A novel DNA double-strand breaks biosensor based on fluorescence resonance energy transfer

Revealing the spatiotemporal behavior of DNA double-strand breaks (DSBs) is crucial for understanding the processes of DNA damage and repair. Traditionally, gamma H2AX and DNA damage response (DDR) factors have been used to detect DSBs using classical biochemical assays, such as antibody-based immunostaining. However, a reliable method to visualize and assess DSB activity real-time in living cells is yet to be established. Herein, we developed a novel DNA double-strand breaks biosensor (DSBS) based on fluorescence resonance energy transfer (FRET) by employing the H2AX and BRCT1 domains. By applying FRET imaging with DSBS, we show that DSBS specifically reacts to drug- or ionizing radiation (IR)-induced gamma H2AX activity, allowing for the quantification of DSB events at high spatiotemporal resolutions. Taken together, we provide a new experimental tool to evaluate the spatiotemporal dynamics of DNA double-strand breaks. Ultimately, our biosensor can be useful for elucidating the molecular mechanisms underlying DNA damage and repair processes.

Automated summary

Understanding the spatiotemporal behavior of DNA double-strand breaks (DSBs) is crucial for studying DNA damage and repair processes. Traditional methods rely on biochemical assays, such as immunostaining, to detect DSBs. However, a reliable technique for real-time visualization and assessment of DSB activity in living cells is still lacking. In this study, we developed a novel DNA biosensor based on fluorescence resonance energy transfer (FRET), which specifically reacts to gamma H2AX activity induced by drugs or ionizing radiation (IR). Our biosensor enables the quantification of DSB events at high spatiotemporal resolutions, providing a new experimental tool for studying DNA double-strand breaks. This biosensor has the potential to elucidate the molecular mechanisms underlying DNA damage and repair processes.