Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations.
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abstract
We have examined the effects of a T----C point mutation at the terminal nucleotide of the Mu ends in a mini-Mu plasmid on the early steps in the in vitro transposition reaction. These mutations inhibit the introduction of nicks at the Mu ends in a reaction with Mu A, HU, and integration host factor proteins. The presence of the point mutation at either the left end or the right end is sufficient to block the nicking reaction at both ends, indicating that the reaction is normally concerted. Addition of Mu B and ATP, however, dramatically stimulates the reaction of mutant mini-Mu plasmids carrying the mutation at one end but not at both ends. The data suggest that the Mu B protein mediates its effect through direct interaction with Mu A and that Mu B may play a role in an earlier step in the transposition process than previously proposed. In the presence of Mu B, two products are observed with the left end or right end mutant mini-Mu plasmids, a normal protein-DNA intermediate (Type 1 complex) which contains nicks at both Mu ends and an abortive product composed of free relaxed plasmid which is nicked only at the wild-type end. Furthermore, stable protein-DNA complexes characteristic of the first step in the in vitro transposition reaction are not observed in the absence of nicking or when only one end is a nicked; the introduction of nicks at both Mu ends is a prerequisite for stable transpososome assembly.
