Two-wavelength fluorescence assay for DNA repair

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA damage is a constant threat to genomic integrity.
• Cellular DNA repair mechanisms are crucial for preventing mutations and disease.
• Accurate measurement of DNA repair capacity is essential for understanding cellular health and disease progression.

Purpose of the Study:

• To develop a simple, reliable, and quantitative assay for measuring cellular DNA repair capacity.
• To assess the DNA repair efficiency of HEK293 cells following UV irradiation.

Main Methods:

• Developed a host cell reactivation assay utilizing UV-irradiated pEGFP and undamaged pEYFP plasmids.
• Co-transfected HEK293 cells with both plasmids to monitor gene expression via fluorescence.
• Quantified EGFP fluorescence relative to EYFP fluorescence at different time points to determine DNA repair capacity.

Main Results:

• The assay allows simultaneous monitoring of two fluorescent proteins (EGFP and EYFP) by measuring fluorescence at distinct wavelengths.
• HEK293 cells demonstrated the ability to repair approximately 1.4 UV lesions per 1000 nucleotides of DNA within a 12-hour period.
• The relative fluorescence measurement effectively compensates for variations in transfection efficiency and experimental variables.

Conclusions:

• The developed assay provides a robust method for quantifying cellular DNA repair capacity.
• HEK293 cells exhibit significant DNA repair activity against UV-induced DNA damage.
• This assay can be a valuable tool for research in DNA repair, toxicology, and cancer biology.