Fig. 3

EMSA analysis of the entire res site and its predicted subsites (boxI, II and III). (a) Detection of DNA-protein complexes formed upon His-tagged SGI1 resolvase (Res) binding to the full res site. All binding reactions contained 5.6 ng 5’FAM-labelled 122-bp probe representing the predicted res site in SGI1ΔIn. Lanes 1–9 – increasing amount of purified Res: 0, 0.075, 0.225, 0.45, 0.9, 1.35, 1.8, 2.25, 2.7 µg, Lanes 10–11 – 1.2 and 1.5 µg Res and 1.7 µg (~ 300-fold) unlabelled probe that was added to the binding reaction 10 min prior to the labelled probe, Lane 12 – 2.7 µg protein preparate obtained from BL21 (DE3)/pET16b strain with no Res protein (negative control). Open arrowhead points to unbound probe, numbered filled arrowheads indicate shifted complexes. (b) Res binding to boxI. (c) Res binding to boxII. (d) Res binding to boxIII. (e) Res binding to the half of boxI. (f) Res binding to the boxI-boxII region of res site. (g) Res binding to the boxII-boxIII region of res site. In panels b-g, all reactions contained 8.0 ng of 5’FAM-labelled probes (see panel h) and increasing amounts of Res protein as follows: Lanes 1–7 – 0, 0.7, 1.4, 2.1, 2.8, 3.5 and 4.9 µg. Open arrowheads point to the unbound probe, filled arrowheads indicate the shifted DNA-protein complexes. (h) Sequence of the restored res site. The predicted boxes are highlighted in red, coordinates refer to base positions in SGI1ΔIn/wt SGI1starting with the first nucleotide of wt SGI1 DRL. Start codon of the res gene and the 5 bp that have been duplicated upon In104 insertion in wt SGI1 are indicated. Horizontal lines (a–g) represent the sequence included in the 5’FAM-labelled probes used in EMSAs on panels a-g (see also Suppl. Table S4). Original gels are presented in (Supplementary Fig. S7).