Transposition of bacteriophage Mu uses two DNA cleavage sites and six transposase recognition sites, with each recognition site divided into two half-sites. The recognition sites can activate transposition of non-Mu DNA sequences if a complete set of Mu sequences is not available. We have analyzed 18 sequences from a non-Mu DNA molecule, selected in a functional assay for the ability to be transposed by MuA transposase. These sequences are remarkably diverse. Nonetheless, when viewed as a group they resemble a Mu DNA end, with a cleavage site and a single recognition site. Analysis of these "pseudo-Mu ends" indicates that most positions in the cleavage and recognition sites contribute sequence-specific information that helps drive transposition, though only the strongest contributors are apparent from mutagenesis data. The sequence analysis also suggests variability in the alignment of recognition half-sites. Transposition assays of specifically designed DNA substrates support the conclusion that the transposition machinery is flexible enough to permit variability in half-site spacing and also perhaps variability in the placement of the recognition site with respect to the cleavage site. This variability causes only local perturbations in the protein-DNA complex, as indicated by experiments in which altered and unaltered DNA substrates are paired.Some of the most fundamental of biological processes occur within nucleoprotein complexes. These processes include recombination, replication, transcription, and RNA splicing. The nucleoprotein complexes are often large and elaborate, containing features that permit sophisticated regulation. As a result, dissecting the chemistry of interactions within a complex is a challenging task, but one that is critical to understanding biological function.Transposition of bacteriophage Mu, like that of other transposons, occurs within complexes called transpososomes. Transpososomes mediate at least two sequential chemical reactions, on a pathway toward transferring the transposon DNA from one site to another. The two reactions are: (i) DNA cleavage, in which a nick is introduced precisely at the end of the transposon, on the 3Ј strand and (ii) DNA strand transfer, in which the nicked strand is joined to a separate DNA molecule called the target (1, 2). The reaction sites on the transposon DNA, or "donor DNA," are defined by specific DNA sequences, but Mu target sites are not very sequence-specific (3).Transpososomes contain multiple subunits of a transposase protein, bound to DNA sequences from both of the transposon's ends (1). These protein-DNA complexes are also called "synaptic complexes" because they bring together the two ends of the transposon DNA. The phage Mu transposase, MuA, is monomeric in solution but forms a tetramer upon binding to specific DNA recognition sites near the transposon ends (4 -6).Each end of the Mu transposon has three MuA recognition sites: R1, R2, and R3 on the right end and L1, L2, and L3 on the left, not all of which are essential for transposition...
