On the Measurement of DNA Double-Strand Breaks by Neutral Elution

Abstract

DNA double-strand breaks unquestionably increase the rate at which DNA is eluted from cell lysates on a filter, in the neutral elution assay introduced by Bradley and Kohn in 1979 (1). Double-strand breaks are generally agreed to be one of the most biologically important types of DNA damage formed by ionizing radiation, and the neutral elution assay has been widely used to detect such breaks. The purpose here is to bring attention to the possibility that neutral elution may not always give a useful measure of the number of double-strand breaks. This letter may be regarded as a follow-up to a comment by Gunner Ahnstrom in 1985 (2), which has not received sufficient attention. If is it assumed that the rate of DNA elution under nondenaturing conditions is controlled fundamentally by double-strand breaks, then according to several studies, the number varies with dose D of ionizing radiation as aD + fD2 (3-9). The presence of the quadratic term is confirmed in neutral elution measurements on cells irradiated by ionizing radiation, with the assay calibrated with '25I-labeled DNA under the assumption that each 125I decay forms a double-strand break (10, 11). Low-speed neutral sucrose gradient sedimentation, on the other hand, shows a linear relation between radiation-induced double-strand breaks and dose (12-17). Therefore at least one of these methods must be giving wrong answers, and it is appropriate to discuss the underlying theories for the two approaches.