RAD51 D-loop structures reveal the mechanism of eukaryotic RAD51-mediated strand exchange

Abstract

Strand exchange is a key step in homologous recombination, enabling template-based repair of DNA double-strand breaks. Eukaryotic RAD51 forms an ATP-dependent helical presynaptic filament on single-stranded DNA (ssDNA), which then searches for homologous double-stranded DNA (dsDNA), and catalyzes the strand exchange to form a D-loop in an ATP hydrolysis-independent manner. The molecular mechanism by which RAD51 facilitates dsDNA unwinding and pairing remains unclear. Here, we present cryo-EM structures of RAD51 mini-filaments bound to homologous dsDNA, capturing five intermediates from dsDNA recruitment to D-loop formation. These structures, together with molecular dynamics simulations, suggest a stepwise mechanism: the conserved N-terminal domain (NTD) recruits and bends the dsDNA, weakening base pairing near the exchange site. Subsequent engagement with positively-charged regions, including the loop L2 and loop Arg303-Arg306, further bends the homologous dsDNA, thereby not only positioning it closer to the strand exchange site but also inducing local base-pair opening. Additionally, the loop L2 (Met278 and Phe279) inserts between strands, and the secondary DNA binding sites (S2 sites) capture the displaced strand to prevent strand reannealing. Together, our findings provide detailed insight into a spatially coordinated mechanism of strand exchange by RAD51.

Fig. 1. Cryo-EM analysis of RAD51 mini-filament assembly and strand exchange activity.

a Representative cryo-EM micrograph depicting both mini-filaments and extended RAD51 filaments formation using the SEAD mutant. The mini-filaments are highlighted in the red circles. The experiment was repeated at least three times with similar results. b DNA strand exchange activity of the mouse RAD51-SEAD mutant is comparable to that of the wild-type (WT) protein. (i) Schematic representation of the strand exchange reaction. An asterisk (*) indicates the Cy5 fluorescent dye. (ii) DNA strand exchange activity of RAD51-SEAD. The results were graphed. Data represent the mean ± standard error of the mean (s.e.m.) from three independent experiments (n = 3). Source data are provided as a Source Data file. c Cryo-EM density map (left) and atomic model (right) of the RAD51 presynaptic mini-filament. The N-terminal domain (NTD) is colored orange and the bound ssDNA is shown in red. d Cryo-EM density map (left) and atomic model (right) of the RAD51 postsynaptic mini-filament. The NTD, ssDNA, and the paired complementary strand are colored orange, red, and blue, respectively.

Fig. 2. Cryo-EM structures of the D-loop complexes with various homologous dsDNA.

a–e Cryo-EM reconstructions of RAD51 D-loop complexes assembled with donor dsDNA substrates containing 4-bp, 8-bp, another 8-bp, 8-bp, and 12-bp mismatched bubbles, respectively. For each panel, a schematic of the ssDNA and the corresponding dsDNA mismatched bubble is shown on the left, and the corresponding cryo-EM density map is shown on the right. RAD51 protomers are labeled from N + 1 to N + 9 along the filament axis. In the density maps, the N-terminal domain (NTD), displaced strand, and complementary strand are colored orange, green, and blue, respectively. The global resolution of each reconstruction is indicated in the respective panel.

Fig. 3. Cryo-EM structures of RAD51 D-loop complexes capturing distinct heteroduplex base-pair triplet states.

a–e D-loop complex containing a ⅓–1 base-pair triplet heteroduplex, (f–i) D-loop complex with a 2 and ⅔ base-pair triplet, (j–m) D-loop complex with a ⅓–3–⅔ base-pair triplet. (a, f, j) Nucleotide sequences of the 27-nt ssDNA and the corresponding 48-bp donor dsDNA. The invading ssDNA is shown in red, and the homologous regions (8 bp or 12 bp) on the dsDNA are highlighted. The green bases represent the displaced strand. b, g, k Atomic models of RAD51–DNA complexes illustrating progressive heteroduplex formation at different triplet stages. Nucleotide sequences are labeled to show specific base-pairing events. c, h, l Ribbon diagrams of the RAD51 D-loop complexes highlighting the heteroduplex core. The ssDNA, displaced strand, and complementary strand are colored red, green, and blue, respectively. Key RAD51 residues involved in DNA interaction are shown as stick-and-ball: S2 site residues in gray, L2 loop residues in yellow, N-terminal domain (NTD) residues in orange, and Arg235 in cyan. Corresponding RAD51 protomers are labeled as superscripts. d, i, m Close-up views of protein–DNA interactions near the strand separation site. – (d) Interactions of Met278^N+6,Phe279^N+6, and Arg306^N+7 with the just-separated displaced strand. – (i) Interactions of Arg235^N+6, M278^N+7, F279^N+7, and Arg306^N+8 with the just-separated displaced and complementary strands. – (m) Interactions involving Arg235^N+6, M278^N+7, F279^N+7, M278^N+8, F279^N+8, and Arg306^N+8 with the just-separated displaced and complementary strands.

Fig. 4. Molecular dynamics (MD) simulations illustrating dsDNA conformational changes during RAD51-mediated D-loop initiation.

a The hydrogen bond distance variation between two specific nucleotide base pairs during the initiation of D-loop formation. The blue line indicates the distance variation for the base-pair 1 (A–T base pair hydrogen bond distance between the N1 atom of adenine and the N3 atom of thymine); the purple line indicated the distance variation for the base-pair 2 (A–T base pair hydrogen bond distance between the N1 atom of adenine and the N3 atom of thymine). Source data are provided as a Source Data file. b Snapshots of RAD51–dsDNA interaction at four key time points (1 ps, 10 ns, 19 ns, and 47 ns). The base-pair opening occurs around 19 ns. RAD51 is rendered as a surface colored by electrostatic potential (blue, positive; red, negative). dsDNA is shown in a cartoon representation. Regions of base-pair opening are highlighted with blue circles, and the positively charged Arg303–Arg306^N+7 region is outlined with a green dashed box. c–f Top panels: Cartoon schematic representations showing nucleotide base-pair interactions at key simulation time points (1 ps, 10 ns, 19 ns, and 47 ns), depicting the progressive disruption of base pairs during dsDNA bending. Bottom panels: Structural snapshots illustrating the corresponding dsDNA bending conformations at 1 ps, 10 ns, 19 ns, and 47 ns, respectively.

Fig. 5. Molecular dynamics (MD) simulations illustrating dsDNA conformational changes during RAD51-mediated D-loop propagation.

a The hydrogen bond distance variation between two specific nucleotide base pairs during the propagation of D-loop formation. The blue line indicates the distance variation for the first pair (A–T base pair hydrogen bond distance between the N1 atom of adenine and the N3 atom of thymine); the purple line indicated the distance variation for the second pair (A–T base pair hydrogen bond distance between the N1 atom of adenine and the N3 atom of thymine). Source data are provided as a Source Data file. b Snapshots of RAD51–dsDNA interaction at four key time points (1 ps, 145 ns, 151 ns, and 180 ns). The base-pair opening occurs around 151 ns. RAD51 is rendered as a surface colored by electrostatic potential (blue, positive; red, negative). The dsDNA is shown in cartoon representation. Regions of base-pair opening are highlighted with blue circles, and the positively charged Arg303–Arg306 region is outlined with a green dashed box. c–f Top panels: Cartoon schematic representations showing nucleotide base-pair interactions at key simulation time points (1 ps, 145 ns, 151 ns, and 180 ns), depicting the progressive disruption of base pairs during dsDNA bending. Bottom panels: Structural snapshots illustrating the corresponding dsDNA bending conformations at 1 ps, 145 ns, 151 ns, and 180 ns, respectively.

Fig. 6. Structure-based mutagenesis data of D-loop formation and propagation.

a Quantification of ssDNA-binding activity of RAD51 variants P83G, K284A, and R306A. The percentage of bound ssDNA was calculated by the decrease in the percentage of free ssDNA. The results were plotted. Data represent the mean ± standard error of the mean (s.e.m.), n = 3 independent experiments. Source data are provided as a Source Data file. b D-loop formation assay of RAD51 variants. (i) Schematic illustration of the D-loop assay. The asterisk (*) marks the Cy5-labeled oligonucleotide. (ii) Quantification of D-loop formation by each RAD51 variant. Data represent the mean ± s.e.m. from four independent experiments (n = 4). Under DNA-saturating conditions, RAD51 variants exhibit intrinsically reduced D-loop formation activity compared to wild-type RAD51. Source data are provided as a Source Data file.

Fig. 7. Proposed mechanism of D-loop formation and propagation.

a Structural Illustration of D-loop Formation: This panel depicts the dynamic transition from a straight B-form dsDNA molecule to a bent conformation that displays the initial base-pair opening, as derived from our MD simulations. The initial straight model (from Fig. 2a) is overlaid in magenta for structural comparison. b Structural Illustration of D-loop Propagation: This image utilizes snapshots of cryo-EM D-loop intermediates to demonstrate the extension of homologous pairing. It contrasts the initial 4 bp heteroduplex structure (Fig. 2c, left) with the expanded 12-bp pairing (Fig. 2e, right). The overlay highlights the overall movement and propagation of the heteroduplex along the filament. c Filament Core and Functional Elements: This provides a simplified cartoon representation of the RAD51 presynaptic filament, highlighting the key functional elements involved in strand exchange: the NTD (blue), the L2 loop (orange), and the Arg303-Arg306 segment (green) of the structural core. d Proposed Stepwise Mechanism of Formation and Propagation: This comprehensive schematic integrates our cryo-EM structures and MD simulations into a unified model for RAD51-mediated D-loop activity. The process proceeds in the 3’ to 5’ direction (relative to the invading ssDNA, red strand): 1. dsDNA Recruitment: The RAD51 filament recruits homologous dsDNA. The N + 1 and N + 2 NTDs initiate engagement and minor unwinding (supported by Fig. 2a). 2. dsDNA Unwinding: Subsequent engagement by additional NTDs and positively charged regions (L2, Arg303-Arg306) induces pronounced dsDNA bending. This bending promotes local base-pair opening at the exchange site (supported by Fig. 2b and MD simulation, Fig. 4). 3. Strand Sequestration: Hydrophobic L2 residues (Met278 and Phe279) insert between the separated strands to prevent re-annealing. Concurrently, the displaced strand (green) is sequestered by the positive S2 sites. 4. Propagation: Once the initial D-loop forms (Fig. 2c), the cycle repeats in the 3’ to 5’ direction. This involves the sequential detachment of the 3’-tilted duplex from one NTD and its re-engagement with the next set of positively charged regions on adjacent protomers. This action drives further base-pair disruption and heteroduplex extension (derived from Fig. 2d, e, and MD simulation, Fig. 5).