Abstract
Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps.
Highlights
Transposable elements drive genome evolution in many ways – increasing DNA content, rearranging and mutating genes, as well as altering gene regulation [1]
The resulting branched strand transfer joint is resolved by target-primed replication, which is initiated by the PriA primosome and completed by the Pol III holoenzyme, and results in duplication of the Mu genome after every round of integration
Our study finds that during its non-replicative pathway, the gaps created by Mu insertion are repaired by the primary machinery for double-strand break repair in E. coli, not by gap-filling polymerases as previously thought
Summary
Transposable elements drive genome evolution in many ways – increasing DNA content, rearranging and mutating genes, as well as altering gene regulation [1]. A unique aspect of Mu is that, depending on the phase of its life cycle, it moves using either replicative or non-replicative modes of DNA transposition [3]. Most of our knowledge of Mu transposition is derived for the replicative pathway, where during lytic growth, Mu amplifies its genome by repeated transpositionreplication events which exploit the host replication apparatus [4,5]. In vitro experiments have established that in this pathway, the Mu transposase (MuA protein) mediates single-strand cleavages at Mu ends followed by strand transfer of the cleaved ends into target DNA; the latter reaction is greatly assisted by MuB protein (Figure 1). At the end of the lytic cycle, Mu genomes are packaged into phage heads such that they include host sequences (flaps) from both sides of a Mu insertion
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