A novel imaging biosensor for the detection of reversed replication forks and four-way junctions in human cells

Replication fork reversal and recombination-dependent replication protect perturbed forks by forming four-way DNA junctions. Despite their central role, detecting these intermediates in intact cells has historically required electron microscopy of bulk extracted DNA. Here, we describe a genetically encoded biosensor for four-way junctions based on nuclear-targeted bacterial RuvA tagged with GFP or Spot-tag. Combined with SIRF, RuvA selectively accumulates at nascent DNA following hydroxyurea- or camptothecin-induced replication stress. Recruitment requires fork-reversal factors and is abolished by a non-binding mutant (K84E/K119E), confirming specificity. The biosensor dynamically tracks reversed fork abundance, capturing MRE11-mediated fork degradation in BRCA2- or RAD52-deficient cells and its functional rescue. Finally, RuvA reveals RAD51-dependent four-way junctions at nucleolar rDNA arrays in unperturbed and stressed cells. This tool converts a bulk population measurement into a visualizable readout with single-cell and subnuclear resolution, enabling direct spatial analysis of DNA structures in situ.