Hairpin formation in Tn5 transposition.

The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3' hydrolytic nick by transposase. The free 3'OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3' and 5' strands without massive protein/DNA rearrangements.