Extended Data Fig. 4: Secretion, enzymatic activity, and protein structure of RuvC.
a, Verification of α-RuvC antibody specificity. Total proteins from the bacterial culture of GMI1000, ΔruvC, and ComRuvC strains were subjected to WB with α-RuvC antibody. b, The secretion of RuvC is partially dependent on the type II and IV secretion system. The bacterial culture (Total), supernatant preparations (SPs) and cell pellets (CPs) were subjected to western blotting. β-tubulin was detected as a control. c, RuvC exists in extracellular membrane vesicles (MVs). The extracellular membrane vesicles were precipitated by ultracentrifuge and analyzed. d, Schematic diagram of the common Holliday junction substrate HJ98. e, His-RuvC fusion proteins were purified from E. coli and separated by SDS-PAGE. Protein expression was induced by IPTG and purified by affinity chromatography with Ni-NTA agarose beads. f, The cleavage activity of RuvC towards cruciform DNA HJ98 was positively correlated with the concentration. His-RuvC protein with increasing concentrations was applied to the reaction. The untreated samples were the blank control group. The band intensity was quantified by ImageJ and the percentage of cleaved substrate relative to the input DNA is shown at the bottom. g, The enzymatic efficiency of RuvC to cruciform DNA HJ98. His-RuvC protein (200 ng/μL) was incubated with HJ98 DNA (100 nmol/μL) in a 20 μL reaction mix. h, Topological diagram of the RuvC secondary structure. Predicted regions required for the enzyme activity are shaded in gray. i, Two perpendicular views of the overall 3D structure of RuvC and its mutation variant. Three conserved acidic residues in the enzymatic region were mutated to alanine (A) as shown on the right. j, RuvC does not affect the cell viability of R. solanacearum. Data are presented as mean values ± SD (n=3 biological replicates). Different letters represent significant differences according to one-way ANOVA analysis with two-sided test. The above experiments were repeated three times with similar results.
