DNA Recognition Sites Activate MuA Transposase to Perform Transposition of Non-Mu DNA

Ilana Goldhaber-Gordon,Tanya L Williams,Tania A Baker

doi:10.1074/jbc.m110341200

Abstract

Mu transposition occurs within a large protein-DNA complex called a transpososome. This stable complex includes four subunits of MuA transposase, each contacting a 22-base pair recognition site located near an end of the transposon DNA. These MuA recognition sites are critical for assembling the transpososome. Here we report that when concentrations of Mu DNA are limited, the MuA recognition sites permit assembly of transpososomes in which non-Mu DNA substitutes for some of the Mu sequences. These "hybrid" transpososomes are stable to competitor DNA, actively transpose the non-Mu DNA, and produce transposition products that had been previously observed but not explained. The strongest activator of non-Mu transposition is a DNA fragment containing two MuA recognition sites and no cleavage site, but a shorter fragment with just one recognition site is sufficient. Based on our results, we propose that MuA recognition sites drive assembly of functional transpososomes in two complementary ways. Multiple recognition sites help physically position MuA subunits in the transpososome plus each individual site allosterically activates transposase.

Highlights

Transposons are found in all the biological kingdoms, and some perform specialized functions
We focused on the 5386-base pair ␾X174 RFI, but two molecules unrelated to ␾X174, pBR322 or pUC19, worked about well
We find that Mu DNA can activate MuA to transpose non-Mu DNA

Summary

EXPERIMENTAL PROCEDURES

Proteins—In some experiments (see Figs. 7, B, C, and 8) the MuA truncation 77– 663 was substituted for full-length MuA. The reactions were incubated at 30 °C for 20 – 60 min unless otherwise indicated They were stopped by addition of 0.2 volumes of a stop solution (ϳ0.1% bromphenol blue, 8% SDS, 50 mM EDTA, 30% glycerol) and electrophoresed through a 0.9% HGTagarose gel, in 1ϫ TAB buffer (40 mM Tris, pH 8, 3.6 mM EDTA, 27 mM sodium acetate). Gel Mobility Shift Assays—Complexes were assembled under standard transposition conditions, in the absence of MgCl2, target DNA, and MuB They were electrophoresed through a 2% MetaPhor (FMC Bioproducts)-agarose gel in 0.5x TBE buffer (44.5 mM Tris 8.5, 44.5 mM Borate, 1 mM EDTA)

RESULTS

Activation by MuA Recognition Sites

DISCUSSION