Extended Data Fig. 1: RecA-catalysed strand-exchange reaction.

a, Schematic of the strand-exchange reaction. RecA is shown as yellow spheres, with the letters D and T respectively indicating ADP and ATP-bound forms of RecA. The ssDNA is indicated by dark-brown lines, the donor dsDNA by double lines that are coloured green for the complementary strand and red for the homologous strand. The role of ATP hydrolysis, which is activated on ssDNA binding, is incompletely understood. ATP hydrolysis reverts the RecA–RecA relationship to a state that is inactive in ssDNA binding³. However, the primary ssDNA probably does not diffuse far because (i) the RecA protomers, which can remain associated in a concentration-dependent manner through the αN helix of one RecA interacting with the helicase domain of the 5′ RecA (oligomerization motifs shown in b), would be topologically wrapped around the ssDNA; and (ii) ATP exchanges for ADP due to the high ratio of ATP to ADP in the cell²⁸. In effect, ATP hydrolysis by the synaptic filament and subsequent exchange of ADP for ATP may serve to dissociate dsDNA while seeming not to affect ssDNA binding²⁹, even though the ADP state cannot bind to ssDNA. In the absence of strand exchange, this probably results in the donor dsDNA rebinding stochastically at a different register (shown hypothetically as a shifted dsDNA), continuing the search for homology. ATP hydrolysis by a fully exchanged postsynaptic filament results in the release of the new heteroduplex and the displaced homologous strand of the donor, whereas partial homology results in postsynaptic filaments that contain D-loops and other joint ssDNA–dsDNA molecules, with the ssDNA portions that have not exchanged reconstituted with RecA after ATP rebinding. ATP hydrolysis is not an inherent requirement for strand exchange (except for the release of products), as short dsDNA molecules can exchange in the presence of the non-hydrolysable ATP analogues^30,31,32. With longer, physiologically relevant substrates, however, ATP hydrolysis is needed for bypassing heterology and for the extension of initial joint molecules, or branch migration^33,34. This presumably involves the release of the portions of the donor duplex that have not exchanged, followed by their resampling in a new round of the reaction. With long DNA substrates, about 100 ATP molecules are hydrolysed per base pair exchanged in vitro³⁵. The direction of branch migration with ATP hydrolysis has been reported to be in the 5′ to 3′ direction with circular ssDNA³⁴. With linear ssDNA, however, the reverse polarity is suggested by the finding that ssDNA with 3′-end homology reacts more efficiently than that with 5′-end homology³⁶. This has been attributed to RecA polymerizing on ssDNA preferentially in the 5′-to-3′ direction³⁷. It is not clear to what extent the directionality of branch migration with ATP hydrolysis is related to the local opening of dsDNA without ATP hydrolysis, which this study finds occurs preferentially in the 3′-to-5′ direction of the mini-filament. Because the mini-filament consists of fused RecA protomers, it does not reflect the effects a preferential polarity of RecA polymerization might have on the directionality of strand exchange. Also, our strand exchange reactions do not include the single-stranded DNA binding protein SSB that is involved in strand exchange in vivo and may sequester released DNA strands. b, RecA monomer structure from the presynaptic mini-filament³. The αN oligomerization motif that interacts with the 5′ RecA, and the site on the helicase domain that interacts with the αN of the 3′ RecA are coloured red. The CTD is black. ATP is shown in sticks. As reported³, ssDNA binding cooperates with ATP binding to induce the conformational change from the inactive to the active filament states. The active filament conformation has a distinct RecA–RecA relationship that is stabilized by the ATP becoming sandwiched between adjacent RecAs, and by two of the three nucleotides in each triplet binding to flanking RecAs. Even though the presynaptic filament binds to primary ssDNA with an overall stoichiometry of 3 nt per RecA, each nucleotide triplet is bound by three RecAs, and, conversely, each RecA contacts three nucleotides³. c, Electrophoretic mobility shift assay evaluating different lengths of non-homologous dsDNA binding to the presynaptic mini-filament of 9-RecA–(dT)₂₇–ATPγS. dsDNA, with lengths ranging from 18 bp to 67 bp, was added at a 1.2 molar excess to the mini-filament as described in Methods. Top, overlay of the gel scanned at the two wavelengths for the two different fluorophores. Signal from Alexa Fluor 647-ssDNA is shown in red and signal from Alexa Fluor 488-dsDNA is shown in green. Middle, signal from Alexa Fluor 488-dsDNA alone. Bottom, signal from Alexa Fluor 647-ssDNA alone. Whereas the presynaptic filament formed readily (lane 2), short dsDNA had no detectable signal under these conditions (lanes 3–7, 18–34 bp). A weak signal was detected at 48 bp of DNA (lane 8) and increased further at 67 bp (lane 9), the longest dsDNA we tested in this series. d, Concentration titration of the non-homologous 67 bp dsDNA used in the cryo-EM analysis binding to the presynaptic mini-filament. Top, overlay of the gel scanned at the two wavelengths (coloured as in c). A clear trend of increased binding is evident as the dsDNA concentration increased from 1.2 molar excess to 14 molar excess (lane 3–6) to the presynaptic filament. To confirm that the green signal is from the binding of dsDNA and not a single strand that dissociated from the dsDNA, we also tested Alexa Fluor 488-labelled 67-nt ssDNA at the same nucleotide concentration as the (dT)₂₇ (lane 7, 0.63 μM), or at the same molar ratio to (dT)₂₇ (lane 8, 1.6 μM). DNA molecular weight markers are marked as in c. e, f, Concentration titrations with 120-bp non-homologous dsDNA (e) and 67-bp partially homologous dsDNA (f) used in the cryo-EM analyses, performed as in d. Because we could not procure Alexa Fluor 488-labelled 120 nt DNA, we instead used the corresponding 6 FAM-labelled DNA (Sigma). The experiments of c–f were repeated three times with similar results (Supplementary Fig. 1). The DNA molecular weight markers are marked to the right of each top panel, in units of thousands of base pairs (Kbp).