Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

Tanya L Williams,Tania A Baker

doi:10.1074/jbc.m308156200

Abstract

Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.

Highlights

The successful relocation of mobile genetic elements, as with all DNA rearrangement processes, requires the precise spatial and temporal organization of DNA components within the active sites of large nucleoprotein complexes
Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues
We propose that the 3؅flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining

Summary

Flanking DNA Slows Mu Transposition

A second phage-encoded protein, MuB, can contribute to the Mu transpososome. MuB interacts with the C-terminal domain of transposase [29, 30] and stimulates transposition in multiple ways. The in-line mechanism of phosphoryl transfer dictates that, between the cleavage and joining reaction steps, an organizational change must occur within the two active sites such that the proximity of each 3ЈOH shifts from near the flanking DNA to near the target DNA. We show that the presence of the flanking DNA strand that was attached to the 3ЈOH transposon DNA end before cleavage (the 3Ј-flanking strand) can slow one or more reaction steps that occur after cleavage. We demonstrate that MuB enhances the rate of transposition by stimulating the step after cleavage that can otherwise be inhibited by the 3Ј-flanking strand Based on these results, we suggest that release of the 3Ј-flanking strand from both active sites is required after cleavage for efficient and productive binding of the target DNA within the two active sites. A model is proposed for the conversion from cleavage to joining in which MuB allosterically stimulates a conformational change that accomplishes this DNA movement within the active sites

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION