The transfer of genetic information between DNA molecules promoted by homologous recombination (HR) is essential in the repair of DNA double-strand breaks (Kowalczykowski, 2015; Wright et al., 2018) and to overcome replicative stress during chromosomal DNA synthesis (Chakraborty et al., 2023; Triplett et al., 2024). The molecular apparatus of HR is also responsible for the crossover reactions that generate genetic diversity in meiosis (Brown and Bishop, 2014). HR deficiency causes genetic instability that predisposes to cancer (Triplett et al., 2024; Prakash et al., 2015; Matos-Rodrigues et al., 2021), and its synthetic lethality with other DNA repair pathways is exploited to induce cancer cell death in therapy (Lord and Ashworth, 2017; Dibitetto et al., 2024).

In eukaryotic cells, the reaction of HR is performed by RAD51 (Baumann et al., 1996; Sung, 1994), a member of a highly conserved family of ATPases that includes bacterial RecA and archaeal RadA (Lin et al., 2006; Chintapalli et al., 2013). RAD51 polymerises on the single-stranded (ss) DNA overhangs that form upon long-range resection of DNA ends (Cejka and Symington, 2021). The RAD51 nucleoprotein filament invades the donor DNA duplex and searches for sequence homology within a three-stranded synaptic filament intermediate (Renkawitz et al., 2014; Greene, 2016). When homology is found, the invading strand of the filament becomes stably paired to the complementary sequence of the donor DNA, yielding a structure known as a displacement loop (D-loop) (Shibata et al., 1979; Jain et al., 1995; Piazza et al., 2019; Figure 1A), which ultimately progresses to strand exchange. Although cellular HR is controlled by a complex network of recombination mediators such as BRCA2 (Bell et al., 2023), the RAD51 paralogues (Greenhough et al., 2023; Taylor et al., 2015; Rawal et al., 2023), and FIGNL1 (Ito et al., 2023; Carver et al., 2025), RAD51 can catalyse the biochemical reaction of strand exchange independently.

Figure 1 with 6 supplements see all

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The RAD51/Reca/RadA protein family has been extensively investigated to elucidate the mechanistic basis for the reaction of strand exchange. Low-resolution electron microscopy (EM) and X-ray crystallography have demonstrated conservation in the helical parameters of the nucleoprotein filament (Ogawa et al., 1993; Yu et al., 2001), the structure of the ATPase domain (Story et al., 1992; Pellegrini et al., 2002), and the protomer self-association into a filament (Conway et al., 2004; Wu et al., 2004). CryoEM analysis of the RAD51 nucleoprotein filament has revealed further conservation with RecA in the mode of interaction with DNA, such as the observation that DNA stretching results from its separation into B-form nucleotide triplets (Short et al., 2016; Xu et al., 2017; Appleby et al., 2023a; Chen et al., 2008). Furthermore, single-molecule experiments have determined the kinetic parameters of filament nucleation and growth (Galletto et al., 2006; Candelli et al., 2014), have shown that a minimum length of eight homologous nucleotides is required for the formation of stable synaptic joints (Hsieh et al., 1992; Qi et al., 2015) and that homologous pairing takes place in steps of three nucleotides (Ragunathan et al., 2011; Lee et al., 2015).

High-resolution information about the RAD51 synaptic state is still missing, limiting our mechanistic understanding of the strand-exchange reaction. In this study, we have carried out the biochemical reconstitution, cryoEM structure, and biophysical validation of a D-loop of human RAD51. The structure elucidates the key steps of strand exchange, including the mechanism of filament engagement with the donor DNA, strand invasion and pairing with the complementary strand, and capture of the exchanged strand. Comparison with the synaptic state of RecA filaments (Yang et al., 2020) reveals both conserved and distinct features of the strand-exchange reaction, shedding light on the molecular evolution of the biochemical reaction of HR.

EM analysis of filamentous RAD51 relies normally on helical averaging, which is unsuitable for reconstruction of an aperiodic structure such as a D-loop. We therefore sought to prepare a D-loop sample of human RAD51 that is amenable to single-particle cryoEM. We designed a 32-nucleotide (nt) poly(dT) with a central, unique 10-nt-long sequence complementary to a 50-nt donor double-stranded (ds) DNA; the complementary sequence of the donor DNA was incorporated in a 10-nt mismatch bubble to favour D-loop formation (Figure 1—figure supplement 1A and Supplementary file 1). The length of the complementary sequence was decided based on evidence that RAD51 requires a homologous tract of at least eight nucleotides for stable capture of the donor DNA (Qi et al., 2015). As RAD51 can bind both ss- and dsDNA, we defined experimentally appropriate stoichiometry and buffer conditions for D-loop reconstitution. Electrophoretic mobility shift assay (EMSA) with fluorescently labelled oligos showed successful D-loop formation (Figure 1—figure supplement 1B and Supplementary file 1).

We collected an initial cryoEM dataset of 2792 movies of the RAD51 D-loop sample and processed the data by single-particle analysis. 2D classification showed clear evidence of the presence of synaptic particles and yielded a D-loop reconstruction at 3.31 Å resolution (Figure 1—figure supplement 2 and Supplementary file 2). To improve data homogeneity and resolution, we collected a larger dataset of 8960 movies of a D-loop sample with ss- and dsDNA that had been biotinylated at both ends and capped with monomeric streptavidin (Figure 1—figure supplement 1C; Appleby et al., 2023b), yielding a structure of the D-loop at 2.64 Å (Figure 1—figure supplement 3 and Supplementary file 2). The final single-particle reconstruction based on 102,596 particles comprises a nucleoprotein filament of 9 RAD51 protomers and 26 nucleotides of ssDNA, bound to a dsDNA containing the central bubble region flanked by 15- and 16-bp-long segments of donor DNA. The cryoEM map ranges in resolution between 2.5 Å and 2.9 Å for the homologous pairing region of the D-loop and 3 Å and 6 Å for the flanking dsDNA arms (Figure 1B).

Unwinding and strand invasion of donor DNA

Biochemical and structural analyses of pre- and post-synaptic filaments have revealed that RAD51 loops L1 and L2 mediate DNA binding, with prominent roles for amino acids L1 R235 and L2 V273 in partitioning filament DNA into nucleotide triplets (Xu et al., 2017; Matsuo et al., 2006). However, L2 residues Q272 to P283 remained disordered in these filament structures (Figure 2A).

Figure 2

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In our D-loop structure, the L2 sequences of four contiguous RAD51 protomers – E, F, G, and H (Figure 2B) – have become ordered and traceable in the density map, folding in random-coil conformation with 3₁₀-helix elements. The structure shows that filamentous RAD51 achieves strand separation by sequential L2-loop insertion of adjacent RAD51 protomers between the strands of the donor DNA (Figure 2C and D). Insertion of the RAD51-H L2 loop at the 5’-arm junction causes a drastic widening of the minor groove, progressing to full separation of the complementary and exchanged strands upon L2-loop insertion by RAD51-G (Figure 2C, D, G, and H). In total, donor DNA unwinding by RAD51-H and -G exposes a continuous csDNA stretch of five nucleotides for homologous pairing: A22-C25 of csDNA base pair with G9-T12 of isDNA, physically organised as a base-pair triplet of (A22-A24)_cs with (T10-T12)_is and one unstacked C25_cs:G9_is base pair, while G26_cs remains unpaired (Figures 1F, 2C, D, G, and H).

Successive L2-loop insertions extend the unwinding of donor DNA, with each insertion exposing a triplet of csDNA nucleotides for homology pairing with the invading strand (Figure 2C). Thus, L2-loop insertions by RAD51-F and -E present (A19-C21)_cs for base pairing with (G13-T15)_is, and (G16-T18)_cs for base pairing with (A16-T18)_is, respectively (Figure 2C–F). After completion of homologous pairing, the strands of the donor DNA rejoin in double-helical form at the 3’-arm junction (Figures 1G and 2C).

The L2 loop acts as a physical spacer, directly separating the unwound strands of the donor DNA. Its sequence is dominated by hydrophobic amino acids (Figure 2A) that make extensive contacts with the phosphoribose backbone of the esDNA and culminate in stacking of F279’s phenylalanine side chain against the last base pair of the 5’-arm duplex (RAD51-H) or the aromatic bases of nucleotides in the exchanged strand (RAD51-G, F, E) (Figure 2E–H). The L2 loop displays a considerable degree of conformational heterogeneity, likely because each loop engages in both common and unique contacts with the DNA. This is best exemplified by the L2 loops of RAD51-E and -H that interact with the duplex arms of the donor DNA in different ways. RAD51-H L2 initiates donor DNA unwinding by buttressing the F279 side chain against G27_cs:C15_is, the last base pair of the 5’-arm duplex (Figure 2H). Conversely, contact with RAD51-E L2 does not alter the conformation of the 3’-arm, as its F279 side chain stacks against C26 of the exchanged strand (Figure 2E).

The asymmetric binding of RAD51-H and -E L2 loops at the boundaries between the arms of the donor DNA and the three-stranded synaptic DNA suggests that strand invasion and pairing follows a preferred direction. Thus, invasion by filament DNA likely begins at the 3’-end of the homology region in the complementary strand of the donor DNA and extends in the 5’-direction, as incrementally more nucleotides of csDNA are exposed by L2-loop insertions and become available for homologous pairing.

Capture of the exchanged strand

Strand invasion leads to displacement of the exchanged strand in the donor DNA. The structure of the D-loop shows that the esDNA is sequestered in a basic channel running parallel to the ssDNA-binding groove of the RAD51 nucleoprotein filament (Figure 3A and B). One side of the channel is formed by RAD51 residues 303–313 (Figure 3C), that fold in a beta hairpin on the inside of the filament groove, whereas the opposite side is formed by the C-terminal half of the L2-loop sequence (Figure 3B and D). In the filament, the beta hairpins of successive protomers align to generate a continuous ssDNA-binding stripe (Figure 3D). The esDNA is bound in the basic channel so that its aromatic bases remain largely exposed to solvent whereas the phosphodiester backbone faces the protein (Figure 3B and D).

Figure 3

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Three adjacent RAD51 protomers – F, G, and H – engage the exchanged strand in the D-loop (Figure 3D). The majority of contacts comprise charged interactions between four invariant RAD51 residues R303, K304, R306, and K313 and the phosphate groups of the esDNA (Figure 3E–G). In addition to the electrostatic interactions, R306 is part of a GRG motif at the tip of the beta hairpin that comes into close contact with the phosphoribose backbone of the esDNA and makes stacking interactions with the aromatic bases of nucleotides A19_es and T25_es in RAD51-H and -G, respectively (Figure 3E and F). The interaction of the 305-GRG-307 motif with esDNA resembles that of L1-loop 234-GRG-236 with csDNA in the homologous duplex of the D-loop.

Of the three RAD51 protomers F, G, and H that bind the esDNA, RAD51-G makes the most extensive set of contacts, spanning five nucleotides, from A21_es to T25_es, involving all four basic residues R303, K304, R306, and K313 (Figure 3F). Binding of RAD51-G to the esDNA is further strengthened by phosphate-group contacts of L2-loop K284 with C22_es and of R130 with C23_es and T24_es. RAD51-H contacts four nucleotides, from A17_es to A20_es, with R303, K304, and 305-GRG-307 (Figure 3E), whereas RAD51-F makes a single long-range interaction between K313 and T25_es (Figure 3G). In addition, R303 and R306 of RAD51-F engage the phosphate groups of T27, A28, and T29 of the 3’-arm duplex, possibly to guide the 3’-arm in the appropriate direction as it exits the filament groove, towards the RAD51-A NTD (Figure 3G).

In total, the filament interacts with 9 of the 11 nucleotides of unpaired esDNA via contiguous interactions of the two RAD51 protomers G and H (5 and 4 nt, respectively). This compares with donor DNA opening by L2-loop insertion, which progresses by exposure of three nucleotides per RAD51 protomer.

Filament binding of donor DNA arms

The D-loop structure shows that the nucleoprotein filament interacts with the double-stranded arms of the donor DNA via the N-terminal domains of RAD51 protomers A and D (Figure 4A and B). Although the position of the RAD51-A and -D NTDs relative to the donor DNA is broadly similar, their DNA-binding interfaces differ significantly in the extent and mode of their engagement with the 3’- and 5’-arm duplexes, respectively.

Figure 4

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The RAD51-D NTD engages the minor groove of the 5’-arm DNA (Figure 4C). The side chains of K39, K40, and K64 contact the phosphate groups of 10-GCTC-13 and 34-CG-35 in the upper and lower rungs of the minor groove, respectively, while K70 and K73 contribute long-range electrostatic interactions to the binding (Figure 4C and E). The association of RAD51-D NTD with 5’-arm is closer and more extensive than that of RAD51-A with the 3’-arm, and includes a direct hydrogen bond between the main-chain nitrogen of G65 and the phosphate group of T12 (Figure 4C and E). In contrast, RAD51-A NTD binds in the major groove of the 3’-arm DNA, using electrostatic interactions mediated by the same set of lysine residues as in RAD51-D (Figure 4D). However, the RAD51-A association with the 3’-arm involves a more limited set of direct contacts with the phosphate groups of C5 and 31-TTG-33 across the major groove (Figure 4D and E).

The interaction of the NTD with the arms of the donor DNA highlights a lack of direct contacts with the bases and the predominantly electrostatic nature of the association. Such plasticity in the NTD-DNA interaction might be required for effective D-loop formation, as the direction at which the 3’-arm exits the filament upon D-loop formation is a function of the number of nucleotides involved in homologous pairing.

Structure-based mutagenesis of RAD51 residues important for D-loop formation

The atomic model of the RAD51 D-loop has revealed several new protein-DNA interfaces, including those involved in strand separation, capture of the exchanged strand, and anchoring of the flanking DNA arms to the filament. To assess the relative importance and contribution of RAD51 residues involved in D-loop formation, we performed alanine mutagenesis of F279 (strand separation); R303, K304, R306, K313 (esDNA capture); K39, K40, K64, K70, and K73 (arm duplex binding) (Figure 5A).

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The designed mutations are distant from the binding site of ssDNA in filamentous RAD51 and therefore are not predicted to reduce the ability of the mutant protein to form nucleoprotein filaments on ssDNA. As expected, EMSA analysis showed that the F279 and NTD mutants retained wild-type ability to form filaments on ssDNA (Figure 5—figure supplement 1A). However, RAD51 mutants R303A, K304A, R306A, and K313A showed a clear decrease in ssDNA binding (Figure 5—figure supplement 1A), suggesting that, in addition to their role in esDNA capture, they might mediate the initial contact of the incoming ssDNA during pre-synaptic filament nucleation. All RAD51 mutants showed a moderate decrease in affinity for dsDNA, with the exception of K304 (Figure 5—figure supplement 1B).

We evaluated the ability of the alanine mutants to perform DNA-strand exchange using an in vitro assay that measures the increase in fluorescent signal when a fluorescently labelled 60-nt oligo that is quenched in duplex form is strand-exchanged with the unlabelled sequence (Figure 5B; Huang et al., 2011). The results of the strand-exchange assay revealed that all mutants decreased the efficiency of the reaction, but with varying degrees of impairment (Figure 5B). The L2-loop F279A showed a modest impact, in agreement with earlier findings (Matsuo et al., 2006), whereas the esDNA-capture mutants R303A, K304A, R306A, and K313A displayed a more pronounced effect. Notably, the NTD mutations showed the largest negative impact on strand exchange: the double lysine-to-alanine substitutions of K39, K40 and K70, K73 reduced strand exchange to a fraction of the yield for the wild-type protein.

To provide further support for the role of the RAD51 NTD in D-loop formation, we developed a single-molecule D-loop assay using fluorescence imaging with optical tweezers (Heller et al., 2014). In the assay, RAD51 filaments were reconstituted on an Alexa 488-labelled 49-nt ssDNA designed to anneal at position 10.6 kb of λ DNA (Figure 5C). The filaments interacted with doubly biotinylated λ DNA held between streptavidin-coated beads and D-loop formation was monitored by the presence of a fluorescent signal at the expected locus on λ DNA. Wild-type RAD51 filaments were capable of D-loop formation at the expected site, as assessed by kymograph and binding events analysis (Figure 5D and E and Figure 5—figure supplement 2). In contrast, filaments of the KK39,40AA RAD51 double mutant failed to show measurable binding events; a fivefold increase in the concentration of the double mutant protein showed non-specific binding and no stable D-loop formation at the expected λ DNA site (Figure 5E and Figure 5—figure supplement 2).

These findings validate our model for the D-loop structure and highlight the important role of the RAD51 NTD in the strand-exchange reaction, by anchoring the donor dsDNA to the nucleoprotein filament in the correct orientation for the strand invasion and homology search. The biochemical behaviour of our esDNA-capture site mutants agrees with the impaired D-loop formation of a yeast RAD51 protein bearing alanine mutations at amino acids corresponding to R130, K303, and K313 of human RAD51 (Cloud et al., 2012).

The architecture of the RAD51 D-loop structure reveals the mechanism of strand invasion and homology search within a synaptic RAD51 filament. The close engagement of the RAD51-D NTD with the 5’-arm duplex, coupled to minor-groove widening by the RAD51-H L2 loop that initiates DNA unwinding, indicates that synapsis formation starts by interaction of the filament with the 5’-arm of the donor DNA. In comparison, the interaction of the RAD51-A NTD with the 3’-arm is more limited, and insertion of the L2 loop of RAD51-E does not remodel the proximal end of the 3’-arm duplex. Furthermore, the first isDNA:csDNA base pair formed at the 3’-arm junction is a T:G mismatch that is unlikely to stabilise the initial strand pairing.

Our findings form the basis for a mechanistic model of strand exchange by a RAD51 filament (Figure 5F). In the model, RAD51-D NTD binding directs the donor duplex towards the filament groove, where L2-loop insertions by RAD51-H and -G initiate strand separation and capture of the exchanged strand. Such unwinding exposes five nucleotides of csDNA, of which four are available for pairing with the invading strand of the filament. Initial base pair formation in the presence of homology would drive further donor DNA unwinding by L2-loop insertions of RAD51-F and -E; each insertion exposes a triplet of csDNA nucleotides, in agreement with experimental evidence that homologous pairing progresses in triplet steps (Qi et al., 2015; Lee et al., 2015). Concomitant capture of the exchanged strand by RAD51-G after insertion of a third L2 loop by RAD51-F would lead to sequestration of nine esDNA nucleotides, contributing to the formation of a stable synaptic intermediate.

Successful homology pairing drives the engagement of the 3’-arm of the donor dsDNA with the NTD of the RAD51 molecule three protomer positions away towards the 3’-end of the filament, completing D-loop formation. The observed plasticity in the interaction of the NTD with dsDNA would facilitate capture of the 3’-arm DNA emerging at slightly different angles from the filament axis, depending on the exact number of paired nucleotides. Overall, the structural features of the RAD51 D-loop provide a strong indication that strand pairing and exchange begins at the 3’-end of the complementary strand in the donor DNA and progresses with 3’-to-5’ polarity (Figure 5F).

The observation of an unrealised base pair involving G26_cs at the 3’-end of the unwound csDNA, which was designed to base pair with C8_is, indicates that the mechanism of unwinding of the 5’-arm prevents base pairing of the first available csDNA nucleotide, likely due to physical constraints. Further unwinding of the 5’-arm to allow G26_cs pairing was not observed, possibly because the presence of three consecutive G:C base pairs in the 5’-arm duplex acted as a GC clamp.

The presence of a T18_is:G16_cs mismatch as the last base pair at the 5’-end of the paired csDNA is intriguing (Figure 1—figure supplement 5). Of note, T18_is and G16_cs do not form a ‘wobble’ base pair (Hunter et al., 1987), indicating that fulfilment of hydrogen bonding potential is not critical. Rather, mismatch pairing that completes a base-pair triplet might occur regardless of perfect homology, to achieve a full complement of base stacking and protein-DNA interactions with RAD51 L1 and L2 loops. This observation provides a potential mechanism for how RAD51 can tolerate mismatches at certain positions of the homologous sequence during strand exchange.

The observed 3’-to-5’ polarity of exchange of the complementary strand by RAD51 is opposite to the 5’-to-3’ polarity of bacterial RecA (Figure 5—figure supplement 3), which was determined based on cryoEM structures of RecA D-loops (Yang et al., 2020). Comparison of RAD51 and RecA D-loop structures reveals that their different polarity follows from the opposite position occupied by their dsDNA-binding domains in the filament: in a vertically oriented filament with the 5’-end of the ssDNA at the top and the 3’-end at the bottom, the RAD51 NTDs line the bottom of the filament groove whereas the RecA CTDs hang from the top (Figure 5—figure supplement 4). The shared mode of minor-groove binding by RAD51-NTD and RecA-CTD and L2-loop insertion into the minor groove half a turn away leads naturally to opposite polarity in the progression of strand invasion and pairing with the complementary strand of the donor DNA. Our mechanistic interpretation of static D-loop structures awaits full reconciliation with earlier efforts to determine strand-exchange polarity for RecA and RAD51 measured under a variety of experimental conditions (Gupta et al., 1998; Baumann and West, 1997; Sung and Robberson, 1995; Murayama et al., 2008).

Altogether, the available evidence suggests that the ability to perform DNA-strand exchange by members of the RecA-family of recombinases results from a process of convergent evolution (Figure 5—figure supplement 5). Thus, an ancestral RecA-fold ATPase protein might first have evolved to bind ssDNA using the L1 and L2 loops, possibly as a means of protecting the exposed DNA template during DNA replication. Independent acquisition of dsDNA binding by RecA and RAD51 via incorporation of carboxy- and amino-terminal domains, respectively, might have further endowed these proteins with the ability to engage simultaneously ss- and dsDNA and catalyse the exchange of homologous DNA strands.

Atomic coordinates and cryoEM maps for the human RAD51 D-loop have been deposited in the PDB and EMDB under accession codes 9I62 and EMD-52646, respectively.

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Author details

1. Luay Joudeh

   Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

   Contribution

   Data curation, Supervision, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Robert E Appleby

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9338-205X

2. Robert E Appleby

   Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

   Present address

   Francis Crick Institute, London, United Kingdom

   Contribution

   Data curation, Funding acquisition, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Luay Joudeh

   Competing interests

   No competing interests declared

3. Joseph D Maman

   Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

   Contribution

   Conceptualization, Supervision, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Luca Pellegrini

   Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review and editing

   For correspondence

   lp212@cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9300-497X

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