Molecular basis of FIGNL1 in dissociating RAD51 from DNA and chromatin

Abstract

Maintaining genome integrity is an essential and challenging process. RAD51 recombinase, the central component of several crucial processes in repairing DNA and protecting genome integrity, forms filaments on DNA, which are tightly regulated. One of these RAD51 regulators is FIGNL1 (fidgetin-like 1), which prevents RAD51 genotoxic chromatin association in normal cells and persistent RAD51 foci upon DNA damage. The cryogenic electron microscopy-imaged structure of FIGNL1 in complex with RAD51 reveals that FIGNL1 forms a nonplanar hexamer and encloses RAD51 N terminus in the FIGNL1 hexamer pore. Mutations in pore loop or catalytic residues of FIGNL1 render it defective in filament disassembly and are lethal in mouse embryonic stem cells. Our study reveals a distinct mechanism for removing RAD51 from bound substrates and provides the molecular basis for FIGNL1 in maintaining genome stability.

Figure 1. FIGNL1 ATPase activity is required for its functionality in cells.

(A) FIGNL1 is required for the viability of mESCs. Fignl1^+/− cells were targeted at the Rosa26 locus with expression cassettes for wild-type Fignl1 (rWT) or mutants K456A in the ATPase Walker A motif (rKA) or D411C for chemical inhibition (rDC) and then selected for Hyg gene expression from the Rosa26 promoter. After confirmation of correct targeting, the second Fignl1 allele was subjected to CRISPR-Cas9 editing using sgRNAs (c+d) to delete the entire Fignl1 coding region. Genomic DNA was screened by PCR using primers -396 and 409 for the undeleted allele. Successful deletion is indicated by a blue asterisk. The number of Fignl1^-/- colonies is indicated, along with the total number of colonies that were screened. (B) Chemical inhibition of the FIGNL1 ATPase is lethal. Covalent modification of FIGNL1 at D411C in the ATPase domain by ASPIR-1 impairs colony formation. (C) Chemical inhibition of FIGNL1 leads to RAD51 accumulation on chromatin (left) but does not impact overall RAD51 levels (right). Fignl1^-/-; rDC or Fignl1^-/-; rWT cells were treated with 0.25 µg/ml ASPIR-1 or DMSO for 24 hr. Chromatin /cytoplasmic fractions or whole cell extracts as indicated were subjected to western blot analysis with antibodies to the indicated proteins. (D) Rad51 heterozygosity rescues survival of the Fignl1 mutant for colony formation in the presence of ASPIR-1 (0.25 µg/ml) for 7 days. Three independent clones were tested (80, 83, 93). (E) Rad51 heterozygosity reduces RAD51 chromatin association in the presence of ASPIR-1 to the level found in untreated Rad51 wild-type cells. Three independent clones were treated with 0.25 µg/ml ASPIR-1 or DMSO for 24 hr and then subjected to chromatin and nuclear fractionation followed by western blot analysis with antibodies to the indicated proteins.

Figure 2. Disassembly of RAD51 filaments is dependent on FIGNL1 ATPase activity

(A) Representative negative-stain electron micrographs of RAD51 filaments on both single-stranded and double-stranded DNA in the absence or present of FIGNL1ΔN or FIGNL1ΔN(E501Q), a mutant in the Walker B motif. Red arrows indicate some of the filaments. Scale bars = 100 nm. (B) Quantification of filaments observed in (A) indicates a decrease in the number of filaments per micrograph upon incubation with FIGNL1ΔN while FIGNL1ΔN^E501Q has reduced effects. n = 18-20 micrographs per condition (C) Image of gel, showing nuclease protection of dsDNA by RAD51 in the presence of increasing concentrations of FIGNL1ΔN or E501Q mutant. (D) Quantification of nuclease protection assays. n = 3, each data point represents the mean ± s.d. WT FIGNL1ΔN data quantification from independent repeats and 0.5µM and 0.9µM data points were shown in C. Filament disruption assays shown in A-D were carried out using 60nt ssDNA and 60bp dsDNA.

Figure 3. CryoEM structures of the FIGNL1-RAD51 complex and FIGNL1 AAA+ hexamer

(A) Top and side views of the cryoEM map and model of the FIGNL1ΔN^E501Q-RAD51 complex in the presence of ATP.Mg²⁺. (B) Top and side views of the cryoEM map (2.9Å) of the FIGNL1 AAA+ hexamer in the presence of ATP.Mg²⁺. (C) Top and side views of the atomic model of the FIGNL1 AAA+ hexamer modelled from the map shown in B.

Figure 4. FIGNL1 coordinates the N-terminus of RAD51 through its central hexameric pore

(A) Extra density observed in the central pore of the FIGNL1 AAA+ hexamer. (B) Sequence conservation plots of the pore loops of FIGNL1 in vertebrates (n=576). Residues 473, 474 and 514 are labelled. (C) The pore loops of FIGNL1 form two helical staircases enclosing the RAD51 N-terminus (D) The pore loop residues intercalate with the RAD51 N-terminal residues. (E) Conservation plot of the N-termini of RAD51 in 548 vertebrates. Numbering refers to the human RAD51 sequence. (F) Nuclease protection of ssDNA by RAD51 or RAD51 with N-terminal 20 a.a. deleted (RAD51ΔN), in the presence of increasing concentrations of FIGNL1ΔN (G) Quantification of ATPase activity of FIGNL1 alone or incubated with RAD51 or RAD51ΔN. (H) Dose-response ATPase activity of FIGNL1ΔN in the presence of increasing concentrations of the RAD51 N-terminal peptide or with the first 5 amino acids replaced by alanine (n = 4).

Figure 5. Mutation of pore loop 1 confers loss of RAD51 filament disassembly and cell lethality

(A) Representative micrographs of RAD51-DNA filaments treated by FIGNL1ΔN bearing mutations in pore loop 1 K483E/W484A (PL mutant) with ss or ds-DNA. Scale bars = 100 nm. (B) Quantification of experiments shown in (A), highlighting the loss of filament disassembly by FIGNL1ΔN bearing PL mutation. n = 20, data are shown as mean ± s.d. WT FIGNL1ΔN data is for comparison and is as shown in Fig. 2. (C) Nuclease protection of dsDNA coated by RAD51 upon treatment with wildtype or PL mutant of FIGNL1ΔN. (D) Quantification of RAD51 + FIGNL1ΔN^PL experiments shown in c, n = 3, data is shown mean ± s.d. WT FIGNL1ΔN data quantification from independent repeats shown in Fig. 3 and 0.5µM and 0.9µM data points shown in C. (E) FIGNL1 pore loop mutant (PL) is not compatible with cell survival. An expression cassette for the mutant (rPL^Myc) was targeted to Rosa26 locus as in Fig. 1A. No Fignl1^-/- colonies were obtained after CRISPR-Cas9 gene editing in Fignl1^+/− cells with gRNAs c and d as in Fig. 1A. Western blotting for the Myc tag confirms that the PL mutant is expressed. (F) An integrated structural model of FIGNL1 engaged on a RAD51-dsDNA filament using our cryoEM structure in conjunction with AlphaFold3 prediction of FRBD binding RAD51. FIGNL1 is colored by subunit as in Fig. 3. RAD51 protomers are colored green, except the target RAD51 engaged with FIGNL1 AAA+ pore, which is colored dark blue. For clarity only a single FRDB is shown from one subunit of the FIGNL1 hexamer, and only a single N-terminus from RAD51. Below is a proposed model of FIGNL1 recruitment and RAD51 filament disassembly. FIGNL1 is recruited to the RAD51 filament via its FRBD domain and forms a hexamer, enclosing the N-terminus of RAD51, which stimulates the ATPase activity of FIGNL1 and promotes translocation of the N-terminus in the hexamer pore, leading to the unfolding/removal of RAD51 from the filament, promoting disassembly. (G) SDS-PAGE gels showing RAD51 degradation by proteases in the presence of FIGNL1ΔN and its mutants with ATP. (H) Quantification of RAD51 band intensity over time is shown below. Data points are mean ± s.d, n = 3.