A bend, flip and trap mechanism for transposon integration

Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities. DOI: http://dx.doi.org/10.7554/eLife.15537.001
eLife digest The complete set of DNA in a cell is referred to as its genome. Most genomes contain short fragments of DNA called transposons that can jump from one place to another. Transposons carry sections of DNA with them when they move, which creates diversity and can influence the evolution of a species. Transposons are also being exploited to develop tools for biotechnology and medical applications. One family of transposons – the Mariner/Tc1 family – has proved particularly useful in these endeavours because it is widespread in nature and can jump around the genomes of a broad range of species, including mammals. DNA transposons are cut out of their position and then pasted at a new site by an enzyme called transposase, which is encoded by some of the DNA within the transposon. DNA is made up of strings of molecules called bases and Mariner/Tc1-family transposons can only insert into a new position in the genome at sites that have a specific sequence of two bases. However, it was not known how this target sequence is chosen and how the transposon inserts into it. Morris et al. have now used a technique called X-ray crystallography to build a three-dimensional model of a Mariner/Tc1-family transposon as it inserts into a new position. The model shows that, as the transposon is pasted into its new site, the surrounding DNA bends. This causes two DNA bases in the surrounding DNA to flip out from their normal position in the DNA molecule, which enables them to be recognised by the transposase. Further experiments showed that this base-flipping is dynamic, that is, the two bases continuously flip in and out of position. Furthermore, Morris et al. identified which parts of the transposase enzyme are required for the transposon to be efficiently pasted into the genome. Together these findings may help researchers to alter the transposase so that it can insert the transposon into different locations in a genome. This will hopefully lead to new tools for biotechnology and medical applications. DOI: http://dx.doi.org/10.7554/eLife.15537.002