinations based on the migratory patterns of complexed DNA in polyacrylamide gels has shown that delta369 specifically binds the Tn5 end sequences as a monomer while full-length Tnp in complex represents a heterodimer.