MainInsertion sequence (IS) elements are transposable DNA sequences found in prokaryotic genomes and are categorized into approximately 30 families, including IS110 (ref. 3). The transposition cycle of IS110 family elements involves two sequential recombination events: excision of the element from the host genome to form a circular double-stranded DNA intermediate and insertion of this circular form into a new target site4,5,6,7,8,9,10 (Fig. 1a,b and Extended Data Fig. 1a). Although insertion has been the focus of recent structural and functional studies1,2, the mechanism by which IS110 elements are excised under native conditions has remained poorly understood. Fig. 1: Biological and biochemical analyses of the IS621-mediated excision reaction.a, Schematic of the life cycle of the IS621 element. The CT core dinucleotide sequences are shown as green diamonds. b, Sequences of the DNA substrates for excision (left and right halves) and insertion (donor and target). c, Schematic of the IS621 sites in the E. coli Mach1 genome. Black arrows indicate PCR primers used to detect circular intermediates in e and post-excision sites in f. d, Transcription profiles of linear IS621 elements in the E. coli Mach1 genome. Mapped reads from the IS621 sites 1, 2 and 3 are overlaid. Reads that could not be assigned to specific loci due to sequence similarity were classified as nonspecific. Predicted transcription start sites (TSSs) are indicated by triangles. CPM, counts per million. e, Formation of circular intermediates in E. coli Mach1. The region spanning the LD–RD junction was amplified by PCR from E. coli Mach1 genomic DNA. E. coli BL21(DE3) was used as the control. f, Formation of post-excision sites in E. coli Mach1. The regions spanning the LT–RT junctions were amplified by PCR from E. coli Mach1 genomic DNA. In e and f, DNA was visualized with SYBR Gold. Data shown are representative of three technical replicates. For gel source data for e and f, see Supplementary Fig. 6. g, Schematic showing base pairing between the TBL/DBL in the wild-type bRNA and the DNA substrates before and after the top-strand exchange during excision and insertion. Non-canonical base pairs are indicated by red lines, and DNA cleavage sites are marked with yellow triangles. TS, top strand; BS, bottom strand. h,i, Recombination efficiencies of excision and insertion in vitro. DNA substrates were incubated with the IS621 recombinase and the engineered bRNA (RTG/RDG extensions and the G53A substitution) containing pre-top strand exchange (pre-TSE) and post-top strand exchange (post-TSE) HSG sequences (h) and various HSG sequences (i) at 37 °C for 2 h. Recombination efficiencies were quantified by qPCR. In h, data are shown as mean ± s.d. (n = 3 technical replicates). In i, the differences in the number of HSG–DNA base pairs before and after top-strand exchange are displayed on each cell. The efficiencies for the pre-TSE and post-TSE HSG sequences are highlighted with solid and dashed yellow-green boxes. Data are shown as mean (n = 3 technical replicates). TSE, top-strand exchange.Unlike typical transposable elements11, IS110 elements, such as IS621 from Escherichia coli6, comprise a left end (LE), a recombinase gene and a right end (RE), with conserved CT (cytidine–thymidine) core dinucleotide sequences at both boundaries (Fig. 1a,b and Extended Data Fig. 1a). The IS110 recombinase has a distinctive domain architecture: a RuvC-like nuclease domain with a non-canonical DEDD catalytic motif (hereafter, RuvC for simplicity), a coiled-coil domain that mediates dimerization and a Tnp domain harbouring a conserved catalytic serine residue1,6 (Extended Data Fig. 1b). During transposition, the IS110 recombinase catalyses recombination between the circular intermediate (donor DNA (dDNA), containing left donor (LD) and right donor (RD) sequences) and the genomic target site (target DNA (tDNA), containing left target (LT) and right target (RT) sequences), generating the LT–RD and LD–RT junctions at the insertion site, referred to as the left half (LH) and right half (RH), respectively (Fig. 1b and Extended Data Fig. 1a). Excision is the reverse process: recombination of the LH and RH regenerates the circular donor and the original target site.We recently established that the IS621 circular intermediate expresses a non-coding bridge RNA (bRNA) from a promoter reconstituted across the RE–LE junction1. The bRNA comprises two programmable DNA-binding loops: a target-binding loop (TBL) and a donor-binding loop (DBL), each containing guide segments that hybridize with tDNA and dDNA, respectively, enabling the IS621 recombinase to catalyse sequence-specific recombination between diverse DNA substrates1 (Extended Data Fig. 1c). Cryo-electron microscopy (cryo-EM) structures of the IS621 recombinase–bRNA complex bound to insertion substrates (referred to as the insertion complex) showed a tetrameric synaptic complex in which composite RuvC–Tnp active sites catalyse stepwise strand cleavage and exchange2 (Extended Data Fig. 1d). Furthermore, we and others demonstrated that the bridge recombinase orthologue ISCro4 can mediate programmable rearrangement of the human genome12,13.Studies on the IS110 family member ISEc21 showed that a non-coding RNA derived from the upstream non-coding region co-purifies with the recombinase and is essential for circular intermediate formation, with a minimum number of target-derived flanking bases required on each side of the element for excision to proceed14. In a related IS110 family member, ISPpu9, multiple distinct circular intermediates are generated through recombination between different combinations of terminal sequences, in a process requiring an active recombinase and specific nucleotides at the element ends15. However, it remains unclear how the bridge recombinase–bRNA complex mediates excision under native conditions in bacterial cells, how the left and right element junctions are recognized, and how the bRNA is produced before circular intermediate generation, given that bRNA expression is itself driven by circularization. Here, we biochemically and structurally characterized the IS621 excision reaction, providing a resolution to this chicken-and-egg problem.Excision of the IS621 elements in E. coli