Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution

Abstract

During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a single-molecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.

Keywords: BRCA2; RAD51; homologous recombination; optical tweezers; single-molecule fluorescence.

Fig. 1.

Visualization and quantification of RAD51 nucleation on ssDNA using dual optical tweezers, wide-field fluorescence microscopy, and microfluidics. (A) A four-channel microfluidics device was used as a platform for the assembly of the single-molecule assay. Typical experiments proceeded in five steps: (1) trapping of two streptavidin-coated microspheres, (2) tethering of a single dsDNA between the spheres, (3) force-induced conversion of dsDNA into ssDNA (DNA force–extension curves were acquired and checked), (4) incubation in a flow channel with fluorescent RAD51 in solution, and (5) visualization of the RAD51–DNA complex in buffer without RAD51 (occasionally, complete photobleaching of the fluorescent signal was used for RAD51 quantification). To measure long-time behavior, multiple cycles of 4 and 5 were performed on the same DNA substrate. (B) Fluorescence images taken after subsequent RAD51 incubation–detection cycles with the same ssDNA construct; cumulative incubation time is indicated. Indicated are a RAD51 nucleus that binds and unbinds before the next incubation cycle (1) and a nucleus that remains bound and shows an increase in fluorescence intensity due to growth (2). (C) RAD51 concentration dependence of nucleation rate. ●, Experimental data; red line, power law fit (k_nucl = k_o[RAD51]ⁿ) yielding an exponent n of 1.5 ± 0.3. The number of incubation measurements was 26 at 7.5 nM, 30 at 12.5 nM, 20 at 25 nM, 29 at 50 nM, and 62 at 75 nM. (D) Histogram of nucleus sizes (RAD51 concentration, 12.5 nM; incubation time, 77 s; total, 105 data points). (E) Fluorescence intensity time traces of individual RAD51 nuclei bound to ssDNA. After incubation with fluorescent RAD51, a fluorescence image was taken of the same ssDNA every 30 s, in the absence of RAD51 in solution (Inset). From such images, fluorescence intensity time traces were determined for individual RAD51. ●, RAD51 nucleus consisting of two fluorophores, detaching between 300 and 330 s; ○, RAD51 nucleus consisting of three fluorophores remaining ssDNA bound for at least 9 min. (F) Bar diagram showing how stable nucleus fraction depends on nucleus size. Stable nucleus fraction was defined as the probability of staying bound to the ssDNA for longer than 6 min. n = 9, 34, 7 for n ≤ 3, 3 < n ≤ 6, n > 6, respectively. Error bars represent normalized counting errors.

Fig. 2.

sm-FRAP allows detection of RAD51 growth on ssDNA. Conditions: RAD51 concentration, 12.5 nM; duration of an incubation period, 77 s. (A) Fluorescence image showing three individual fluorescent RAD51 nuclei on ssDNA. Subsequent continuous laser illumination resulted in complete photobleaching of the nuclei. (B) Fluorescence image of the same ssDNA–RAD51 complex after an additional incubation period. Fluorescent image shows the appearance of three distinct fluorescent patches. (C) Superposition of A and B allows the distinguishing of new nucleation events from RAD51 growth. In the yellow circle, we show that two of the fluorescent patches obtained from consecutive incubations colocalize exactly. (D) Line profile and Gaussian fitting of A and B confirm the colocalization of the two patches within 20 nm (fitted locations indicated by X_c). This confirms the direct separate detection of RAD51 nucleation and growth on ssDNA.

Fig. 3.

Selectivity of RAD51 binding. All incubations were performed in buffer containing 75 nM fluorescent RAD51. (A) Fluorescence image of an ssDNA molecule held at a tension of 20 pN and incubated for 15 s. (B) Fluorescence image of a dsDNA molecule after 8 min of incubation. (C) Rate of nucleation (nucleation events per second per nucleotide) versus applied tension for ssDNA (●) and dsDNA (○); error margins represent SEM. Nucleation on ssDNA is not affected by tension, whereas it increases strongly with tension on dsDNA. The dotted red line represents a fit to the Arrhenius model (k(F)=k(0) Exp[-F·δx/k_BT]), yielding δx = 0.45$n^{\ast }\left(t_{\text{inc}}\right)$; 0.05 nm for dsDNA and k(0) = (4 ± 3)·10⁻⁹ nucleation events per second per base pair. (D) Rate of filament growth (RAD51 monomers per second per nucleus) versus applied tension for ssDNA (●) and dsDNA (○); error margins represent SEM. Fitting to the Arrhenius equation (dashed line) yielded δx = 0.27 ± 0.03 nm and k(0) = (3 ± 1)·10⁻³ RAD51 monomers per second per nucleus.