Fig. 2. Complex formation with disintegration substrates and disintegration activity. (A) The indicated substrates were mixed with either IHF, transposase or IHF + transposase as described in Materials and methods, and analyzed on a native 5% polyacrylamide gel. Positions of S-92 and S-53 higher order complexes are indicated along with the bPEC, IHF complex and unreacted substrate (URS). The set of bands within the bracket (lane 4) has not been characterized and could be indicative of an extremely low level of transpososome formed in the absence of IHF. (B) PEG precipitated S-53 complex or S(T-D)-92 PEC was incubated in reaction buffer either without a divalent metal ion or with MgCl₂ (5 mM) or MnCl₂ (0.18 mM) as indicated. Reactions were terminated by phenol extraction and the purified DNA analyzed on an 8% denaturing gel. S-53 DNA was labeled at the 5′ terminus of the transferred strand with ³²P and thus it was only possible to detect SD and cleaved end products. S(T-D)-92 DNA was 5′ end labeled with ³²P on both transferred and non-transferred strands and transposase-directed cleavage generates a flanking donor fragment of 31 nt and a transposon fragment of 59 nt. FD is flanking donor DNA.