Probing DNA superstructure in human quiescent lymphocytes by X-ray-induced double-strand breakage

Abstract

Human quiescent lymphocytes were lysed onto neutral sucrose gradients in order to sediment subsequently the nuclear DNA released within nucleoids. The position of nucleoids in the centrifuge tubes was detected fluorometrically by using the dye, ethidium bromide, and the height of the fluorescence peak was taken as a measure of DNA content. X-irradiation of lymphocytes, before their lysis, altered the DNA content of nucleoids and their sedimentation rate in accord with the view that (1) nuclear DNA is attached along its length at distances corresponding to 1.7 x 10(10) g/mol, and that (2) X-ray-induced double-strand breakage releases DNA fragments at random. Incubation at 37 degrees C of irradiated lymphocytes restored the amount of attached DNA as it would be expected from an intracellular repair process for DNA double-strand breaks.