Two-Wavelength Fluorescence Assay for DNA Repair

• Published in: Analytical biochemistry 2000-12, Vol.287 (2), p.313-318
• Main Authors: Roguev, Assen, Russev, George
• Format: Article
• Language: English
• Subjects: Cell Line; DNA Damage; DNA Repair; Green Fluorescent Proteins; HEK293 cells; host cell reactivation assay; Humans; Luminescent Proteins - genetics; Spectrometry, Fluorescence - methods; Ultraviolet Rays
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4865

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.