Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of t he UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced gr een fluorescent protein (EGFP). As a reference we used the plasmid pEYFP ca rrying 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 w ith a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This ma kes it possible to monitor the expression of the two genes simultaneously b y measuring the fluorescence at two wavelengths. HEK293 cells were cotransf ected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At differe nt time intervals after transfection the fluorescence of EGFP was determine d relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used t o calculate the number of UV lesions in DNA and hence the repair capacity o f the host cells. It was found that HEK293 cells were able to repair approx imately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average. (C ) 2000 Academic Press.