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-loop1,2. 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.

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Fig. 1: Cryo-EM analysis of the strand-exchange reactions with non-homologous dsDNA.
Fig. 2: Cryo-EM analysis of the strand-exchange reaction with 67-bp dsDNA containing a 10-nt region of homology.
Fig. 3: Structure of D-loopDG.
Fig. 4: Structure of D-loopDH.

Data availability

The refined coordinates and corresponding cryo-EM maps—including the consensus and focused reconstructions, composite maps used in refinement and the maps in Fig. 1e–j and 2d–k—have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under accession codes 7JY6 and EMD-22522 (strand exchange reactions containing non-homologous 120 bp dsDNA), 7JY8 and EMD-22524 (strand exchange reactions containing homologous 67 bp dsDNA), 7JY9 and EMD-22525 (D-loopDG complex) and 7JY7 and EMD-22523 (D-loopDH complex).

References

  1. 1.

    Bell, J. C. & Kowalczykowski, S. C. Regulation and mechanism of a molecular search engine. Trends Biochem. Sci. 41, 491–507 (2016).

    CAS  Google Scholar 

  2. 2.

    Cox, M. M. Motoring along with the bacterial RecA protein. Nat. Rev. Mol. Cell Biol. 8, 127–138 (2007).

    CAS  Google Scholar 

  3. 3.

    Chen, Z., Yang, H. & Pavletich, N. P. Mechanism of homologous recombination from the RecA–ssDNA/dsDNA structures. Nature 453, 489–494 (2008).

    ADS  CAS  Google Scholar 

  4. 4.

    Zlotnick, A., Mitchell, R. S. & Brenner, S. L. RecA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor. J. Biol. Chem. 265, 17050–17054 (1990).

    CAS  Google Scholar 

  5. 5.

    Mazin, A. V. & Kowalczykowski, S. C. The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange. Proc. Natl Acad. Sci. USA 93, 10673–10678 (1996).

    ADS  CAS  Google Scholar 

  6. 6.

    Wittung, P., Bazemore, L. R., Takahashi, M., Nordén, B. & Radding, C. Second-site RecA–DNA interactions: lack of identical recognition. Biochemistry 35, 15349–15355 (1996).

    CAS  Google Scholar 

  7. 7.

    Mazin, A. V. & Kowalczykowski, S. C. The function of the secondary DNA-binding site of RecA protein during DNA strand exchange. EMBO J. 17, 1161–1168 (1998).

    CAS  Google Scholar 

  8. 8.

    Story, R. M., Weber, I. T. & Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature 355, 318–325 (1992).

    ADS  CAS  Google Scholar 

  9. 9.

    Greene, E. C. DNA sequence alignment during homologous recombination. J. Biol. Chem. 291, 11572–11580 (2016).

    CAS  Google Scholar 

  10. 10.

    Aihara, H. et al. An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing. J. Mol. Biol. 274, 213–221 (1997).

    CAS  Google Scholar 

  11. 11.

    Kurumizaka, H., Ikawa, S., Sarai, A. & Shibata, T. The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing. Arch. Biochem. Biophys. 365, 83–91 (1999).

    CAS  Google Scholar 

  12. 12.

    Hsieh, P., Camerini-Otero, C. S. & Camerini-Otero, R. D. The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA. Proc. Natl Acad. Sci. USA 89, 6492–6496 (1992).

    ADS  CAS  Google Scholar 

  13. 13.

    Forget, A. L. & Kowalczykowski, S. C. Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature 482, 423–427 (2012).

    ADS  CAS  Google Scholar 

  14. 14.

    Qi, Z. et al. DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160, 856–869 (2015).

    CAS  Google Scholar 

  15. 15.

    Gourves, A. S., Defais, M. & Johnson, N. P. Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA. J. Biol. Chem. 276, 9613–9619 (2001).

    CAS  Google Scholar 

  16. 16.

    Cazaux, C. et al. Investigation of the secondary DNA-binding site of the bacterial recombinase RecA. J. Biol. Chem. 273, 28799–28804 (1998).

    CAS  Google Scholar 

  17. 17.

    Min, J. H. & Pavletich, N. P. Recognition of DNA damage by the Rad4 nucleotide excision repair protein. Nature 449, 570–575 (2007).

    ADS  CAS  Google Scholar 

  18. 18.

    Scrima, A. et al. Structural basis of UV DNA-damage recognition by the DDB1–DDB2 complex. Cell 135, 1213–1223 (2008).

    CAS  Google Scholar 

  19. 19.

    Jain, S. K., Cox, M. M. & Inman, R. B. Occurrence of three-stranded DNA within a RecA protein filament. J. Biol. Chem. 270, 4943–4949 (1995).

    CAS  Google Scholar 

  20. 20.

    Lee, A. J., Sharma, R., Hobbs, J. K. & Wälti, C. Cooperative RecA clustering: the key to efficient homology searching. Nucleic Acids Res. 45, 11743–11751 (2017).

    CAS  Google Scholar 

  21. 21.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  Google Scholar 

  22. 22.

    Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Google Scholar 

  23. 23.

    Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Google Scholar 

  24. 24.

    Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    CAS  Google Scholar 

  25. 25.

    Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015).

    CAS  Google Scholar 

  26. 26.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    CAS  Google Scholar 

  27. 27.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  Google Scholar 

  28. 28.

    Tran, Q. H. & Unden, G. Changes in the proton potential and the cellular energetics of Escherichia coli during growth by aerobic and anaerobic respiration or by fermentation. Eur. J. Biochem. 251, 538–543 (1998).

    CAS  Google Scholar 

  29. 29.

    Lee, J. Y., Qi, Z. & Greene, E. C. ATP hydrolysis promotes duplex DNA release by the RecA presynaptic complex. J. Biol. Chem. 291, 22218–22230 (2016).

    CAS  Google Scholar 

  30. 30.

    Menetski, J. P., Bear, D. G. & Kowalczykowski, S. C. Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc. Natl Acad. Sci. USA 87, 21–25 (1990).

    ADS  CAS  Google Scholar 

  31. 31.

    Rosselli, W. & Stasiak, A. Energetics of RecA-mediated recombination reactions: without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle. J. Mol. Biol. 216, 335–352 (1990).

    CAS  Google Scholar 

  32. 32.

    Stasiak, A. Z., Rosselli, W. & Stasiak, A. RecA–DNA helical filaments in genetic recombination. Biochimie 73, 199–208 (1991).

    CAS  Google Scholar 

  33. 33.

    Rosselli, W. & Stasiak, A. The ATPase activity of RecA is needed to push the DNA strand exchange through heterologous regions. EMBO J. 10, 4391–4396 (1991).

    CAS  Google Scholar 

  34. 34.

    Jain, S. K., Cox, M. M. & Inman, R. B. On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. III. Unidirectional branch migration and extensive hybrid DNA formation. J. Biol. Chem. 269, 20653–20661 (1994).

    CAS  Google Scholar 

  35. 35.

    Cox, M. M. Why does RecA protein hydrolyse ATP? Trends Biochem. Sci. 19, 217–222 (1994).

    CAS  Google Scholar 

  36. 36.

    Konforti, B. B. & Davis, R. W. The preference for a 3′ homologous end is intrinsic to RecA-promoted strand exchange. J. Biol. Chem. 265, 6916–6920 (1990).

    CAS  Google Scholar 

  37. 37.

    Konforti, B. B. & Davis, R. W. DNA substrate requirements for stable joint molecule formation by the RecA and single-stranded DNA-binding proteins of Escherichia coli. J. Biol. Chem. 266, 10112–10121 (1991).

    CAS  Google Scholar 

  38. 38.

    Kurumizaka, H. et al. A possible role of the C-terminal domain of the RecA protein. A gateway model for double-stranded DNA binding. J. Biol. Chem. 271, 33515–33524 (1996).

    CAS  Google Scholar 

  39. 39.

    Aihara, H., Ito, Y., Kurumizaka, H., Yokoyama, S. & Shibata, T. The N-terminal domain of the human RAD51 protein binds DNA: structure and a DNA binding surface as revealed by NMR. J. Mol. Biol. 290, 495–504 (1999).

    CAS  Google Scholar 

  40. 40.

    Conway, A. B. et al. Crystal structure of a Rad51 filament. Nat. Struct. Mol. Biol. 11, 791–796 (2004).

    CAS  Google Scholar 

  41. 41.

    Short, J. M. et al. High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy. Nucleic Acids Res. 44, 9017–9030 (2016).

    ADS  CAS  Google Scholar 

  42. 42.

    Xu, J. et al. Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange. Nat. Struct. Mol. Biol. 24, 40–46 (2017).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff of the MSKCC cryo-EM facility, the NYSBC Simons Electron Microscopy Center and the HHMI cryo-EM facility for help with data collection. This work was supported by the HHMI and by National Institutes of Health grant CA008748.

Author information

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Authors

Contributions

H.Y. designed and carried out the DNA binding experiments, prepared RecA mini-filaments and strand-exchange reactions, and collected, processed and analysed the cryo-EM data; C.Z. carried out the initial cryo-EM characterization of the RecA mini-filaments; A.D. carried out the DNA-binding experiments, prepared RecA proteins and strand-exchange reactions and collected cryo-EM data; and N.P.P. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Nikola P. Pavletich.

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The authors declare no competing interests.

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Peer review information Nature thanks Charles Bell, Eric Greene and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

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 analogues30,31,32. With longer, physiologically relevant substrates, however, ATP hydrolysis is needed for bypassing heterology and for the extension of initial joint molecules, or branch migration33,34. 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 cf 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 Fig. 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 9-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 before polishing resulted in similar classes, except for classes with low-quality 2D projections that were discarded. d, Left, 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 coloured by local resolution estimated with the RELION3 post-processing program. The resolution range is mapped to the colours 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 Fig. 1a, primary ssDNA is coloured in brown, S2 ssDNA is in red. The 9-RecA protein is coloured uniformly khaki for simplicity. Fourth and fifth, cartoon representations of duplexes A to I in the 5′- and 3′-tilt conformations, respectively coloured 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 labelled 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 CTDA, and for the same reason the penultimate CTDB 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 RecAI was overall poorly ordered, duplexI-containing particles could readily be identified, as CTDI points towards the mid-portion of the filament, and its density is considerably better defined than that of CTDA and CTDB that point away from the 3′ end of the filament. Masks and maps of the 3D classification for all 28 combinations of duplex pairs are shown in Supplementary Fig. 2 and their details are listed in Supplementary Data 2. f, Histogram of the number of duplexes per particle. The chart shows the percentage of particles that have the indicated number of duplexes for this dataset. The dataset was collected once. Also see Supplementary Data 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 CTDA to CTDC point into empty solvent and the dsDNA can approach from a roughly half-spherical volume (left). CTDA is even more accessible as it has no neighbouring RecA 3′ to it. Moving towards the 5′ end, the CTDs become increasingly encumbered by the presence of RecA protomers 3′ to them. Thus, CTDD becomes slightly hindered by the RecAA L2 loop that is 47 Å away (right; Cα–Cα distance from CTD Gly288 to L2 loop Gly200 in a direction that would approximately bisect the axis of dsDNA). CTDE is more encumbered because, in addition to the RecAB L2 loop, it is within 35 Å of the RecAA L1 loop (right panel; Cα-Cα distance from CTD Gly288 to L1 loop Glu158). Additionally, CTDF is obstructed not only by the RecAC L2 and RecAB L1 loops, but also by the helicase domain of RecAA, which is within 35 Å (right; Cα–Cα distance from CTD Gly288 to helicase Ala131). CTDG and CTDH are encumbered the most, by the full turn of filament 3′ to them (left). Their DNA-binding tip is 28 Å away from the N-terminal helices of RecAA and RecAB, respectively (Cα–Cα distance from CTD Gly288 to Lys19 of αN), a distance that is only fractionally larger than the approximately 20 Å width of a DNA duplex. The terminal CTDI is similarly close to the N-terminal helix of RecAC, although the absence of a 5-neighbouring RecA would substantially increase its accessibility to dsDNA compared to those of CTDG and CTGH. The figure shows the molecular surface of the 9-RecA filament with the aforementioned structural elements coloured for each RecA as in Fig. 1a and labelled. 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 coloured brown. The homologous ssDNA is not shown for clarity. The view in the right panel is rotated by 180°, roughly half a turn of the filament, about the vertical axis to show the environment of CTDD, CTDE and CTDF that are obscured in the left view.

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

ad, Select multi-duplex combinations containing duplexC 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 FSC procedure are shown on top of each map. Maps are coloured by the duplex tilt (5′ tilt, cyan; 3′ tilt, purple), with S2 DNA in red. Duplex positions are labelled. 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 Fig. 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 duplexH or duplexI. Top, maps of classes with short duplexes. Bottom, maps of classes from the same 3D classification but with regular, long duplexes. The black circle highlights the difference in duplex length. Maps are coloured by the duplex tilt (5′, cyan; 3′, purple), with S2 DNA in 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 first duplex in common. Pairs of duplexes starting at RecAC are omitted, as they are shown in Fig. 1d. The dataset 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 RecAC are omitted as they are shown in Fig. 1l. The dataset was collected once.

Extended Data Fig. 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 dataset. b, Starting from the left, consensus reconstruction map of this dataset coloured by local resolution estimated with the RELION3 post-processing program. The resolution range is mapped to the colours in the inset below the map. Next, cartoon representation of the refined model of the consensus refinement. Primary ssDNA is coloured in brown, S2 ssDNA red, and RecA is khaki. Cartoon representation of duplexes A to I in the 5′-tilted (cyan) and 3′-tilted (purple) conformations, 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 duplexF and duplexG 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, duplexB and duplexI 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 combinations of duplex pairs are shown in Supplementary Fig. 3 and their details listed in Supplementary Data 4. Paired-duplex 3D classification showed a distribution that is 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 dataset was collected once. Also see Supplementary Data 3 for more details.

Extended Data Fig. 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 coloured green and red for the complementary and homologous strands, respectively. The directions of the DNA strands, and every tenth nucleotide are labelled. 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 combinations of duplex pairs are shown in Supplementary Fig. 4 and their details listed in Supplementary Data 6. d, Chart of the percentage of particles that have the indicated number of duplexes for this dataset. The dataset was collected once. Also see Supplementary Data 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 RecAC are shown in Fig. 2c. See Supplementary Data 6. The dataset was collected once. f, Reconstruction after 3D refinement of the minor class from the classification of the 5′/3′ tilted duplexD–duplexG 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 owing to limited numbers of particles. This suggests that the D-loop forms preferentially in the 5′/3′-tilt conformation. h, Reconstruction after 3D refinement of the major class from the 3D sub-clasification of the 5′/5′ tilted duplexD–duplexG 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 owing to extensive heterogeneity and limited numbers of particles.

Extended Data Fig. 7 Flowcharts of focused reconstructions for the datasets with partially homologous 67-bp dsDNA, non-homologous 120-bp dsDNA, D-loopDG and D-loopDH.

ad, Data processing of partially homologous 67-bp dsDNA reaction (a), non-homologous 120-bp dsDNA reaction (b), 9-RecA–D-loopDG complex (c) and 9-RecA–D-loopDH complex (d). The consensus reconstruction map and the two focused refinement maps for each dataset are coloured by local resolution estimated with the RELION3 postprocess program. The resolution range is mapped to the colours 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, labelled 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, labelled 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 grey curve, labelled 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 Fig. 8 Models and density of D-loopDG and D-loopDH.

Discussion of RecA–DNA contacts. The L2 loop-duplex contacts involve non-equivalent RecA protomers owing to the different duplex orientations. Thus, duplexD abuts the L2 loop of the adjacent RecAC and additionally stacks with Gly204 backbone atoms, whereas duplexG abuts the L2 loop of RecAE, 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 duplexG 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 duplexD and 14.6 Å for duplexG. 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 (duplexD) or guanidine N3 (duplexG) 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 snugly 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 (duplexD) or guanine (duplexG). 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 duplexG, but the corresponding contact is not made to duplexD, which is farther away owing to its 5′ tilt. Among the CTD–duplex contacts, the K286N and K302N mutations were shown to cause defects in UV-damage repair in vivo and in binding to and pairing with dsDNA, although they were interpreted as affecting the secondary DNA-binding activity38. The S2 site contacts to the homologous strand are overall more extensive and the density stronger at the duplexD-proximal two thirds of the strand than near duplexG. The contacts start immediately after the opening of duplexD by L2C (these Ade28 contacts are discussed in the main text). The base group of the next residue, Cyt27, is sandwiched between the L2C Met202 and L2D Pro206 side chains, while its ribose group packs against the E207D–R226D salt bridge (Fig. 3g, bottom). Cyt26 then packs on one side with the L2D 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 β6E (Fig. 3g, middle). The next two nucleotides stack together and as a pair fit snugly into a tight gap between the backbones of β6E and L2D, as if they are pinched (Fig. 3g, middle). Here, β6E side chain and backbone groups of Ile228 and Gly229 pack with Cyt24, while L2D backbone groups from Phe203 and Gly204 pack with Cyt23. In addition, the phosphodiester group of Cyt24 hydrogen-bonds to the backbone amide of L2D Asn205, and that of Cyt23 to the side chain of β6E Arg226. Thereafter, Cyt22 has RecA contacts and relative position very similar to that of Cyt27, five nucleotides away in the 3′ filament direction. The one difference is the Cyt27–L2C Met202 packing is replaced by Cyt22–L2D Phe203, owing to the alternate conformation that L2C adopts as it book-ends duplexD (Fig. 3g, top). The Cyt22 base is also within contact distance of β6E Lys245, where the K245N mutation was reported to affect homologous pairing11. The D-loopDH structure recapitulates the key aspects of D-loopDG. These include the conformations of the L2C and L2F loops and their stacking with their respective duplexD and duplexH, 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-loopDG, whereas 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 duplexH. The D-loopDH 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 duplexD (Ade31) packs with L2C, while its phosphodiester backbone is contacted by Arg226 of β6D. The opposing, flipped-out base of the complementary strand has poor density and does not seem to make any RecA contacts. And, as with D-loopDG, the three nucleotides just before duplexH (Thy20-Thy19-Ade18) are poorly ordered, and make few contacts as they follow an alternative path around their respective L2F. 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 RecAA, RecAB, RecAC, RecAE, RecAF, RecAH, and RecAI CTD domains (grey) do not exhibit any conformational changes, whereas those of RecAD and RecAG rotate (curved arrow) in opposite directions, by −3° and 13° respectively. b, Cartoon representation showing the superposition of RecAD and RecAG on their CTD domains to highlight the different tilts of duplexD and duplexG relative to their already rotated CTD domains. Coloured as in Fig. 3d (which shows the superposition of the RecA protomers on their helicase domains). The rest of the RecAs are coloured yellow and grey. c, d, Density of duplexD and duplexG from the D-loopDG maps used in REFMAC5, as described in Methods, in the same orientation as Fig. 3b, c. e, f, Density of the interactions of duplexD and duplexG with their respective CTDs from the D-loopDG REFMAC5 refinement the same orientation as Fig. 3e, f. g, Density of the S2 site structural elements and all DNA from the D-loopDG maps used in REFMAC5. Orientations as in Fig. 3g. h, Density of the DNA only from D-loopDH maps used in REFMAC5 in the same orientation as Fig. 4b. RecA protomers and their density are omitted for clarity.

Extended Data Fig. 9 RAD51, the eukaryotic RecA homologue, probably 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 domain39. Although the RAD51 NTD is structurally unrelated to the RecA CTD, it occupies an analogous position at the filament periphery40,41,42, 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, A side-by-side comparison of the RecA D-loopDG 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 3-RAD51 segment bound to 9 nt of ssDNA (PDB: 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 a root-mean-square deviation (r.m.s.d.) of 1.9 Å for 201 of 245 RecA Cα atoms, three-protomer segments superimpose with an r.m.s.d. of 2.2 Å for 531 Cα atoms). For the side-by-side comparison, the two filaments were superimposed on the central, 3-protomer segment (RecAD–RecAF). The proteins and cofactors are coloured uniformly grey, except their respective CTD and NTD domains are coloured rainbow as in Fig. 1a, the primary ssDNA is brown for both, and the rest of the RecA DNA molecules are coloured as in Fig. 3a. The NTD and CTD domains are labelled. b, Magnified views of the RecA DloopDG and the 9-RAD51 model superimposed on the three 3′-most RecAA–RecAC, focusing on the RecA duplexG in an orientation similar to a (right view rotated 90° about the vertical axis). This superposition brings duplexG of RecA in close proximity to the RAD51 NTDA, which is nearly one turn of the filament away (in the 3′ direction) from CTDG owing to the different locations of the RAD51 NTDs. The backbone amide nitrogen atoms reported to be involved in dsDNA binding39 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 duplexG of the superposition.

Extended Data Table 1 Cryo-EM data collection, model refinement and validation statistics

Supplementary information

Supplementary Information

Supplementary Discussion: Additional discussion of 3D classification of particles containing duplex pairs, and of dsDNA opening.

Reporting Summary

Supplementary Figures

This file contains Supplementary Figures 1-4.

Supplementary Data

Supplementary Data 1: Excel spreadsheet with the pattern of duplexes of the 67-bp non-homologous dsDNA strand exchange reaction.

Supplementary Data

Supplementary Data 2: Excel spreadsheet with a summary of 5’- and 3’-tilt of duplexes, and of whether the pairs are connected by S2 density from 3D classification of the 67-bp non-homologous dsDNA strand exchange reaction. First page of the spreadsheet is the analysis of only S2-connected pairs, used to make Figure 1d and Extended Data Figure 4b. Second page of the spreadsheet is the analysis of all duplex pairs and whether the duplexes have 5’- or 3’-tilts, used to make Figure 1c.

Supplementary Data

Supplementary Data 3: Excel spreadsheet with the pattern of duplexes of the 120-bp non-homologous dsDNA strand exchange reaction.

Supplementary Data

Supplementary Data 4: Excel spreadsheet with a summary of 5’- and 3’-tilt of duplexes, and of whether the pairs are connected by S2 density from 3D classification of the 120-bp non-homologous dsDNA strand exchange reaction. First page of the spreadsheet page is the analysis of only S2-connected pairs, used to make Figure 1l and Extended Data Figure 4c. Second page of the spreadsheet is the analysis of all duplex pairs and whether the duplexes have 5’- or 3’-tilts, used to make Figure 1k.

Supplementary Data

Supplementary Data 5: Excel spreadsheet with the pattern of duplexes of the 67-bp partially-homologous dsDNA strand exchange reaction.

Supplementary Data

Supplementary Data 6: Excel spreadsheet with a summary of 5’- and 3’-tilt of duplexes, and of whether the pairs are connected by S2 density from 3D classification of the 67-bp partially-homologous dsDNA strand exchange reaction. First page of the spreadsheet page is the analysis of only S2-connected pairs, used to make Figure 2c and Extended Data Figure 6e. Second page of the spreadsheet is the analysis of all duplex pairs and whether the duplexes have 5’- or 3’-tilts, used to make Figure 2b.

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Yang, H., Zhou, C., Dhar, A. et al. Mechanism of strand exchange from RecA–DNA synaptic and D-loop structures. Nature 586, 801–806 (2020). https://doi.org/10.1038/s41586-020-2820-9

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