Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells.
DNA breaks in eukaryotic cells can be detected by alkaline electrophoresis of cells embedded in agarose. DNA containing breaks extends in the direction of the anode forming an image resembling the tail of a comet. We have adapted this procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m-2 and incubation for as little as 5 min. We have also used SCGE to study UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, we have incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examining the consequent breaks both by SCGE and by alkaline unwinding in order to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10(9) daltons are detected.
Gedik
C M
CM
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, UK.
Ewen
S W
SW
Collins
A R
AR
D004249:DNA Damage; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000528:radiation effects*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm