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Structural mechanism governing the directionality of IS110 bridge recombination

ABSTRACT: Bridge recombinases from the IS110 family of transposons, such as IS621, associate with a bridge RNA (bRNA) to mediate programmable recombination between donor and target DNAs. While insertion is mediated by the recombinase–bRNA complex, it remains unknown how these elements are excised from genomes to form circular intermediates. Here, we show that bRNA is weakly expressed from IS621 loci in the Escherichia coli genome and that the IS621 recombinase–bRNA complex mediates excision less efficiently than insertion. Furthermore, we determine the cryo-electron microscopy structures of the IS621 recombinase–bRNA complex bound to excision DNA substrates, providing mechanistic insights into the excision reaction. Similar to the previously reported donor- and target-bound insertion complex, the excision complex comprises two recombinase dimers, each accommodating target- and donor-binding loops of the bRNA. However, DNA recognition differs notably between the two complexes. While the donor and target DNA form a bent U-shape during insertion, the excision substrates adopt linear conformations and bind across both bRNA loops, forming an X-shaped structure, which reduces the efficiency of top-strand exchange and contributes to the naturally observed bias in reaction direction against excision. Despite such differences, the efficiencies of both reactions are similarly modulated by base pairing between the bRNA handshake guides and the top strands of DNA. Overall, this study provides mechanistic insights into the complete IS110 transposition cycle, and facilitates the optimal design of programmable bridge editing applications.

ORGANISM(S): Escherichia coli

PROVIDER: GSE328474 | GEO | 2026/08/12

REPOSITORIES: GEO

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Project description:Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605 elements undergo ‘peel-and-paste’ transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB-family genes that are evolutionarily related to the CRISPR-associated effectors Cas9 and Cas12. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that IscB and TnpB are essential to prevent loss of the donor IS element and potential transposon extinction as a consequence of the TnpA transposition mechanism. We first performed phylogenetic analysis of IscB/TnpB proteins and selected a family of related IS elements from Geobacillus stearothermophilus that we predicted would be mobilized by a common TnpA homolog. After reconstituting transposition using a heterologous expression system in E. coli, we found that IS elements were readily lost from the donor site due to the activity of TnpA in rejoining the flanking sequences back together upon excision. However, these IS elements also encode non-coding RNAs that guide TnpB and IscB nucleases to precisely recognize and cleave these excision products, leading either to elimination of the excision product or re-installation of the transposon through recombination. Indeed, under experimental conditions in which TnpA and TnpB-RNA complexes were co-expressed together with a genomically integrated IS element, transposon retention was significantly increased relative to conditions expressing TnpA alone. Remarkably, both TnpA and TnpB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between transposase and nuclease. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.

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Structural mechanism governing the directionality of IS110 bridge recombination

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