Structure of RADX and mechanism for regulation of RAD51 nucleofilaments

Abstract

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed ssDNA. To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (EM) from maps in the 2-3 Å range. The structure reveals the molecular basis for RADX oligomerization and binding of ssDNA binding. The binding of RADX to RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the growing end of RAD51 filaments.

Keywords: Biological Sciences; Biophysics and Computational Biology; DNA replication; RAD51; cryo-EM; replication fork.

Figure 1.. RADX oligomerization is stabilized and preferentially forms trimers upon binding ssDNA.

(a) RADX is primarily monomeric at 50 nM but dimerizes at 100 nM. (b) Addition of ssDNA to monomeric RADX leads to a significant increase in the population of trimers, from 4% to 33%. (c),(d) Addition of different lengths of ssDNA leads to varying distribution of oligomers. (e) DNA binding affinity of RADX determined by fluorescence polarization anisotropy show there is no dependence on the length of the substrate. (f) Tryptophan quenching assay measuring the DNA footprint of RADX shows a footprint length of 19–27 nucleotides, consistent with the mass photometry results in panel (c).

Figure 2.. Optimization of RADX for cryo-EM.

(a) Negative stain EM micrograph image of the RADX-dT25 complex. 2D classes showing monomeric RADX in a variety of configurations is shown below the micrograph, indicating heterogeneity remains an issue. (b) Cryo-EM micrographs of RADX-dT25 complex without cross-linking. 2D classes under the micrographs show that without cross-linking RADX has very poorly resolved 2D classes due to its flexibility. (c) Cryo-EM micrograph of RADX-dT25 complex crosslinked with BS3. 2D class averages show the resolution improves significantly upon cross-linking, allowing for 3D reconstruction.

Figure 3.. High resolution structure shows RADX is comprised of four independent OB-fold domains.

(a) Structure of the RADX trimer bound to ssDNA (pink) with the three protomers in cyan, blue and purple. (b) The structure of the RADX tetramer with the fourth RADX protomer RADX-D (violet). (c) Ribbon diagram of a RADX protomer showing the location of each of the four OB-fold domains.

Figure 4. RADX oligomerization is stabilized by multiple inter-domain interfaces.

(a) The D1–D4 interaction between two RADX protomers A and B (teal and blue, respectively). RADX oligomerizes primarily via hydrogen bond and salt-bridge interactions between domains 1 and 4. The inset shows the residues involved in hydrogen bonding colored according to the protomer of origin, with the hydrogen bonds shown as red dashed lines. (b) Three of the sites of RADX oligomerization mutations in the central beta sheet of OB4 displayed in red in the inset. Mutation of these residues are likely to perturb the beta sheet and the entire D4 domain, which could contribute to effects observed in functional assays.

Figure 5.. RADX binding of ssDNA is coupled to oligomerization.

(a) The DNA binding site of RADX-A. The inset shows the residues involved in hydrogen bonding or pi-stacking interactions (in red) with the DNA (dark grey). H-bonds are shown as black dashed lines. (b) The full DNA interaction surface is shown in red in a surface representation of RADX-A (teal) with bound DNA (grey). The inset shows residues involved in hydrogen bonding or pi-stacking in any of the three RADX protomers (red). The yellow residues indicate the sites of mutation for the DNA binding Ob2M mutant, and the mutation site is seen to overlap only partially with the DNA binding site as shown by the two residues labelled in black (W279, K305).

Figure 6.. RADX is unique but well conserved.

(a) Analysis of sequence conservation by ConSurf plotted onto the structure of RADX. The color code of the conservation scale is given below the structure. Regions of the protein with well-defined secondary structure are conserved. (b) Analysis of sequence conservation by ConSurf focused on the large loop in OB4 (S566-I676), delineated by grey stars. The loop has very poor sequence conservation, apart from small regions which include Ser and Thr residues, highlighted within red dashed boxes. (c) The high scored of 12.1 from the Dali server suggests RADX OB1 (blue)is a homolog of RPA70N domain (pink). Their structural similarity is reflected in the RMSD over all Ca atoms of 2.5 Å.

Figure 7.. RADX modulation of RAD51 filaments correlates with end-binding.

(a) (left) Negative stain EM micrograph showing anti-RADX Ab bound gold nanoparticles localized at the ends of RAD51 filaments. (right) 2D class averages of dataset containing RADX crosslinked to RAD51 filaments reveal populations of free RADX and RADX bound to RAD51 filaments. (b) 3D reconstructions of free RADX and RADX bound to RAD51 filaments. The extra volume assigned to RADX is circled in red. (c) The residues mutated in the RAD51 binding-deficient QVPK mutant displayed on the structure of RADX. These residues form a surface-exposed patch, consistent with the proposal that these are in RAD51 binding site. (d) Model of a RADX trimer bound to a RAD51 filament generated via manual docking. The RADX is shown in green, the RAD51 filament in blue (8BQ2) and the ssDNA in both models is shown in red.

Figure 8.. Model for the mechanism of RADX action.

Upon replication fork stalling, single stranded DNA is exposed (a), which is then bound by RPA to protect the ssDNA from damaging agents (b). RPA is replaced on ssDNA with RAD51 by the action of BRCA2, a mediator protein (c). RAD51 binds cooperatively to form filaments on ssDNA. RADX is recruited to ssDNA where it binds the ends of the growing RAD51 filament (d). It promotes filament disassembly by either i) blocking expansion at the end of the filament as RAD51 hydrolyses ATP and detaches from ssDNA or ii) both blocking filament expansion and accelerating the ATP hydrolysis rate of RAD51(e).