Abstract
DNA damage occurs when cells encounter exogenous and endogenous stresses such as long periods of desiccation, ionizing radiation and genotoxic chemicals. Efforts have been made to detect DNA damage in vivo and in vitro to characterize or quantify the damage level. It is well accepted that single-stranded DNA (ssDNA) is one of the important byproducts of DNA damage to trigger the downstream regulation. A recent study has revealed that PprI efficiently recognizes ssDNA and cleaves DdrO at a specific site on the cleavage site region (CSR) loop in the presence of ssDNA, which enables the radiation resistance of Deinococcus. Leveraging this property, we developed a quantitative DNA damage detection method in vitro based on fluorescence resonance energy transfer (FRET). DdrO protein was fused with eYFP and eCFP on the N-terminal and C-terminal respectively, between which the FRET efficiency serves as an indicator of cleavage efficiency as well as the concentration of ssDNA. The standard curve between the concentration of ssDNA and the FRET efficiency was constructed, and application examples were tested, validating the effectiveness of this method.