Extended Data Fig. 6: Synaptic complex formation.

(a) Interactions between RuvC.3 and Tnp.2. Similar interactions are observed between RuvC.1 and Tnp.4. (b) Interface between the IS621–TBL–tDNA and IS621–DBL–dDNA dimeric complexes. Nucleotides tT3*–tT5* in the target DNA and nucleotides dA3*–dA6* in the donor DNA are labeled as t3*–t5* and d3*–d6*, respectively. (c) Interactions between RuvC.1 and DBL-SL. (d) In vitro DNA recombination activities of IS621 in complex with the WT bRNA or the ΔDBL-SL bRNA mutant, in which nucleotides C137–G147 were replaced with GAAA. The tDNA (38 bp) substrate was labeled with Cy5 at the 5′ end of the top strand. The Cy5-tDNA (38 bp) and non-labeled dDNA (100 bp) were incubated with the IS621–bRNA complex at 37 °C for 1 h, and then the reaction was analyzed using an 18% TBE–urea gel. Recombination between the Cy5-tDNA and dDNA yields a 69-bp Cy5-labeled product. Experiments were repeated at least three times with similar results. (e) In vitro DNA recombination activities of IS621 in complex with the WT bRNA or separated TBL (nucleotides 31–104) and DBL (nucleotides 99–177). The tDNA (38 bp) substrate was labeled with Cy5 at the 5′ end of the top strand. The Cy5-tDNA (38 bp) and non-labeled dDNA (44 bp) (containing 6-nt mismatches in their top strands) were incubated with the IS621–bRNA complex at 37 °C for 1 h, and then the reaction was analyzed using an 18% TBE–urea gel. Recombination between the Cy5-tDNA and dDNA yields a 49-bp Cy5-labeled product. Experiments were repeated at least three times with similar results. (f) Base-pairing between tDNA and dDNA. In (a), (c), and (f), cryo-EM density maps are shown as grey semi-transparent surfaces. (g) DNA recombination activities of the WT IS621 and the active-site mutants in the in vitro recombination assays. The tDNA (38 bp) substrate was labeled with Cy5 at the 5′ end of the top strand. The Cy5-tDNA (38 bp) and non-labeled dDNA (100 bp) were incubated with the IS621–bRNA complex at 37 °C for 1 h, and then the reaction was analyzed using an 18% TBE–urea gel. Recombination between the Cy5-tDNA and dDNA yields a 69-bp Cy5-labeled product. Experiments were repeated at least three times with similar results. dRuvC, D11A/E60A/D102A/D105A. (h) DNA recombination activities of the WT IS621 and the active-site mutants in the bacterial recombination assays. Data are shown as mean ± SD for three biological replicates.