Radiation-Induced Double-Strand Scission of the DNA of Mammalian Metaphase Chromosomes

The molecular weight distribution of double-stranded mammalian (hamster) DNA was determined by ultracentrifugation of isolated metaphase chromosomes previously layered onto sucrose gradients containing high salt concentrations to dissociate the protein and nucleic acid components. In untreated controls the distribution (as determined by counting the incorporated radioactivity in the resultant fractions) exhibited a peak at <tex-math>$225\times 10^{6}$</tex-math> daltons. Inclusion of mercaptoethanol and hydroxylamine into the gradients produced no significant change of these sedimentation patterns. Gamma-radiation-induced reduction in the number and weight average molecular weights was used to calculate a value of <tex-math>$1.05\times 10^{11}$</tex-math> double-strand breaks/gram rad, equivalent to about 600 eV/break. No significant difference was observed for chromosomes irradiated either before or after isolation from intact mitotic cells. Irradiation in the presence of cystamine resulted in at least a sevenfold reduction in the apparent double-strand scission. The observed sedimentation patterns were compared with those generated by a theoretical computer simulation of radiation-induced degradation which assumed random selection and breakage of molecules. These results suggested that at least 80 to 90% of the isolated DNA was distributed approximately normally with a mean molecular weight of about 200× 10⁶ daltons and a standard deviation of about 50× 10⁶ daltons.