The study investigates the structural mechanism of RNA-guided recombination in IS110 family elements, specifically focusing on the IS621 element. The IS621 recombinase, encoded by these elements, forms a synaptic complex with a non-coding bridge RNA (bRNA) and target DNA (tDNA) and donor DNA (dDNA). The bRNA contains two programmable loops that confer specificity to both target and donor DNA. The synaptic complex consists of four IS621 recombinase protomers, each interacting with one of the bRNA's target-binding loop (TBL) or donor-binding loop (DBL). The TBL and DBL base pair with the tDNA and dDNA, respectively, and the catalytic serine residues (S241) form covalent 5′-phosphoserine intermediates adjacent to the CT core dinucleotides in both DNA strands. The top strands of the tDNA and dDNA are cleaved at these sites, followed by strand exchange and the formation of a Holliday junction intermediate. This intermediate is resolved by cleaving the bottom strands, completing the recombination process. The study reveals how the IS621 recombinase and bRNA orchestrate this complex mechanism, providing insights into the programmable DNA recombination capabilities of IS110 elements.The study investigates the structural mechanism of RNA-guided recombination in IS110 family elements, specifically focusing on the IS621 element. The IS621 recombinase, encoded by these elements, forms a synaptic complex with a non-coding bridge RNA (bRNA) and target DNA (tDNA) and donor DNA (dDNA). The bRNA contains two programmable loops that confer specificity to both target and donor DNA. The synaptic complex consists of four IS621 recombinase protomers, each interacting with one of the bRNA's target-binding loop (TBL) or donor-binding loop (DBL). The TBL and DBL base pair with the tDNA and dDNA, respectively, and the catalytic serine residues (S241) form covalent 5′-phosphoserine intermediates adjacent to the CT core dinucleotides in both DNA strands. The top strands of the tDNA and dDNA are cleaved at these sites, followed by strand exchange and the formation of a Holliday junction intermediate. This intermediate is resolved by cleaving the bottom strands, completing the recombination process. The study reveals how the IS621 recombinase and bRNA orchestrate this complex mechanism, providing insights into the programmable DNA recombination capabilities of IS110 elements.