The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells

Abstract

Single cell gel electrophoresis is a sensitive method for detecting DNA strand breaks. Cells embedded in agarose are converted to nucleoids by treating with detergent and high salt. DNA breaks render the nucleoid DNA susceptible to extension by electrophoresis, forming 'comets'. We find that when DNA breakage resulting from H2O2 treatment is examined, freshly isolated normal human lymphocytes are relatively resistant compared with transformed human cells. When incubated after treatment with H2O2, HeLa cells repair most strand breaks within 1 h, and a substantial fraction of the oxidised pyrimidines (detected by converting them to DNA breaks with endonuclease III) within 4 h. However, lymphocytes are less proficient at repair; during incubation for 4 h after treatment with H2O2, no detectable removal of endonuclease III-sensitive sites is seen. While the addition of deoxyribonucleosides promotes completion of repair of UV damage by lymphocytes, it has no significant effect on repair of oxidative damage.