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 player of several crucial processes in repairing and protecting genome integrity, forms filaments on DNA. RAD51 filaments are tightly regulated. One of these regulators is FIGNL1, that prevents persistent RAD51 foci post-damage and genotoxic chromatin association in cells. The cryogenic electron microscopy structure of FIGNL1 in complex with RAD51 reveals that the FIGNL1 forms a non-planar hexamer and RAD51 N-terminus is enclosed 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 unique mechanism for removing RAD51 from DNA 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) RAD51 accumulates in the chromatin fraction in Fignl1^−/−; rDC but not Fignl1^−/−; rWT cells upon ASPIR-1 treatment. Cells are treated with 0.25 µg/ml ASPIR-1 or DMSO for 24 hr and then subjected to chromatin and cytoplasmic fractionation followed by 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 FIGNL1∆N or FIGNL1DN(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 FIGNL1∆N while FIGNL1DN(E501Q) has little 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 FIGNL1∆N or E501Q mutant. (D) Quantification of nuclease protection assays. n = 3, each data point represents the mean ± s.d. WT FIGNL1∆N data quantification from independent repeats and 0.5µM and 0.9µM data points 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 FIGNL1DN^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 (RAD51DN), in the presence of increasing concentrations of FIGNL1DN (G) Quantification of ATPase activity of FIGNL1 alone or incubated with RAD51 or RAD51∆N. (H) Dose-response ATPase activity of FIGNL1∆N 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 FIGNL1∆N bearing mutations in pore loop 1 (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 FIGNL1∆N bearing PL mutation. n = 20, data are shown as mean ± s.d. WT FIGNL1∆N 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 FIGNL1∆N. (D) Quantification of RAD51 + FIGNL1∆N^PL experiments shown in c, n = 3, data is shown mean ± s.d. WT FIGNL1∆N 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 K483E/W484A (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. (F) A proposed model of RAD51 filament disassembly by FIGNL1. 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/removing of the RAD51 from the filament, promoting disassembly. (G) SDS-PAGE gels showing RAD51 degradation by proteases in the presence of FIGNL1DN and its mutants with ATP. (H) quantification of RAD51 band intensity over time.