Intermediates in excision repair by human cells: use of S1 nuclease and benzoylated naphthoylated cellulose to reveal single-strand breaks

Abstract

Treatment of human lymphoid cells with methyl methanesulfonate (MMS) results in discontinuities in cellular DNA. Discontinuities can be detected by sedimentation through neutral sucrose gradients after digestion with S1 nuclease on top of the gradient and by benzoylated naphthoylated diethylaminoethylcellulose (BND-cellulose) chromatography. DNA with discontinuities is sensitive to shear which produces single-stranded regions and causes the DNA to adhere to BND-cellulose. The shear-sensitive and S1-sensitive sites produced by MMS treatment are probably identical. Treatment of cells with acetoxyacetylaminofluorene (AAAF) does not result in detectable discontinuities either at doses which produce the same number of adducts as observed after MMS treatment or at doses which inhibit DNA excision repair. Addition of aromatic adducts to DNA can also result in adherence to BND-cellulose but such DNA is not S1 sensitive. We conclude that MMS- and AAAF-induced damage are repaired by different pathways in human cells and that DNA with discontinuities is a readily detectable intermediate only in the repair of MMS damage by the apurinic pathway.