Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange

Abstract

The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) and generates a helical filament that is able to search for and invade homologous double-stranded DNA (dsDNA), thus leading to strand separation and formation of new base pairs between the initiating ssDNA and the complementary strand within the duplex. Here, we used cryo-EM to solve the structures of human RAD51 in complex with DNA molecules, in presynaptic and postsynaptic states, at near-atomic resolution. Our structures reveal both conserved and distinct structural features of the human RAD51-DNA complexes compared with their prokaryotic counterpart. Notably, we also captured the structure of an arrested synaptic complex. Our results provide new insight into the molecular mechanisms of the DNA homology search and strand-exchange processes.

Figure 1. Structures of RAD51 presynaptic and post-synaptic complexes

(a) Cryo-EM density map superimposed with the (b) atomic model of the presynaptic complex. (c) Cryo-EM density map superimposed with the (d) atomic model of the post-synaptic complex. The invading ssDNA is in red and the complementary strand from the homologous dsDNA is in blue. RAD51 protomers are in orange, golden, lime green, cyan, medium purple, magenta as the order and AMP-PNP-Mg²⁺ is in yellow.

Figure 2. Structure analysis of RAD51 protomer-protomer interface

(a) AMP-PNP is buried in two neighboring RAD51 protomers coupling with ssDNA binding. Key residues involved in nucleotide binding and nucleic acid coupling are labeled in spheres. (b) The comparison of RAD51 and RecA in their nucleotide binding core. RAD51 is in limegreen and green for two protomers, respectively. RecA is in grey. Key residues involved in nucleotide binding and nucleic acid coupling are labeled in spheres for RecA. (c) The comparison of HsRAD51 and ScRad51 protomer interface. Green and salmon are used to present HsRAD51 and ScRad51, respectively, and AMP-PNP in the HsRAD51 presynaptic filament is shown in red. (d) The other two conserved protomer-protomer interfaces in eukaryotic Rad51 orthologs.

Figure 3. Interaction of RAD51 with DNA in the presynaptic and post-synaptic complexes

(a) In the presynaptic complex, each nucleotide triplet connects with three RAD51 protomers that are shown in green, cyan and purple following the direction from 5′ to 3′. (b) The EM density in corresponding view of (a) showing that ssDNA interacts with three RAD51 residues that are conserved in RecA. DNA is in red color. (c) The adjacent triplets in a presynaptic complex are separated and stabilized by V273 in Loop 2 of the middle RAD51. RAD51 mainly interacts with the backbone of ssDNA from the front view. (d) The EM density in corresponding view of (c) showing the interaction between V273 and ssDNA. DNA is in red color. (e) The complementary strand in the post-synaptic complex is observed in a similar extended state with ssDNA. R235 in Loop 1 facilitates the separation of the neighboring triplets from the back view of (b). Key residues are labeled with the potential hydrogen bonds highlighted. (f) The EM density in the corresponding view of (e) showing the interaction of R235 and the complementary strand in blue color. The EM density is shown at a high threshold so the complementary strand phosphate backbone is relatively weak. The invading strand is in red color.

Figure 4. Capture of the arrested state in Rad51-mediated DNA strand exchange

(a) Substrate design. Shown are the 72-mer invading strand that bears four 18-nt repeats and the 18-bp duplex that bears 13 nucleotides of homology to each of the repeats in the invading strand. The non-homologous nucleotides are in red. (b) The difference maps calculated by subtracting the presynaptic complex atomic model from the arrested synaptic complex 3D reconstruction with 15σ (orange color) and by subtracting the post-synaptic complex atomic model from the arrested synaptic complex 3D reconstruction with 9σ (green color) are superimposed on the atomic model of a post-synaptic complex, in which RAD51 protomers are colored grey, the invading strand is in red, and the complementary strand is in blue. Two orthogonal views are shown, with six consecutive protomers labeled by numbers. (c) A schematic interpretation of the arrested synaptic state in one hexameric RAD51 repeat. In (a) and (c), I-strand, C-strand, and D-strand stand for the invading strand, complementary strand, and displaced strand, respectively.

Figure 5. Model of RAD51-mediated DNA strand exchange with depiction of the intermediate state

(a) The interaction of RAD51 and three DNA strands in the intermediate state. The displaced strand is located in proximity to the C-terminal part of RAD51's potential secondary DNA binding site. The invading strand (red) and the complementary strand (blue) are stabilized by V273 and R235, respectively. (b) Interaction of R235 with the complementary strand and the various stretch ratios in different length of DNA compared to the B-form DNA. (c) A hypothetical model of DNA molecule transition during the synaptic reaction.