Figure 2. Base flipping after the first nick.

Transpososomes were assembled and treated with KMnO₄. KMnO₄ oxidizes thymine bases in distorted DNA, particularly if they are in an extra-helical position [23]. Oxidation converts the thymine to cis-thymine glycol which, upon piperidine treatment, undergoes further degradation leading to cleavage of the DNA strand. After quenching the DNA was analyzed on a DNA sequencing gel. A The substrate was either uncleaved or pre-nicked. The nucleotide sequence of the transposon end is given on the left, with the arrowhead indicating the location of the transposon end. UC, uncleaved substrate; N, pre-nicked substrate, T'ase, transposase; IHF, integration host factor; *, This band is an artifact that appears to be caused by a heterogeneity at the 5′-end of the DNA strand. Since the label is located at the 3′-end of the DNA strand, this artifact does not contribute to the sequencing ladder or the permanganate footprints. B As in part A except that the transpososomes were assembled on the pre-nicked substrate with the wild type and mutant proteins.