Fig 2: Proposed model for RuvC-mediated scanning and site-specific cleavage. When RuvC binds to the HJ, it fluctuates between a loosely bound PD complex that allows the HJ junction to maintain its dynamic iso-IR↔iso-IIR exchange and the more tightly bound, shallow-angle OpR state. Branch migration can occur at this stage, allowing RuvC to scan for the cleavable cognate sequence. Once a cognate sequence is found, the OpR state undergoes a interconversion, wherein each cleavage site is snap-locked by a stacking amino acid, disallowing branch migration and adoption of the PD complex and iso-IR conformation.

Fig 3: Holliday junction design to observe conformational dynamics by smFRET. (A) Schematic and sequence of the HJxhR design. The nucleotides highlighted in yellow represent the cognate cleavage sequence for RuvC. The nucleotides highlighted in gray represent the region of the junction that can potentially branch migrate due to sequence homology. The brown arrows indicate the cleavage sites. The 5′ ends of the r, x and b-strand are labeled with biotin, Cy5 and Cy3, respectively. (B) Schematic of our smFRET assay under TIRF illumination to monitor the dynamics of HJxhR molecules. The HJ transitions between two isomeric states iso-I and iso-II. The cognate sequences are shown in yellow in the bent, cleavage-incompetent iso-I and continuous, cleavage-competent iso-II strands. (C) Single molecule FRET probability distribution of HJ7nR in presence of 5 mM Mg2+. (D) Single molecule FRET histogram of HJxhR construct in presence of 5 mM Mg2+. (E) Representative single-molecule time trajectory showing donor (green) and acceptor (red) intensity in the top panel, FRET (blue) and HMM fitted to the data (magenta) in the bottom panel of HJ7nR. (F) Representative single-molecule time trajectory showing donor (green) and acceptor (red) intensity in the top panel, FRET (blue) and HMM fitted to the data (magenta) in the bottom panel of HJxhR. Fast transitions highlighted in gray are attributed to conformer exchange at a different branchpoint position. Representative cartoons indicate the conformational states associated with particular FRET values.

Fig 4: Holliday junction cleavage by RuvC. (A) Superimposed Cy3 (green) and Cy5 (red) scans of a denaturing urea–polyacrylamide gel showing RuvC-mediated cleavage of HJxhR in the presence of varying concentrations of RuvC (0, 50, 100, 200 and 400 nM respectively from lanes 3–7). The top bands represent the full-length 20-nt-long DNA; the bottom bands are the cleaved 10-nt-long DNA product. The schematics on the right-hand side represent the relative length of DNA corresponding to each band. The Cy3-labeled b strand is not cleaved due to the absence of a cleavage sequence. (B) Quantification of cleavage using a single molecule assay. Normalized ratios of number of colocalized Cy3–Cy5 spots and number of all Cy5 spots before and after cleavage were calculated to estimate the fraction of cleaved HJs regardless of slide to slide variation in the number of initial HJ molecules. The fraction of resolved HJs are plotted for the presence and absence of RuvC. Without RuvC, this ratio remains close to zero. In the presence of 10 mM Mg2+ and 400 nM RuvC incubated at 37°C for 1 h, this ratio becomes 0.40±0.05, representing RuvC-mediated cleavage. The inset shows a schematic of the experiment before and after cleavage in the presence of RuvC. (C) Quantitative analysis of a time course of RuvC-mediated cleavage of HJxhR under varying conditions as indicated, at 400 nM RuvC. Saturation curves are fit to the data. As expected, the fraction of HJxhR molecules cleaved by RuvC decreases with decreasing temperature and decreasing Mg2+ concentration.

Fig 5: Single molecule cluster analysis (SiMCAn) of smFRET trajectories reveals dynamic behaviors unique to the cleavage-competent RuvC-HJ complex. FRET probability distributions of (A) 5 mM Mg2+ without RuvC, (B) 5 mM Mg2+ plus 400 nM RuvC (C) 5 mM Ca2+ without RuvC, and (D) 5 mM Ca2+ plus 400 nM RuvC. Multi-peak Gaussian functions are used to fit each histogram. N indicates the number of molecules used to generate the histogram. Associated structural cartoons depict the conformational states corresponding to each FRET population. (E) Summary of SiMCAn results, showing bar graphs with occupancy of the four clusters found in four experimental conditions. Clusters S1, S2 and S3 are distributed over all different conditions. By contrast, the R-cluster is predominantly found in the 5 mM Mg2+ plus 400 nM RuvC condition, indicating that the R-cluster is associated with the RuvC- HJxhR complex. (F) Bar graph showing the fraction of molecules from of each experimental condition contributing to the four clusters. More than 80% of the molecules in the R-cluster belong to the condition where both Mg2+ and RuvC are present. (G) FRET efficiency histogram calculated from the traces belonging to the R-cluster, shown with multi-peak Gaussian fits. Associated cartoons represent different RuvC-HJ complex conformations corresponding to different FRET states. (H) Transition Occupancy Density Plots (TODPs) from R-cluster traces showing the fraction of HJs that undergo conformational transitions from a given initial FRET state to a specific final FRET. Three main sets of bidirectional transitions are observed, representing iso-IIR ↔ OpR, iso-IR ↔ iso-IIR, and iso-IR ↔ OpR behavior, respectively. (I) Representative fluorescence intensity trace of Cy3 (green) and Cy5 (red) fluorophore, FRET efficiency (blue) and HMM fitting (magenta) from the 5 mM Mg2+plus 400 nM RuvC condition (top panel). Three additional FRET efficiency traces show different types of behaviors under the same conditions (bottom panel), with percentages delineating the fractions of traces displaying such behavior. Associated cartoons represent most likely conformations corresponding to each FRET value. The orange background represents iso-IR ↔ OpR behavior and the blue background represents iso-IIR ↔ OpR behavior. Sudden visits to non-prevalent FRET states, assigned as RuvC’s PD complex (marked by black arrows) and conformer exchange at a different branchpoint position (marked by gray background), are observed in the iso-IR ↔ OpR behavior and absent from the iso-IIR ↔ OpR behavior.