A novel DNA damage detection method based on a distinct DNA damage response system

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA damage arises from various environmental and internal cellular stresses.
• Single-stranded DNA (ssDNA) is a critical signaling molecule in DNA damage response pathways.
• The Deinococcus PprI protein's ability to bind ssDNA and cleave DdrO protein is crucial for radiation resistance.

Purpose of the Study:

• To develop a quantitative in vitro method for detecting DNA damage.
• To leverage the ssDNA-binding property of PprI and its interaction with DdrO for damage detection.
• To establish a fluorescence resonance energy transfer (FRET)-based assay for ssDNA quantification.

Main Methods:

• Engineered a DdrO protein construct with N-terminal eYFP and C-terminal eCFP fusion proteins.
• Utilized FRET efficiency between eYFP and eCFP as a readout for DdrO cleavage.
• Developed a standard curve correlating FRET efficiency with ssDNA concentration.

Main Results:

• Demonstrated that FRET efficiency directly reflects DdrO cleavage, which is dependent on ssDNA presence.
• Successfully constructed a standard curve for quantifying ssDNA concentrations.
• Validated the method's effectiveness through application examples.

Conclusions:

• The developed FRET-based assay provides a sensitive and quantitative method for detecting DNA damage in vitro.
• This assay can accurately measure ssDNA concentrations, serving as a reliable biomarker for DNA damage.
• The findings offer a valuable tool for research in DNA repair, radiation biology, and genotoxicity testing.