Abstract
Cut-and-paste transposons are important tools for mutagenesis, gene-delivery and DNA sequencing applications. At the molecular level, the most thoroughly understood are Tn5 and Tn10 in bacteria, and mariner and hAT elements in eukaryotes. All bacterial cut-and-paste transposases characterized to date are monomeric prior to interacting with the transposon end, while all eukaryotic transposases are multimers. Although there is a limited sample size, we proposed that this defines two pathways for transpososome assembly which distinguishes the mechanism of the bacterial and eukaryotic transposons. We predicted that the respective pathways would dictate how the rate of transposition is related to transposase concentration and genome size. Here, we have tested these predictions by creating a single-chain dimer version of the bacterial Tn5 transposase. We show that artificial dimerization switches the transpososome assembly pathway from the bacterial-style to the eukaryotic-style. Although this had no effect in vitro, where the transposase does not have to search far to locate the transposon ends, it increased the rate of transposition in bacterial and HeLa cell assays. However, in contrast to the mariner elements, the Tn5 single-chain dimer remained unaffected by over-production inhibition, which is an emergent property of the transposase subunit structure in the mariner elements.