A bend, flip and trap mechanism for transposon integration

Abstract

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

A single active site in the mariner transposase cleaves DNA strands of opposite polarity

“…The minor shift in electrophoretic mobility between the pre- and post-cleavage PECs suggests that their protein content remains unchanged. Since the dimeric stroichiometry of the post-cleavage PEC already received strong support (30–32), this experiment is consistent with a dimeric pre-cleavage PEC. Indeed, the dimeric stoichiometry of the PEC is also consistent with the monomeric stoichiometry of SEC1 into which it dissociates (above and (28)).…”

“…A dimeric model for the mariner transpososome was supported by several crystal structures (30–32). However, until now, aspects of the model have remained hypothetical.…”

“…The crystal structure of the Mos1 strand transfer complex suggests that the A metal ion activates the 3′-hydroxyl at the end of the transposon for the integration step (Figure 6B, right element) (32). This is the same as in Tn 5 and the retroviral integrases (50,51).…”

“…The resulting sequence provides a 187 amino-acid linker between the C-terminus of the first subunit and N-terminus of the second subunit, which includes, in addition to the 18 amino-acid sequence above, a TrxA tag, six histidines, a thrombin recognition site, a S-tag and an enterokinase recognition site. This provides ample sequence length to span the ∼60 Å between the opposite ends of the two subunits predicted from the Mos1 PEC structure (30–32), without introducing significant strain. Other expression vectors include pRC1113 for the MBP Hsmar1-D155A mutant; pRC1144 and pRC1145 for the single-chain dimers with a D155A mutation in the first and second subunits, respectively; pRC1146 and pRC1147 for the single-chain dimers with a R104A mutation in first and second subunits, respectively.…”

“…More recently, a series of crystal structures of Mos1 complexes assembled with pre-cleaved or partially cleaved substrates have revealed a dimeric PEC (30–32). However, structural studies do not capture dynamics and cannot rule out models in which subunits change their roles or positions during the reaction (33).…”