Mechanism of strand exchange from RecA-DNA synaptic and D-loop structures

Abstract

The strand-exchange reaction is central to homologous recombination. It is catalysed by the RecA family of ATPases, which form a helical filament with single-stranded DNA (ssDNA) and ATP. This filament binds to a donor double-stranded DNA (dsDNA) to form synaptic filaments, which search for homology and then catalyse the exchange of the complementary strand, forming either a new heteroduplex or-if homology is limited-a D-loop^1,2. How synaptic filaments form, search for homology and catalyse strand exchange is poorly understood. Here we report the cryo-electron microscopy analysis of synaptic mini-filaments with both non-complementary and partially complementary dsDNA, and structures of RecA-D-loop complexes containing a 10- or a 12-base-pair heteroduplex. The C-terminal domain of RecA binds to dsDNA and directs it to the RecA L2 loop, which inserts into and opens up the duplex. The opening propagates through RecA sequestering the homologous strand at a secondary DNA-binding site, which frees the complementary strand to sample pairing with the ssDNA. At each RecA step, there is a roughly 20% probability that duplex opening will terminate and the as-yet-unopened dsDNA portion will bind to another C-terminal domain. Homology suppresses this process, through the cooperation of heteroduplex pairing with the binding of ssDNA to the secondary site, to extend dsDNA opening. This mechanism locally limits the length of ssDNA sampled for pairing if homology is not encountered, and could allow for the formation of multiple, widely separated synapses on the donor dsDNA, which would increase the likelihood of encountering homology. These findings provide key mechanistic insights into homologous recombination.

Extended Data Figure 1 |. RecA-catalyzed 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 colored 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 binding. However, the primary ssDNA likely 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 exchanging for ADP due to the high ratio of ATP to ADP in the cell. In effect, ATP hydrolysis by the synaptic filament and subsequent exchange of ADP for ATP may serve to dissociate dsDNA while appearing to not affect ssDNA binding, even though the ADP state cannot bind to ssDNA. In the absence of strand exchange, this likely 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, while 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 analogs^–. With longer, physiologically relevant substrates, however, ATP hydrolysis is needed for bypassing heterology and for the extension of initial joint molecules, or branch migration^,. 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 hydrolyzed per base pair (bp) exchanged in vitro. The direction of branch migration with ATP hydrolysis has been reported to be in the 5’ to 3’ direction with circular ssDNA. With linear ssDNA, however, the reverse polarity is suggested by the finding that ssDNA having 3’-end homology reacs more efficiently than that with 5’-end homology. This has been attributed to RecA polymerizing on ssDNA preferentially in the 5’ to 3’ direction. 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. Since the mini-filament consists of fused RecA protomers, it does not reflect the effects a preferential polarity of RecA polymerization may 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 filament. 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 colored red. The CTD is black. ATP is shown in sticks. As reported, 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 getting 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 nts per RecA, each nucleotide triplet is bound by three RecAs, and, conversely, each RecA contacts three nucleotides. c, Electrophoretic mobility shift assay evaluating different lengths of non-homologous dsDNA binding to the presynaptic mini filament of nine-RecA-(dT)₂₇-ATPγS. dsDNA, with lengths ranging from 18 bps to 67 bps, 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 Alex 647-ssDNA is shown in red and signal from Alex 488-dsDNA is shown in green. Middle, signal from Alexa 488-dsDNA alone. Bottom, signal from Alex647-ssDNA alone. While 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 bps 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 (colored 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 488-labeled 67 nucleotide 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 488-labeled 120 nt DNA, we instead used the corresponding 6 FAM-labeled 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).

Extended Data Figure 2 |. Cryo-EM analysis of the strand exchange reaction with non-homologous 67 bp dsDNA.

a, Sequences of the 27 nt ssDNA (left, brown) and the 67 bp non-homologous dsDNA (right, black) used in the strand exchange reaction. b, Micrograph from the reaction containing nine-RecA, (dT)₂₇, ATPγS, and non-homologous 67 bp dsDNA. The micrograph is similar to the rest of the 14,762 micrographs except for variations in particle numbers, ice thickness and other parameters across the grid. c, Representative 2D classes of the particles after polishing. Box size is 279 Å. 2D classifications, performed two to three successive times prior to polishing resulted in similar classes, except for classes with low-quality 2D projections that were discarded. d, Chart shows the gold-standard FSC plot of the consensus reconstruction. Dashed line marks the FSC cutoff of 0.143. Second from the left is the consensus reconstruction map colored by local resolution estimated with the RELION3 post-processing program. The resolution range is mapped to the colors in the inset below the map; the terminal RecA proteins are less ordered than the rest. Third, cartoon representation of the refined model of the consensus refinement. As in Figure 1a, primary ssDNA is colored in brown, S2 ssDNA red. The nine-RecA protein is colored uniformly khaki for simplicity. Fourth and fifth, cartoon representations of duplexes A to I in the 5’- and 3’-tilt conformations, respectively colored cyan and purple, in the same relative orientation as the refined model. Lastly, duplexes with both tilts are superimposed on the protein to highlight the difference in the 5’ and 3’ tilts. The 5’- and 3’-tilted duplexes were combined to generate the masks for the 3D classifications as described in methods. e, The masks used for 3D classification with partial signal subtraction at each duplex are at the top, and the maps of the 3D classes at the bottom. For each RecA position, the classes with duplexes are labeled with percentage and particle numbers (in parentheses). Because of the poor order of the terminal RecAs, and in particular at the 3’ end of the filament where the CTDs extend the farthest away from the filament, we could not reliably classify particles at CTD^A, and for the same reason the penultimate CTD^B was an outlier with a low 4 % duplex occupancy (hereinafter we will be referring to individual RecA protomers with letters, starting with A from the 3’ end of the primary ssDNA). At the 5’ end, even though RecA^I was overall poorly ordered, duplex^I-containing particles could readily be identified, as CTD^I points towards the mid-portion of the filament, and its density is significantly better defined than CTD^A and CTD^B that point away from the filament’s 3’ end. Masks and maps of the 3D classification for all 28 combination of duplex pairs are shown in Supplementary Figure 2 and their details listed in Supplementary Spreadsheet 2. f, Histogram of the number of duplexes per particles. Chart shows the percentage of particles that have the indicated number of duplexes for this dataset. The data set was collected once. Also see Supplementary Spreadsheet 1 for details. g, The accessibility of the CTDs to dsDNA is highest at the 3’ end of the filament, where the DNA-binding tips of CTD^A to CTD^C point into empty solvent and the dsDNA can approach from a roughly half-spherical volume (left panel). CTD^A is even more accessible as it has no neighboring RecA 3’ to it. Moving towards the 5’ end, the CTDs start getting increasingly encumbered by the presence of RecA protomers 3’ to them. Thus, CTD^D gets slightly hindered by the RecA^A L2 loop that is 47 Å away (right panel; C_α-C_α distance from CTD Gly288 to L2 loop Gly200 in a direction that would approximately bisect the axis of dsDNA). CTD^E is more encumbered, as, in addition to the RecA^B L2 loop, is within 35 Å of the RecA^A L1 loop (right panel; C_α-C_α distance from CTD Gly288 to L1 loop Glu158). And, CTD^F is obstructed by not only the RecA^C L2 and RecA^B L1 loops, but also by the helicase domain of RecA^A, which is within 35 Å (right panel; C_α-C_α distance from CTD Gly288 to helicase Ala131). CTD^G and CTD^H are encumbered the most, by the full turn of filament 3’ to them (left panel). Their DNA-binding tip is 28 Å away from the N-terminal helices of RecA^A and RecA^B, respectively (C_α-C_α distance from CTD Gly288 to Lys19 of αN), a distance that is only fractionally larger than the ~20 Å width of a DNA duplex. The terminal CTD^I is similarly close to the N-terminal helix of RecA^C, although the absence of a 5-neighboring RecA would substantially increase its accessibility to dsDNA compared to those of CTD^G and CTG^H. Figure shows molecular surface of the 9-RecA filament with the aforementioned structural elements colored for each RecA as in Figure 1a and labeled. Black dotted lines indicate the shortest RecA-RecA distances (marked) that would approximately bisect the axis of dsDNA bound at each CTD. Primary ssDNA is colored brown. The homologous ssDNA is not shown for clarity. View in right panel is rotated by 180°, roughly half a turn of the filament, about the vertical axis to show the environment of CTD^D, CTD^E and CTD^F that are obscured in the left view.

Extended Data Figure 3 |. Initial 3D classification of multi-duplex classes.

a-d, Select multi-duplex combinations containing duplex^C from the 120 bp dsDNA reaction were 3D classified with partial signal subtraction and masks specific for the duplex combinations as described in methods. Shown here are the reconstructions of select classes after 3D refinement. Duplexes, their connectivity, particle number and map resolution according to the gold-standard fourier shell correlation (FSC) procedure are shown on top of each map. Maps are colored by the duplex tilt (5’ tilt cyan, 3’ purple), S2 DNA red. Duplex positions are labeled. A cartoon showing a simplified version of the duplex pattern for each map is shown on the left of each class. Some maps are rotated to give a clear view of obscured duplexes in the top view with rotation axis indicated. The maps are organized by the number of duplexes in each classification.

Extended Data Figure 4 |. Cryo-EM Analysis of S2-connected duplex pairs from non-homologous dsDNA reactions.

a, The 5’ end of the filament, but not the 3’ end produces classes with conspicuously short duplexes. Reconstruction after 3D refinement of paired duplexes that have duplex^H or duplex^I. Top, maps of classes with short duplexes. Bottom, maps of classes from the same 3D classification but with regular, long duplexes. Black circle highlights the difference in the duplex length. Maps are colored by the duplex tilt (5’ cyan, 3’ purple), S2 DNA red. Overall resolution, from gold-standard refinement procedure, and particle numbers are also shown. b, Charts show the number of particles with S2-connected duplex pairs for the non-homologous 67 bp dsDNA dataset. Each chart is of the series that contains the 1^st duplex in common. Pairs of duplexes starting at RecA^C are omitted, as they are shown in Fig 1d. The data set was collected once. c, Charts show the number of particles with S2-connected duplex pairs for the non-homologous 120 bp dsDNA dataset as in b. Pairs of duplexes starting at RecA^C are omitted as they are shown in Fig 1l. The data set was collected once.

Extended Data Figure 5 |. Cryo-EM analysis of the strand exchange reaction with non-homologous 120 bp dsDNA.

a, Sequences of the 27 nt ssDNA (top, brown) and the 120 bp non-homologous dsDNA (bottom, black) used for this data set. b, Starting from the left, consensus reconstruction map of this dataset colored by local resolution estimated with the RELION3 post-processing program. The resolution range is mapped to the colors in the inset below the map. Next, cartoon representation of the refined model of the consensus refinement. Primary ssDNA is colored in brown, S2 ssDNA red, and RecA is khaki. Cartoon representation of duplexes A to I in the 5’-tilted (cyan) and 3’-tilted conformations (purple), followed by the superposition of the two. Last, all cartoons superimposed highlighting the tilt difference, and the relative organization of the DNA elements on the filament. c, Masks and maps of the 3D classification for individual duplexes as in Extended Data Fig 2e. The overall duplex occupancy of up to 39 % is higher, with 1.9 duplexes per particle on average, as expected from the presence of nearly twice the number of binding sites corresponding to a duplex compared to the 67-bp non-homologous dsDNA reaction. The mid-filament duplex^F and duplex^G were again outliers with low relative occupancy (22 % and 13 %, respectively), consistent with the crowded filament mid-portion having low accessibility for dsDNA. And, as before, duplex^B and duplex^I at the poorly-ordered filament termini resulted in apparently low occupancy (14 % and 10 %, respectively). Masks and maps of the 3D classification for all 28 combination of duplex pairs are shown in Supplementary Figure 3 and their details listed in Supplementary Spreadsheet 4. Paired-duplex 3D classification showed a distribution qualitatively very similar to that of the 67 bp dsDNA reaction, including the preference for 5’ tilts at the 3’ but not 5’ end of the filament. d, Chart of the percentage of particles that have the indicated number of duplexes for this dataset. The data set was collected once. Also see Supplementary Spreadsheet 3 for more details.

Extended Data Figure 6 |. Cryo-EM analysis of the strand exchange reaction with partially-homologous 67 bp dsDNA.

a, Sequences of the primary 27-nt ssDNA (top, brown) and of the 67-bp donor dsDNA (bottom) containing a 10-nt segment of homology to the ssDNA. The dsDNA region of homology is colored green and red for the complementary and homologous strands, respectively. The directions of the DNA strands, and every 10^th nucleotide are labeled. Dots indicate complementarity. b, Consensus reconstruction as in Extended Data Fig. 2d, except complementary DNA can now be seen and is included in the model (green). c, Masks and maps of the 3D classification for individual duplexes as in Extended Data Fig. 2e. Masks and maps of the 3D classification for all 28 combination of duplex pairs are shown in Supplementary Figure 4 and their details listed in Supplementary Spreadsheet 6. d, Chart of the percentage of particles that have the indicated number of duplexes for this dataset. The data set was collected once. Also see Supplementary Spreadsheet 5 for details. e, Charts show the number of particles with the indicated S2-connected duplex pairs for this dataset. Pairs of duplexes starting at RecA^C are shown in Fig 2c. See Supplementary Spreadsheet 6. The data set was collected once. f, Reconstruction after 3D refinement of the minor class from the classification of the 5’/3’ tilted duplex^D-duplex^G particles. This class essentially has no heteroduplex density. g, The same 3D sub-clasification analysis of the class with 5’/5’-tilted duplexes (10,703 particles) identified only 1,297 particles with some heteroduplex density. Their reconstruction, shown in the figure, had overall weak density for both the homologous and complementary strands, and the connections to both duplexes were very weak. We presume that this class was still heterogeneous, but it could not be further sub-classified due to limited numbers of particles. This suggests that the D-loop forms preferentially in the 5’/3’-tilt conformation. h, Figure shows reconstruction after 3D refinement of the major class from the 3D sub-clasification of the 5’/5’ tilted duplex^D-duplex^G particles. The density for the complementary strand of the heteroduplex is even weaker than that of g, and it is discontinuous. We could not further sub-classify this class, presumably due to extensive heterogeneity and limited numbers of particles.

Extended Data Figure 7 |. Flowcharts of focused reconstructions for the data sets with partially-homologous 67-bp dsDNA, non-homologous 120 bp dsDNA, D-loop^DG and D-loop^DH.

a-d, Data processing of a, partially homologous 67 bp dsDNA reaction, b, non-homologous 120 bp dsDNA reaction, c, 9-RecA–D-loop^DG complex, and d, 9-RecA–D-loop ^DH complex. The consensus reconstruction map and the two focused refinement maps for each dataset are colored by local resolution estimated with the RELION3 postprocess program. The resolution range is mapped to the colors in the inset next to each map. Bottom, the graphs show gold-standard FSC plots of the consensus reconstruction (blue) and two focused refinement maps (green and red), as well as the FSC curves between the composite map and the refined model (black, labeled pdb), between the composite map combining the first of the two half maps of each reconstruction and the model refined against this map (dashed pink curve, labeled half1-ref), and between the composite map combining the second of the two half maps and the model refined against the first composite half map for validation (dashed gray curve, labeled half2-val). Horizontal dashed line marks the FSC cutoff of 0.143 and the vertical dashed line indicates the resolution of the REFMAC5 refinement.

Extended Data Figure 8 |. Models and density of D-loop^DG and D-loop^DH.

Discussion of RecA-DNA contacts. The L2 loop-duplex contacts involve non-equivalent RecA protomers due to the different duplex orientations. Thus, duplex^D abuts the L2 loop of the adjacent RecA^C and additionally stacks with Gly204 backbone atoms, whereas duplex^G abuts the L2 loop of RecA^E, two RecAs over, and packs with the Met202 side chain (Fig. 3b–c). The CTD-dsDNA interactions are very similar in the two duplexes, except those at duplex^G consistently have slightly longer distances. The contacts from the loop-helix and hairpin motifs expand the minor groove of the DNA to 15.2 Å for duplex^D and 14.6 Å for duplex^G. The loop and the amino-terminus of the loop-helix motif (residues 297 to 302) make a set of hydrogen bonds to backbone phosphate groups of both strands (backbone amide of Lys302 and side chain of Lys297) while the side chain amide group of Gln300 hydrogen bonds to a thymidine O2 (duplex^D) or guanidine N3 (duplex^G) groups. Crucially, the Gln300 side chain is also in a π−π stack with the Trp290 side chain from the hairpin motif (residues 286 to 290 with the sequence Lys-Ala-Gly-Ala-Trp). With both side chains thus rigidified, they fit snuggly in the minor groove and make multiple van der Waals contacts to the ribose groups, with the amino group of the Trp290 side chain also hydrogen bonding to an N3 group of an adenine (duplex^D) or guanine (duplex^G). The tip of the hairpin (Ala-Gly-Ala portion) partially inserts in the minor groove as well, with the preceding Lys286 within contact distance of the phosphodiester backbone. A second hairpin at the end of the long β6 strand of the helicase core is positioned above the adjacent major groove, and Lys232 contacts the phosphodiester backbone of duplex^G, but the corresponding contact is not made to duplex^D, which is farther away due to its 5’ tilt. Among the CTD-duplex contacts, the K286N and K302N mutations were shown to cause defects in UV-repair in vivo and in binding to and pairing with dsDNA, although they were interpreted as affecting the secondary DNA-binding activity. The S2 site contacts to the homologous strand are overall more extensive and the density stronger at the duplex^D-proximal two thirds of the strand than near duplex^G. The contacts start immediately after the opening of duplex^D by L2^C (these Ade28 contacts are discussed in the main text). The base group of the next residue, Cyt27, is sandwiched between the L2^C Met202 and L2^D Pro206 side chains, while its ribose group packs against the E207^D-R226^D salt bridge (Fig. 3g, bottom). Cyt26 then packs on one side with the L2^D Pro206 side chain and Gly204 and Asn205 backbone groups, and on the other side with Cyt25. The Cyt25 phosphodiester group in turn hydrogen bonds to the Ala230 backbone amide and Arg227 side chain groups, both from β6^E (Fig. 3g, middle). The next two nucleotides stack together and as a pair fit snuggly into a tight gap between the backbones of β6^E and L2^D, as if they are pinched (Fig. 3g, middle). Here, β6^E side chain and backbone groups of Ile228 and Gly229 pack with Cyt24, while L2^D backbone groups from Phe203 and Gly204 pack with Cyt23. In addition, the phosphodiester group of Cyt24 hydrogen bonds to the backbone amide of L2^D Asn205, and that of Cyt23 to the side chain of β6^E Arg226. Thereafter, Cyt22 has RecA contacts and relative position very similar to Cyt27, five nucleotides away in the 3’ filament direction. The one difference is the Cyt27-L2^C Met202 packing is replaced by Cyt22 -L2^D Phe203, owing to the alternate conformation that L2^C adopts as it book-ends duplex^D (Fig. 3g, top). The Cyt22 base is also within contact distance of β6^E Lys245, where the K245N mutation was reported to affect homologous pairing. The D-loop^DH structure recapitulates the key aspects of D-loop^DG. These include the conformations of the L2^C and L2^F loops and their stacking with their respective duplex^D and duplex^H, the overall S2 ssDNA backbone conformation, and the β6-L2 pinch of a nucleotide pair, of which it has two (Fig. 4b). One, at Thy26-Thy27, is essentially identical to that of D-loop^DG, while the other, at Thy21-Thy22 has Thy21 in a slightly different conformation, as it is next to the 3 nt spacer that transitions from S2-binding to duplex^H. The D-loop^DH structure also exhibits the same pattern of contacts at the transitions from the duplexes to the opened up homologous strands. The first-opened up base immediately after duplex^D (Ade31) packs with L2^C, while its phosphodiester backbone is contacted by Arg226 of β6^D. The opposing, flipped out base of the complementary strand has poor density and does not appear to make any RecA contacts. And, as with D-loop^DG, the 3 nts just before duplex^H (Thy20-Thy19-Ade18) are poorly ordered, and make few contacts as they follow an alternate path around their respective L2^F. a, On dsDNA binding, the CTD domains undergo a rotation about an axis (red stick) near residue 269 and roughly perpendicular to the direction of the filament. The 9 RecA protomers of the 9-RecA fusion protein were superimposed by aligning their helicase core domains. The RecA^A, RecA^B, RecA^C, RecA^E, RecA^F, RecA^H, and RecA^I CTD domains (gray) do not exhibit any conformational changes, whereas those of RecA^D and RecA^G rotate (curved arrow) in opposite directions, by −3° and 13° respectively. b, Cartoon representation showing the superposition RecA^D and RecA^G on their CTD domains to highlight the different tilts of duplex^D and duplex^G relative to their already rotated CTD domains. Colored as in Figure 3d (which shows the superposition of the RecA protomers on their helicase domains). The rest of the RecAs are colored yellow and gray. c-d, Density of duplex^D and duplex^G from the D-loop^DG maps used in REFMAC5, as described in methods, in the same orientation as Fig 3b and 3c. e-f, Density of the interactions of duplex^D and duplex^G with their respective CTDs from the D-loop^DG REFMAC5 refinement the same orientation as Fig 3e and 3f. g, Density of the S2 site structural elements and all DNA from the D-loop^DG maps used in REFMAC5. Orientations as in Fig 3g. h, Density of the DNA only from D-loop^DH maps used in REFMAC5 in the same orientation as Fig 4b. RecA protomers and their density are omitted for clarity.

Extended Data Figure 9 |. Rad51, the eukaryotic RecA homolog, likely functions similarly to RecA in strand exchange.

Rad51 lacks the RecA CTD but instead contains an N-terminal domain (NTD) that has been implicated in dsDNA binding by chemical shift perturbation data of the isolated NTD domain. While the RAD51 NTD is structurally unrelated to the RecA CTD, it occupies an analogous position at the filament periphery^–, except that it is oriented with its solvent exposed surface pointing to the 5’ end of the filament instead of the 3’ end that the RecA CTD points to. Because of this, the NTDs at the 5’ end of the RAD51 filament are more accessible for initial dsDNA binding compared to those at the 3’ end, the opposite of the RecA filament. a, Figure shows a side-by-side comparison of the RecA D-loop^DG structure (left) and a model of a 9-protomer Rad51-ssDNA presynaptic filament (right). The Rad51 model was constructed from the coordinates of the 4.4 Å cryo-EM structure of a three-Rad51 segment bound to 9 nts of ssDNA (PDBID 5H1B) by successively applying the transformation that relates the middle-Rad51 to the 5’-most Rad51. Because of small differences in the relative orientation of adjacent protomers, the RecA and Rad51 filaments are superimposable only locally (while individual helicase domains superimpose with an r.m.s.d. of 1.9 Å for 201 of 245 RecA C_α atoms, three-protomer segments superimpose with an r.m.s.d. of 2.2 Å for 531 C_α atoms). For the side-by-side comparison, the two filaments were superimposed on the central, 3-protomer segment (RecA^D to RecA^F). The proteins and cofactors are colored uniformly gray, except their respective CTD and NTD domains are colored rainbow as in Figure 1a, the primary ssDNA is brown for both, and the rest of the RecA DNA molecules are colored as in Figure 3a. The NTD and CTD domains are labeled. b, Close-up views of the RecA Dloop^DG and the 9-Rad51 model superimposed on the three 3’-most RecA^A-RecA^C, focusing on the RecA duplex^G in an orientation similar to a (right view rotated 90° about the vertical axis). This superposition brings duplex^G of RecA in close proximity to the RAD51 NTD^A, which is nearly one turn of the filament away, in the 3’ direction, from CTD^G owing to the different locations of the Rad51 NTDs. The backbone amide nitrogen atoms reported to be involved in dsDNA binding are shown as yellow spheres (Ile61, Lys64, Gly65, Ile66 and Ala69). These are located at a loop (shown in thick tube) and the N-terminus of the helix that follows. These structural elements are positioned relative to the DNA duplex analogously to the loop-helix motif of the RecA CTD, although they approach the DNA from the opposite direction. An adjacent Rad51 loop (residues 30–35, also shown as a thick tube) is also in close proximity to the RecA duplex^G of the superposition.

Figure 1 |. Cryo-EM analysis of the strand exchange reactions with non-homologous dsDNA.

a, Nine-RecA–(dT)₂₇–ATPγS structure and model of S2 ssDNA from the 67-bp dsDNA reaction. RecA protomers are colored rainbow (red-orange-yellow-green-blue-violet) starting with red for RecA^A at the primary ssDNA’s 3’ end (labeled), then repeating for RecA^G-RecA^I. Primary ssDNA is brown, and S2 ssDNA red. ATPγS shown as spheres. Filament axis is vertical. The CTD shown in b is boxed. b, Right, unsharpened cryo-EM density (semi-transparent) from the consensus reconstruction of the 67-bp dsDNA reaction overlaid on the structure (protein yellow, rest as in a). Density within 2.6 Å of the primary and S2 ssDNA colored as the model. Left insets, close-up of RecA^D CTD (boxed region in right) density filtered at 6 Å and contoured at a level of 0.005 (top, comparable to consensus map level) or 0.001 (bottom). dsDNA density is cyan. c, Number of particles that have a duplex in the 5’ (cyan) or 3’-tilted (purple-blue) orientation at RecA^B to RecA^I. d, Number of particles that have S2-connected duplex pairs from the series that contains duplex^C in common. e-j, Reconstructions of S2-connected, 5’/3’ tilted classes containing duplex^C in common from the 120-bp dsDNA strand exchange reaction. Duplexes are colored according to their tilt (cyan for 5’ tilt, purple for 3’), and the rest as in b. The schematic depicts the duplexes (parallel lines), their S2 connection (short vertical line), their associated CTD (letters) and the primary ssDNA (long vertical line). Top, orientations as in a unless indicated otherwise. Bottom, close-up views rotated as shown. The number of particles and resolution, from the gold-standard refinement procedure, are indicated. k-l, Duplex tilts and S2-connected pairs (duplex^C series) of the 120-bp dsDNA reaction charted as in c-d. Charts are from data sets collected once.

Figure 2 |. Cryo-EM analysis of the strand exchange reaction with 67 bp dsDNA containing a 10-nt region of homology.

a, Semi-transparent maps of the unsharpened cryo-EM density from the consensus reconstruction (left), and of the masked heteroduplex density from the post-processed 2.4 Å map (right). The density and model of the complementary strand in the heteroduplex are in green, and the rest are colored as in Figure 1b. b, Number of particles that have 5’ (cyan) or 3’-tilted (purple-blue) tilted duplexes. c, Number of particles with S2-connected duplex pairs (duplex^C series). d-j, Representative 3D refined classes that contain patchy heteroduplex density between the indicated S2-connected duplex pairs. The density and schematics, drawn as in Figure 1e, now also show the complementary strand in green. k, Overall (center) and close-up views of the class containing a continuous D-loop spanning RecA^D-RecA^G after 3D refinement. Charts are from a single data set.

Figure 3 |. Structure of D-loop^DG.

a, Cartoon representation and sequence of D-loop^DG. Protein is yellow, DNA is colored as indicated (gray nucleotides in sequence are disordered), and ATPγS is in sticks. b, Close-up view of duplex^D stacking against L2^C. All L2 loop side chains are shown, but only those that contact the duplex are labeled. c, Close-up view of duplex^G stacking against L2^E, rotated ~180° from a. d, Superposition of the helicase cores of RecA^D and RecA^G highlighting differences in duplex^D and duplex^G tilts and L2 loops. Colored as in a, except the CTD domains and L2 loops are colored as their respective duplexes. Cartton is limited to the duplex-proximal DNA portions, and to only one set of RecA protomers outside RecA^D and RecA^G. e-f, Close-up views of the CTD-dsDNA contacts in the minor groove of the DNA. Green dotted lines indicate hydrogen-bond contacts. g, Left, overall view of the S2 ssDNA, colored as in a, except the S2 site β6 and L2 are orange. Right, close-up views of the interactions.

Figure 4 |. Structure of D-loop^DH.

a, Cartoon representation of D-loop^DH and its sequence. Coloring and view as in Fig. 3a, except for a −30° rotation about the horizontal axis. b, Closeup view of the S2 ssDNA and S2 protein residues as in Fig. 3g.