Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

James R Fuller,Phoebe A Rice

doi:10.7554/elife.21777

Abstract

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

Highlights

Transposons are mobile DNA elements that move or copy their DNA sequence from one location to another
We use two methods to increase the flexibility of duplex DNA: DMSO added to the reaction buffer (Escara and Hutton, 1980; Herrera and Chaires, 1989), and/or a single G:G base-pairing mismatch incorporated at the center of the target DNA sequence (Rossetti et al, 2015)
A 2:1 mixture of MuA protein to Mu end DNA in this buffer results in a gel filtration peak shifted to a very high molecular weight that we identify as Cleaved Donor Complex (CDC) based on size and near-stoichiometric reactivity with target DNA (Figure 2)

Summary

Introduction

Transposons are mobile DNA elements that move or copy their DNA sequence from one location to another. They have exhibited a remarkable ability to spread, such that sequences derived from transposons are pervasive in the genomes of prokaryotes and eukaryotes alike (Aziz et al, 2010). MuA, belongs to the large DDE family of recombinases (Baker and Luo, 1994; Rice and Mizuuchi, 1995). This family is named for the amino acid residues in their shared RNase-H-like catalytic domains that bind the divalent metals necessary for catalysis. In addition to the transposases for many common transposons, the DDE family includes retroviral integrases, which use the same reaction mechanism to integrate viral genomes into host chromatin (Fujiwara and Mizuuchi, 1988; Li et al, 2006)

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Conclusion