Effect of mutations in the Mu-host junction region on transpososome assembly

Mu transposition occurs through a series of higher-order nucleoprotein comp lexes called transpososomes. The region where the Mu DNA joins the host DNA plays an integral role in the assembly of these transpososomes. We have cr eated a series of point mutations at the Mu-host junction and characterized their effect on the Mu in vitro strand transfer reaction. Analysis of thes e mutant constructs revealed an inhibition in transpososome assembly at the point in the reaction pathway when the junction region is engaged by the t ransposase active site (i.e. the transition from LER to type 0). We found t hat the degree of inhibition was dependent upon the particular base-pair ch ange at each position and whether the substitution occurred at the left or right transposon end. The MuB transposition protein, an allosteric effector of MuA, was shown to suppress all of the inhibitory Mu-host junction mutan ts. Most of the mutant constructs were also suppressed, to varying degrees, by the substitution of Mg2+ with Mn2+. Analysis of the mutant constructs h as revealed hierarchical nucleotide preferences at positions -1 through +3 for transpososome assembly and suggests the possibility that specific metal ion-DNA base interactions are involved in DNA recognition and transpososom e assembly. (C) 2001 Academic Press.