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

Shitong Zhong1, Shuang Song1, Linjia Wang1

  • 1MOE Key Laboratory of Biosystems Homeostasis & Protection, Institute of Biophysics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.

Microbial Biotechnology
|September 17, 2024
PubMed

Insights

Researchers developed a new in vitro method to detect DNA damage using fluorescence resonance energy transfer (FRET). This technique quantifies single-stranded DNA (ssDNA) levels, a key indicator of cellular stress and damage.

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.

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