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).