Organization and dynamics of the bacteriophage Mu transpososome

Abstract

Transposition of a linear mobile genetic element, such as the bacteriophage Mu, requires a set of spatially and temporally coordinated DNA phosphoryl transfer reactions to move the element from an existing DNA location to a new DNA site. The Mu transpososome, where the DNA cleavage and joining reactions that compose transposition take place, is a large nucleoprotein complex containing multiple, identical transposase subunits. The cleavage and joining reactions are the result of precise manipulation, by the transposase subunits, of the DNA segments surrounding the two transposon ends and the DNA at the new location. The purpose of this work is to provide a description of the arrangement of the catalytic centers within the Mu transpososome and of the dynamic interaction of the DNA components with the active sites. By employing a unique protein-DNA crosslinking strategy developed in this thesis work, the transposase subunits that contribute three essential acidic amino acids (the DDE motif) to the active sites are identified. This work reveals that the transpososome contains two active sites, as defined by the number of required sets of DDE residues, each of which catalyzes cleavage and joining of one transposon DNA end to the new location. The organization of the subunits supplying these active site residues is such that a subunit bound site-specifically to one transposon DNA end contributes DDE residues to the active site that promotes the phosphoryl transfer reactions at the other transposon end.

(cont.) This crosslinking strategy should be able to be extended to determine the subunits supplying other active site residues and to determine the active site arrangement in other transpososomes and integration complexes. Experiments are also described that provide evidence for a cooperative transition that takes place during the conversion of the cleaved DNA intermediate to the joined transposition product. The evidence supports a model for the arrangement of DNA components during this transition. In this model, the DNA strand previously attached to the transposon end must be removed from both active sites after cleavage to allow movement of the new location DNA from an initial 'holding' site to a proper position within the active sites.