Structural mechanism of strand exchange by the RAD51 filament

Reviewer #1 (Public review):

Summary:

The paper describes the cryoEM structure of RAD51 filament on the recombination intermediate. In the RAD51 filament, the insertion of a DNA-binding loop called the L2 loop stabilizes the separation of the complementary strand for the base-pairing with an incoming ssDNA and the non-complementary strand, which is captured by the second DNA-binding channel called the site II. The molecular structure of the RAD51 filament with a recombination intermediate provides a new insight into the mechanism of homology search and strand exchange between ssDNA and dsDNA.

Strengths:

This is the first human RAD51 filament structure with a recombination intermediate called the D-loop. The work has been done with great care, and the results shown in the paper are compelling based on cryo-EM and biochemical analyses. The paper is really nice and important for researchers in the field of homologous recombination, which gives a new view on the molecular mechanism of RAD51-mediated homology search and strand exchange.

Weaknesses:

The authors need more careful text writing. Without page and line numbers, it is hard to give comments.

Reviewer #2 (Public review):

Summary:

Homologous recombination (HR) is a critical pathway for repairing double-strand DNA breaks and ensuring genomic stability. At the core of HR is the RAD51-mediated strand-exchange process, in which the RAD51-ssDNA filament binds to homologous double-stranded DNA (dsDNA) to form a characteristic D-loop structure. While decades of biochemical, genetic, and single-molecule studies have elucidated many aspects of this mechanism, the atomic-level details of the strand-exchange process remained unresolved due to a lack of atomic-resolution structure of RAD51 D-loop complex.
In this study, the authors achieved this by reconstituting a RAD51 mini-filament, allowing them to solve the RAD51 D-loop complex at 2.64 Å resolution using a single particle approach. The atomic resolution structure reveals how specific residues of RAD51 facilitate the strand exchange reaction. Ultimately, this work provides unprecedented structural insight into the eukaryotic HR process and deepens the understanding of RAD51 function at the atomic level, advancing the broader knowledge of DNA repair mechanisms.

Strengths:

The authors overcame the challenge of RAD51's helical symmetry by designing a minifilament system suitable for single-particle cryo-EM, enabling them to resolve the RAD51 D-loop structure at 2.64 Å without imposed symmetry. This high resolution revealed precise roles of key residues, including F279 in Loop 2, which facilitates strand separation, and basic residues on site II that capture the displaced strand. Their findings were supported by mutagenesis, strand exchange assays, and single-molecule analysis, providing strong validation of the structural insights.

Weaknesses:

Despite the detailed structural data, some structure-based mutagenesis data interpretation lacks clarity. Additionally, the proposed 3′-to-5′ polarity of strand exchange relies on assumptions from static structural features, such as stronger binding of the 5′-arm-which are not directly supported by other experiments. This makes the directional model compelling but contradicts several well-established biochemical studies that support a 5'-to-3' polarity relative to the complementary strand (e.g., Cell 1995, PMID: 7634335; JBC 1996, PMID: 8910403; Nature 2008, PMID: 18256600).

Overall:

The 2.6 Å resolution cryoEM structure of the RAD51 D-loop complex provides remarkably detailed insights into the residues involved in D-loop formation. The high-quality cryoEM density enables precise placement of each nucleotide, which is essential for interpreting the molecular interactions between RAD51 and DNA. Particularly, the structural analysis highlights specific roles for key domains, such as the N-terminal domain (NTD), in engaging the donor DNA duplex.

This structural interpretation is further substantiated by single-molecule fluorescence experiments using the KK39,40AA NTD mutant. The data clearly show a significant reduction in D-loop formation by the mutant compared to wild-type, supporting the proposed functional role of the NTD observed in the cryoEM model.

However, the strand exchange activity interpretation presented in Figure 5B could benefit from a more rigorous experimental design. The current assay measures an increase in fluorescence intensity, which depends heavily on the formation of RAD51-ssDNA filaments. As shown in Figure S6A, several mutants exhibit reduced ability to form such filaments, which could confound the interpretation of strand exchange efficiency. To address this, the assay should either: (1) normalize for equivalent levels of RAD51-ssDNA filaments across samples, or (2) compare the initial rates of fluorescence increase (i.e., the slope of the reaction curve), rather than endpoint fluorescence, to better isolate the strand exchange activity itself.

Based on the structural features of the D-loop, the authors propose that strand pairing and exchange initiate at the 3'-end of the complementary strand in the donor DNA and proceed with a 3'-to-5' polarity. This conclusion, drawn from static structural observations, contrasts with several well-established biochemical studies that support a 5'-to-3' polarity relative to the complementary strand (e.g., Cell 1995, PMID: 7634335; JBC 1996, PMID: 8910403; Nature 2008, PMID: 18256600). While the structural model is compelling and methodologically robust, this discrepancy underscores the need for further experiments.

Reviewer #3 (Public review):

Summary:

Built on their previous pioneer expertise in studying RAD51 biology, in this paper, the authors aim to capture and investigate the structural mechanism of human RAD51 filament bound with a displacement loop (D-loop), which occurs during the dynamic synaptic state of the homologous recombination (HR) strand-exchange step. As the structures of both pre- and post-synaptic RAD51 filaments were previously determined, a complex structure of RAD51 filaments during strand exchange is one of the key missing pieces of information for a complete understanding of how RAD51 functions in the HR pathway. This paper aims to determine the high-resolution cryo-EM structure of RAD51 filament bound with the D-loop. Combined with mutagenesis analysis and biophysical assays, the authors aim to investigate the D-loop DNA structure, RAD51-mediated strand separation and polarity, and a working model of RAD51 during HR strand invasion in comparison with RecA.

Strengths:

(1) The structural work and associated biophysical assays in this paper are solid, elegantly designed, and interpreted. These results provide novel insights into RAD51's function in HR.

(2) The DNA substrate used was well designed, taking into consideration the nucleotide number requirement of RAD51 for stable capture of donor DNA. This DNA substrate choice lays the foundation for successfully determining the structure of the RAD51 filament on D-loop DNA using single-particle cryo-EM.

(3) The authors utilised their previous expertise in capping DNA ends using monomeric streptavidin and combined their careful data collection and processing to determine the cryo-EM structure of full-length human RAD51 bound at the D-loop in high resolution. This interesting structure forms the core part of this work and allows detailed mapping of DNA-DNA and DNA-protein interaction among RAD51, invading strands, and donor DNA arms (Figures 1, 2, 3, 4). The geometric analysis of D-loop DNA bound with RAD51 and EM density for homologous DNA pairing is also impressive (Figure S5). The previously disordered RAD51's L2-loop is now ordered and traceable in the density map and functions as a physical spacer when bound with D-loop DNA. Interestingly, the authors identified that the side chain position of F279 in the L2_loop of RAD51_H differs from other F279 residues in L2-loops of E, F, and G protomers. This asymmetric binding of L2 loops and RAD51_NTD binding with donor DNA arms forms the basis of the proposed working model about the polarity of csDNA during RAD51-mediated strand exchange.

(4) This work also includes mutagenesis analysis and biophysical experiments, especially EMSA, single-molecule fluorescence imaging using an optical tweezer, and DNA strand exchange assay, which are all suitable methods to study the key residues of RAD51 for strand exchange and D-loop formation (Figure 5).

Weaknesses:

(1) The proposed model for the 3'-5' polarity of RAD51-mediated strand invasion is based on the structural observations in the cryo-EM structure. This study lacks follow-up biochemical/biophysical experiments to validate the proposed model compared to RecA or developing methods to capture structures of any intermediate states with different polarity models.

(2) The functional impact of key mutants designed based on structure has not been tested in cells to evaluate how these mutants impact the HR pathway.

The significance of the work for the DNA repair field and beyond:

Homologous recombination (HR) is a key pathway for repairing DNA double-strand breaks and involves multiple steps. RAD51 forms nucleoprotein filaments first with 3' overhang single-strand DNA (ssDNA), followed by a search and exchange with a homologous strand. This function serves as the basis of an accurate template-based DNA repair during HR. This research addressed a long-standing challenge of capturing RAD51 bound with the dynamic synaptic DNA and provided the first structural insight into how RAD51 performs this function. The significance of this work extends beyond the discovery of biology for the DNA repair field, into its medical relevance. RAD51 is a potential drug target for inhibiting DNA repair in cancer cells to overcome drug resistance. This work offers a structural understanding of RAD51's function with the D-loop and provides new strategies for targeting RAD51 to improve cancer therapies.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The paper describes the cryoEM structure of RAD51 filament on the recombination intermediate. In the RAD51 filament, the insertion of a DNA-binding loop called the L2 loop stabilizes the separation of the complementary strand for the base-pairing with an incoming ssDNA and the non-complementary strand, which is captured by the second DNA-binding channel called the site II. The molecular structure of the RAD51 filament with a recombination intermediate provides a new insight into the mechanism of homology search and strand exchange between ssDNA and dsDNA.

Strengths:

This is the first human RAD51 filament structure with a recombination intermediate called the D-loop. The work has been done with great care, and the results shown in the paper are compelling based on cryo-EM and biochemical analyses. The paper is really nice and important for researchers in the field of homologous recombination, which gives a new view on the molecular mechanism of RAD51-mediated homology search and strand exchange.

Weaknesses:

The authors need more careful text writing. Without page and line numbers, it is hard to give comments.

We would like to thank the reviewer for their kind words of appreciation of our work.

Reviewer #2 (Public review):

Summary:

Homologous recombination (HR) is a critical pathway for repairing double-strand DNA breaks and ensuring genomic stability. At the core of HR is the RAD51-mediated strand-exchange process, in which the RAD51-ssDNA filament binds to homologous double-stranded DNA (dsDNA) to form a characteristic D-loop structure. While decades of biochemical, genetic, and single-molecule studies have elucidated many aspects of this mechanism, the atomic-level details of the strand-exchange process remained unresolved due to a lack of atomic-resolution structure of RAD51 D-loop complex.

In this study, the authors achieved this by reconstituting a RAD51 mini-filament, allowing them to solve the RAD51 D-loop complex at 2.64 Å resolution using a single particle approach. The atomic resolution structure reveals how specific residues of RAD51 facilitate the strand exchange reaction. Ultimately, this work provides unprecedented structural insight into the eukaryotic HR process and deepens the understanding of RAD51 function at the atomic level, advancing the broader knowledge of DNA repair mechanisms.

Strengths:

The authors overcame the challenge of RAD51's helical symmetry by designing a minifilament system suitable for single-particle cryo-EM, enabling them to resolve the RAD51 D-loop structure at 2.64 Å without imposed symmetry. This high resolution revealed precise roles of key residues, including F279 in Loop 2, which facilitates strand separation, and basic residues on site II that capture the displaced strand. Their findings were supported by mutagenesis, strand exchange assays, and single-molecule analysis, providing strong validation of the structural insights.

Weaknesses:

Despite the detailed structural data, some structure-based mutagenesis data interpretation lacks clarity. Additionally, the proposed 3′-to-5′ polarity of strand exchange relies on assumptions from static structural features, such as stronger binding of the 5′-arm-which are not directly supported by other experiments. This makes the directional model compelling but contradicts several well-established biochemical studies that support a 5'-to-3' polarity relative to the complementary strand (e.g., Cell 1995, PMID: 7634335; JBC 1996, PMID: 8910403; Nature 2008, PMID: 18256600).

Overall:

The 2.6 Å resolution cryoEM structure of the RAD51 D-loop complex provides remarkably detailed insights into the residues involved in D-loop formation. The high-quality cryoEM density enables precise placement of each nucleotide, which is essential for interpreting the molecular interactions between RAD51 and DNA. Particularly, the structural analysis highlights specific roles for key domains, such as the N-terminal domain (NTD), in engaging the donor DNA duplex.

This structural interpretation is further substantiated by single-molecule fluorescence experiments using the KK39,40AA NTD mutant. The data clearly show a significant reduction in D-loop formation by the mutant compared to wild-type, supporting the proposed functional role of the NTD observed in the cryoEM model.

However, the strand exchange activity interpretation presented in Figure 5B could benefit from a more rigorous experimental design. The current assay measures an increase in fluorescence intensity, which depends heavily on the formation of RAD51-ssDNA filaments. As shown in Figure S6A, several mutants exhibit reduced ability to form such filaments, which could confound the interpretation of strand exchange efficiency. To address this, the assay should either: (1) normalize for equivalent levels of RAD51-ssDNA filaments across samples, or (2) compare the initial rates of fluorescence increase (i.e., the slope of the reaction curve), rather than endpoint fluorescence, to better isolate the strand exchange activity itself.

Based on the structural features of the D-loop, the authors propose that strand pairing and exchange initiate at the 3'-end of the complementary strand in the donor DNA and proceed with a 3'-to-5' polarity. This conclusion, drawn from static structural observations, contrasts with several well-established biochemical studies that support a 5'-to-3' polarity relative to the complementary strand (e.g., Cell 1995, PMID: 7634335; JBC 1996, PMID: 8910403; Nature 2008, PMID: 18256600). While the structural model is compelling and methodologically robust, this discrepancy underscores the need for further experiments.

We would like to thank the reviewer for highlighting the importance of our findings to our understanding of the mechanism of homologous recombination.

We agree with the reviewer that the reduced filament-forming ability of some of the RAD51 mutants complicates a straightforward interpretation of their strand-exchange assay. Interestingly, the RAD51 mutants that appear most impaired are the esDNA-capture mutants that do not contact the ssDNA in the structure of the pre-synaptic filament. However, the RAD51 NTD mutants, that display the most severe defect in strand-exchange, have a near-WT filament forming ability.

The reviewer correctly points out that the polarity of strand exchange by RecA and RAD51 is an extensively researched topic that has been characterised in several authoritative studies. In our paper, we simply describe the mechanistic insights obtained from the structural D-loop models of RAD51 (our work) and RecA (Yang et al, PMID: 33057191).The structures illustrate a very similar mechanism of D-loop formation that proceeds with opposite polarity of strand exchange for RAD51 and RecA. Comparison of the D-loop structures for RecA and RAD51 provides an attractive explanation for the opposite polarity, as caused by the different positions of their dsDNA-binding domains in the filament structure. We agree with the reviewer that further investigation will be needed for an adequate rationalisation of the available evidence. We will mention the relevant literature in the revised version of the manuscript.

Reviewer #3 (Public review):

Summary:

Built on their previous pioneer expertise in studying RAD51 biology, in this paper, the authors aim to capture and investigate the structural mechanism of human RAD51 filament bound with a displacement loop (D-loop), which occurs during the dynamic synaptic state of the homologous recombination (HR) strand-exchange step. As the structures of both pre- and post-synaptic RAD51 filaments were previously determined, a complex structure of RAD51 filaments during strand exchange is one of the key missing pieces of information for a complete understanding of how RAD51 functions in the HR pathway. This paper aims to determine the high-resolution cryo-EM structure of RAD51 filament bound with the D-loop. Combined with mutagenesis analysis and biophysical assays, the authors aim to investigate the D-loop DNA structure, RAD51-mediated strand separation and polarity, and a working model of RAD51 during HR strand invasion in comparison with RecA.

Strengths:

(1) The structural work and associated biophysical assays in this paper are solid, elegantly designed, and interpreted. These results provide novel insights into RAD51's function in HR.

(2) The DNA substrate used was well designed, taking into consideration the nucleotide number requirement of RAD51 for stable capture of donor DNA. This DNA substrate choice lays the foundation for successfully determining the structure of the RAD51 filament on D-loop DNA using single-particle cryo-EM.

(3) The authors utilised their previous expertise in capping DNA ends using monomeric streptavidin and combined their careful data collection and processing to determine the cryo-EM structure of full-length human RAD51 bound at the D-loop in high resolution. This interesting structure forms the core part of this work and allows detailed mapping of DNA-DNA and DNA-protein interaction among RAD51, invading strands, and donor DNA arms (Figures 1, 2, 3, 4). The geometric analysis of D-loop DNA bound with RAD51 and EM density for homologous DNA pairing is also impressive (Figure S5). The previously disordered RAD51's L2-loop is now ordered and traceable in the density map and functions as a physical spacer when bound with D-loop DNA. Interestingly, the authors identified that the side chain position of F279 in the L2_loop of RAD51_H differs from other F279 residues in L2-loops of E, F, and G protomers. This asymmetric binding of L2 loops and RAD51_NTD binding with donor DNA arms forms the basis of the proposed working model about the polarity of csDNA during RAD51-mediated strand exchange.

(4) This work also includes mutagenesis analysis and biophysical experiments, especially EMSA, single-molecule fluorescence imaging using an optical tweezer, and DNA strand exchange assay, which are all suitable methods to study the key residues of RAD51 for strand exchange and D-loop formation (Figure 5).

Weaknesses:

(1) The proposed model for the 3'-5' polarity of RAD51-mediated strand invasion is based on the structural observations in the cryo-EM structure. This study lacks follow-up biochemical/biophysical experiments to validate the proposed model compared to RecA or developing methods to capture structures of any intermediate states with different polarity models.

(2) The functional impact of key mutants designed based on structure has not been tested in cells to evaluate how these mutants impact the HR pathway.

The significance of the work for the DNA repair field and beyond:

Homologous recombination (HR) is a key pathway for repairing DNA double-strand breaks and involves multiple steps. RAD51 forms nucleoprotein filaments first with 3' overhang single-strand DNA (ssDNA), followed by a search and exchange with a homologous strand. This function serves as the basis of an accurate template-based DNA repair during HR. This research addressed a long-standing challenge of capturing RAD51 bound with the dynamic synaptic DNA and provided the first structural insight into how RAD51 performs this function. The significance of this work extends beyond the discovery of biology for the DNA repair field, into its medical relevance. RAD51 is a potential drug target for inhibiting DNA repair in cancer cells to overcome drug resistance. This work offers a structural understanding of RAD51's function with the D-loop and provides new strategies for targeting RAD51 to improve cancer therapies.

We thank the reviewer for their positive comments on the significance of our work. Concerning the proposed polarity of strand exchange based on our structural finding, please see our reply to the previous reviewer; we agree with the reviewer that further experimentation will be needed to reach a settled view on this.

Testing the functional effects of the RAD51 mutants on HR in cells was not an aim of the current work but we agree that it would be a very interesting experiment, which would likely provide further important insights into the mechanism of strand exchange at the core of the HR reaction.