Figure 5.

Effect of hairpin loop length and sequence on stable complex formation and catalysis. (A) Complex formation. Complexes with hairpin (3-fold scaled up reactions) and precleaved (1-fold reactions) substrates were assembled for 1 h in the absence of metal ions, and the reaction products were analyzed by native agarose gel electrophoresis and autoradiography. Primarily, the assay detects transpososomes but may also detect assembly and/or disassembly intermediates. The slower migrating complexes (indicated by C1) include transpososomes. (B) Analysis of strand transfer products. Target DNA (pUC19) and MgCl₂ were added to the assembly reactions to allow catalysis (Materials and Methods), and the reactions were incubated for 6 h prior to analysis of strand transfer products (as in Figure 4). (C) Tabulation of data from (A) and (B). Only a minor portion of the substrates were converted to reaction products (in most cases <1%). The levels of the detected products are based on visual inspection and indicated by a scale of four categories: (−), no products; (+), low level; (++), medium level; (+++), high level. Note that the HP4 substrate did not produce appreciable amounts of DEPs in this experiment, whereas in a similar experiment seen in Figure 2B it did. This apparent discrepancy is due to lower level of radioactivity in the substrate.