Live-Cell Visualization of DNA Double-Strand Break Repair Dynamics via Recombinase-Coated Gold Nanoconjugates

Abstract DNA double-strand breaks (DSBs) represent one of the most severe forms of genomic damage, closely linked to cancer, neurodegeneration, and cell death. Real-time monitoring of DSBs in live cells is challenging because repair events are highly dynamic. Current in vivo detection tools rely mainly on fluorescently tagged repair proteins like RecA or γH2AX/RAD51, which can disrupt protein function and compromise DNA repair fidelity. To address these limitations, we developed fluorescence-embedded gold nanoparticles (femAuNPs) conjugated with the recombinant Escherichia coli RecA (rRecA) protein. Biochemical and biophysical characterization revealed that the protein’s native functions, including DNA-binding and ATPase activity, were preserved postconjugation. Live cell imaging using ciprofloxacin to induce DNA DSBs revealed that femAuNP-rRecA localized to form discrete foci corresponding to sites of break. Time-lapse imaging further revealed that these femAuNP-rRecA dynamically colocalized with endogenous RecA-GFP assemblies, appearing rapidly after DSB induction and persisting throughout the repair process. These conjugates exhibit better performance compared to fluorescent-fusion proteins and eliminate the need for genetic manipulation. This study describes a fluorescent nanoparticle-based biosensor for monitoring DNA repair in real-time, highlighting its potential for theranostic applications.