Figure 4.

FAM image of urea-PAGE showing G1-IC and G2-IC integrating prespacers into HP targets to observe full site integration. Both protein complexes integrate full site (FS) only into their cognate target. Removing the 3′-OH abolishes FS integration in both cases as well, showing that the full-site band is the product of a single prespacer being inserted at both ends and not from two independent integration events. Removing the 3′-OH also abolishes spacer-side (SS) integration in the case of G1-IC, but not in G2-IC, which shows a difference in mechanism between the two. Removing all GAGs outside the leader–repeat junction abolishes off-target bands generated by G2-IC (G2-HP-No GAG). The slight variations in the band positions in −OH F and −OH R lanes is most probably due to the remaining secondary structures affecting mobility on a gel (hydroxyl group removed from spacer_Sy_overhangs_5-F (Table S1, −OH F); hydroxyl group removed from spacer_Sy_overhangs_5-R (Table S1, −OH R); and hydroxyl group removed from both these strands (−OH F + R)).