Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

Area of Science:

• Molecular Biology
• Cell Biology
• Biomedical Research

Background:

• Genome integrity is crucial for preventing diseases like cancer and premature aging.
• Current DNA damage assessment methods have limitations in sensitivity, cost, and applicability.
• A need exists for accessible, high-throughput DNA damage detection techniques.

Purpose of the Study:

• To develop an affordable, semi-automatic fluorescence imaging method for sensitive DNA damage assessment.
• To provide a viable alternative to expensive high-throughput screening devices.
• To facilitate mechanistic insights into DNA damage response (DDR) and its role in human diseases.

Main Methods:

• Transformation of a standard fluorescence microscope and PC into a screening tool.
• Development of a semi-automatic image analysis procedure for adherent cells.
• Utilizing phosphorylated histone H2AX (gamma-H2AX) for DNA breakage and RPA/Rad51 for homologous recombination (HR) assessment.

Main Results:

• Demonstrated high sensitivity, quantitative analysis, and speed for thousands of cells daily.
• Successfully assessed low radiation doses, DNA repair kinetics, and spontaneous damage in cancer cells.
• Validated the method's versatility in studying HR events and their dependence on DDR factors.

Conclusions:

• The presented affordable fluorescence imaging approach offers a sensitive, rapid, and simple alternative for DNA damage assessment.
• This method can significantly aid basic and translational research by facilitating mechanistic insights into DNA damage in various diseases.
• The technique's applicability extends to any research area utilizing suitable fluorescence markers for DNA damage or repair studies.