Figure 4.

Transposase binding to the Herves-right (R) end. (a) Herves transposase binds to Herves-R bp 1-30, bp 5-45 and bp 61-90. Electrophoretic mobility shift assay (EMSA) analysis of transposase binding to the overlapping 30 bp fragments (bp 1-30, bp 31-60, bp 61-90, bp 91-110, bp 15-45 and bp 46-75). The asterisk (*) indicates various protein DNA complexes. (b) Herves transposase binding to Herves-R bp 15-45 and bp 61-90. The Herves-R bp 61-90 fragment was used as a probe in EMSAs. The fraction of the transposase-bound probe was quantified using a phosphoimager. A homologous fragment was used as specific competitor at a molar excess of 50-fold, 100-fold and 200-fold, whereas non-specific competition was used as described for Figure 2b. Overlapping fragments (bp 1-30, bp 31-60, bp 91-110, bp 15-45, and bp 46-75) were used as competitors of transposase binding to the probe, at 200-fold molar excess. (c) DNase I protection assay of the Herves-R end. The single-end-labeled Herves-R 1-100 bp fragment (100 nM) was incubated in presence (+, ++) or absence (-) of DNase I or the transposase. The ++ indicates 1.4 μM of transposase or 0.5 units of DNase I, whereas + indicates 850 nM of transposase or 0.25 units of DNase I. ³²P indicates the end of the probe that was labeled. The solid bars on the sides indicate the region of the probe protected by the transposase. The asterisk (*) indicates hypersensitive sites.