Real-time visualization of spatial and temporal coordination in resolvase-mediated Holliday junction binding

Holliday junctions (HJs) are key intermediates in homologous recombination and must be processed with high precision to maintain genome stability. The bacterial resolvase RuvC has served as a model system to study this process. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize RuvC-DNA interactions at the single-molecule level and without the need for labeling, under near-physiological ionic conditions. HS-AFM revealed that RuvC exists as monomeric and dimeric species, and that binding to cruciform DNA mimicking HJs was associated with the dimeric form. Importantly, magnesium ions markedly enhanced the persistence of RuvC-HJ complexes, highlighting a structural role beyond catalysis. Structural predictions further supported that Mg²⁺ promotes a symmetric and coordinated RuvC-HJ interface. Together, these findings show how HS-AFM enables direct dissection of binding dynamics at DNA junctions and reveal a previously underappreciated role of Mg²⁺ in promoting long-lived RuvC-HJ interactions prior to catalysis.