This study elucidates the structural mechanism of bridge RNA-guided recombination in the IS110 family of insertion sequences. The researchers used cryo-electron microscopy to determine the structures of the IS110 recombinase in complex with its bridge RNA (bRNA), target DNA, and donor DNA in three stages of the recombination reaction cycle. The structures reveal that the IS110 synaptic complex comprises two recombinase dimers, one of which binds to the target DNA and the other to the donor DNA, with the bRNA's target-binding and donor-binding loops interacting with the respective DNA strands. A composite RuvC–Tnp active site spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both DNA strands. The structures show that the top strands of the target and donor DNA are cleaved to form covalent 5'-phosphoserine intermediates, followed by exchange and religation to form a Holliday junction intermediate, which is then resolved by cleavage of the bottom strands. The study highlights how the bispecific bRNA confers target and donor DNA specificity to the IS110 recombinases, enabling programmable DNA recombination. The IS621 recombinase, a member of the IS110 family, was analyzed in detail, revealing its structure and the role of its RuvC and Tnp domains in the recombination process. The study also describes the architecture of the bRNA, showing how its target-binding and donor-binding loops recognize specific DNA sequences. The findings provide insights into the mechanism by which the IS621 recombinase and its bRNA facilitate DNA recombination, offering a framework for understanding and engineering programmable DNA rearrangement systems. The study underscores the importance of the bRNA in directing the recombination process and highlights the unique features of the IS110 family elements, including their use of a RuvC–Tnp composite active site and their ability to recognize specific DNA sequences through their bRNA. The results contribute to the broader understanding of transposable elements and their role in genome function and evolution.This study elucidates the structural mechanism of bridge RNA-guided recombination in the IS110 family of insertion sequences. The researchers used cryo-electron microscopy to determine the structures of the IS110 recombinase in complex with its bridge RNA (bRNA), target DNA, and donor DNA in three stages of the recombination reaction cycle. The structures reveal that the IS110 synaptic complex comprises two recombinase dimers, one of which binds to the target DNA and the other to the donor DNA, with the bRNA's target-binding and donor-binding loops interacting with the respective DNA strands. A composite RuvC–Tnp active site spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both DNA strands. The structures show that the top strands of the target and donor DNA are cleaved to form covalent 5'-phosphoserine intermediates, followed by exchange and religation to form a Holliday junction intermediate, which is then resolved by cleavage of the bottom strands. The study highlights how the bispecific bRNA confers target and donor DNA specificity to the IS110 recombinases, enabling programmable DNA recombination. The IS621 recombinase, a member of the IS110 family, was analyzed in detail, revealing its structure and the role of its RuvC and Tnp domains in the recombination process. The study also describes the architecture of the bRNA, showing how its target-binding and donor-binding loops recognize specific DNA sequences. The findings provide insights into the mechanism by which the IS621 recombinase and its bRNA facilitate DNA recombination, offering a framework for understanding and engineering programmable DNA rearrangement systems. The study underscores the importance of the bRNA in directing the recombination process and highlights the unique features of the IS110 family elements, including their use of a RuvC–Tnp composite active site and their ability to recognize specific DNA sequences through their bRNA. The results contribute to the broader understanding of transposable elements and their role in genome function and evolution.