Skip to main content
. 2009 Jul 10;4(7):e6201. doi: 10.1371/journal.pone.0006201

Figure 3. Crosslinking between transposase and the flipped base.

Figure 3

The thymidine residue T+2 on the top strand of the DNA substrate was substituted with the zero-length crosslinking reagent iodouracil which reacts primarily with aromatic amino acid side chains. Transpososomes were assembled on 5′-end labeled uncleaved (UC) and/or pre-nicked (N) substrates. An aliquot was removed to monitor transpososome assembly by EMSA. The remainder was exposed to UV light and analyzed by SDS-PAGE. Autoradiograms are shown. A Transpososome assembly with wild type transposase was monitored by EMSA. As expected, assembly on linear DNA fragments requires the presence of the host protein IHF [29]. B UV crosslinking of the reaction mixtures from part A and analysis by SDS-PAGE. Crosslinking was detected only in the presence of transposase and was more prominent after the first nick. C and D The same as parts A and B respectively, except that the transpososomes were assembled on the pre-nicked substrate using wild type and mutant transposase.