Extended Data Fig. 8: Effects of handshake base-pairing.

(a) Schematics of the TBL/DBL and tDNA/dDNA sequences used for cryo-EM analysis and in vitro recombination assays. The pre- and post-HSB (handshake base-pairing) bRNAs stabilize the synaptic complex in the pre- and post-strand exchange states, respectively. Mutated nucleotides in the pre- and post-HSB bRNAs and their complementary DNA nucleotides are highlighted. (b) Effects of handshake base-pairing on in vitro DNA activity of IS621. Three bRNAs with different HSGs, A81/U82/G166/U167 (WT), G81/C82/A166/U167 (pre-HSB) or A81/U82/G166/C167 (post-HSB), were used for the in vitro DNA recombination experiments. The tDNA (38 bp) and dDNA (44 bp) substrates were labeled with Cy5 and FAM at the 5′ ends of the top and bottom strands, respectively. The Cy5/FAM-tDNA (38 bp) and Cy5/FAM-dDNA (44 bp) were mixed with the non-labeled dDNA (100 bp) and tDNA (102 bp), respectively. The DNA substrates were incubated with the IS621–bRNA complex at 37 °C for 1 h, and the reaction was then analyzed using an 18% TBE–urea gel. Recombination between the Cy5/FAM-dDNA and tDNA and between the Cy5/FAM-tDNA and dDNA yields 60- and 69-bp Cy5-labeled products, respectively. For the results with the labeled tDNA and pre- and post-HSB bRNAs, the band intensities of the product and cleaved DNAs were quantified, and the recombination ratios (product DNA / product DNA + cleaved DNA) were calculated. Experiments were repeated at least three times with similar results. (c) Effects of handshake base-pairing between the DBL-HSG and dDNA on IS621-mediated DNA recombination in E. coli. Data are shown as mean ± SD for three biological replicates.