Abstract
The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposon ends. This is achieved using a hairpin intermediate that requires the DNA to change conformation during the reaction. In Tn5 these changes are controlled in part by a flipped nucleoside that is stacked on a tryptophan residue in a hydrophobic pocket of the transposase. Here we have investigated the base flipping mechanism in Tn10 transposition. As in Tn5 transposition, we find that base flipping takes place after the first nick and is required for efficient hairpin formation and resolution. Experiments with an abasic substrate show that the role of base flipping in hairpin formation is to remove the base from the DNA helix. Specific interactions between the flipped base and the stacking tryptophan residue are required for hairpin resolution later in the reaction. We show that base flipping in Tn10 transposition is not a passive reaction in which a spontaneously flipped base is captured and retained by the protein. Rather, it is driven in part by a methionine probe residue that helps to force the flipped base from the base stack. Overall, it appears that base flipping in Tn10 transposition is similar to that in Tn5 transposition.
Highlights
IntroductionNot as numerous as retrotransposons in higher eukaryotes, cut-and-paste DNA-transposons are a successful group of elements, well represented in all branches of life
Mobile DNA sequences have had a profound influence on evolution
In these enzymes the catalytic core is disrupted by the insertion of an extra sub-domain that in Tn5 transposase interacts with a flipped base at the transposon end [1,2,3,4]
Summary
Not as numerous as retrotransposons in higher eukaryotes, cut-and-paste DNA-transposons are a successful group of elements, well represented in all branches of life They generally encode a single transposase protein with a characteristic DDE triad of amino acid residues in the active site. Most cut and paste transposases, with the exception of the mariner family, use a hairpin intermediate to cleave the second strand of DNA at the transposon end (Figure 1A) In these enzymes the catalytic core is disrupted by the insertion of an extra sub-domain that in Tn5 transposase interacts with a flipped base at the transposon end [1,2,3,4]
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