Single strand breakage and repair in eukaryotic DNA as assayed by S1nuclease

Abstract

A sensitive new approach for measuring the repair of single strand breaks 1n DNA induced by low doses of gamma irradiation was tested 1n cultured fibroblasts from Chinese hamster lung, human afflicted with ataxia telangiectasia or Fanconi's anemia and 1n normal cells of early and late passages. The assay 1s based on the increasing rate of strand separation of DNA duplexes in alkali for molecules with increasing numbers of single strand scissions. DNA strand separation 1s shown to follow the relation, 1n F + -(1/M_n. const) .t^β where F 1s the proportion of double-stranded DNA, detected as S1 nuclease resistant, after alkaline denaturation time, t. M_n 1s the number-average molecular weight of DNA between single strand breaks. β<1 is an empirically determined constant. The results suggest an increase 1n the number-average molecular weight between breaks, Mn, with increasing times for repair. The final level attained corresponds to the Mn of control DNA in unirradiated cells. As few as one break introduced into 109 daltons of single-stranded control cell DNA can be detected. The kinetics, requirements and sensitivities of this assay are described.