Abstract:During cut-and-paste mariner/Tc1 transposition, transposon DNA is cut precisely at its junction with flanking DNA, ensuring the transposon is neither shortened nor lengthened with each transposition event. Each transposon end is flanked by a TpA dinucleotide: the signature target site duplication of mariner/Tc1 transposition. To establish the role of this sequence in accurate DNA cleavage, we have determined the crystal structure of a pre-second strand cleavage mariner Mos1 transpososome. The structure reveals… Show more
“…This is the same as in Tn 5 and the retroviral integrases (50,51). If we now work backward in the reaction pathway: the structure of Mos1 with an uncleaved transferred strand suggests that the B metal ion activates the nucleophilic water for the cleavage step (31). Once again, this is the same as in Tn 10/5 and suggests that mariner conforms to the ping-pong mechanism proposed by Yang (48).…”
The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease.
“…This is the same as in Tn 5 and the retroviral integrases (50,51). If we now work backward in the reaction pathway: the structure of Mos1 with an uncleaved transferred strand suggests that the B metal ion activates the nucleophilic water for the cleavage step (31). Once again, this is the same as in Tn 10/5 and suggests that mariner conforms to the ping-pong mechanism proposed by Yang (48).…”
The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease.
“…The largest displacement (5.7 Å) is at P210 in the turn between β7 and α8 and around helices α8 and α10, which cradle the target DNA (Figure 9b). T 0 in the Mos1 STC is in a different orientation to the thymine (T 57 ) of the flanking target site duplication in the pre -TS cleavage PEC (Figure 9c), which is recognised by base-specific interactions with the WVPHEL motif (Dornan et al, 2015). By contrast, T 0 closely aligns with T 54 of the additional DNA duplex in the post -TS cleavage PEC (Figure 9d), which may represent the target strand before integration.10.7554/eLife.15537.018Figure 9.Structural comparison of the Mos1 STC with the pre- and post-TS cleavage Mos1 paired-end complexes.( a ) Orthogonal views of the Mos1 STC (orange) superimposed on the pre -TS cleavage PEC (PDB ID: 4U7B, green): r.m.s.d.…”
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
“…The Mos1 IRL PEC structure has a similar overall architecture to the Mos1 IRR PEC described previously ( 18 ) and comprises a transposase dimer bound to two IRL DNA molecules in a crossed configuration; one IRL DNA is bound by the DNA-binding domain of one monomer and the catalytic domain of the other monomer, and vice versa. As before, two additional IRL DNA duplexes interact with the catalytic domains, possibly occupying the binding sites of target DNA ( 30 ).…”
Background: Transposases orchestrate movement of DNA transposons around and between genomes.Results: Structural and biochemical approaches are combined to dissect the DNA preferences of two mariner transposases in each step of transposition.Conclusion: Two active mariner transposases preferentially associate with and process one of their transposon ends.Significance: The efficiency of mariner DNA transposition can be improved by optimizing transposon end sequences.
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