An Effect of Elevated Postirradiation pH on the Yield of Double-Strand Breaks in DNA from Irradiated Bacterial Cells

Abstract

Exposure of DNA isolated from irradiated cells of Escherichia coli to a pH of 9.6 caused a marked increase in the yield of double-strand breaks (dsb). The dsb were measured by sedimentation analysis of E. coli chromosomal DNA using neutral sucrose gradients. After incubation for 4 hr at 37 degrees C and pH 9.6 the dsb yields were 95% and 71% higher than when incubation was at pH 7.0 for irradiation under oxic and anoxic conditions, respectively. This effect was not apparent when dsb were induced enzymatically and it was linearly related to radiation dose. After oxic irradiation, the increase in dsb at pH 9.6 was consistent with first-order kinetics over greater than 2 half-lives (t1/2 = 1.6 hr at 37 degrees C). The effect of elevated pH was largely additive to a previously reported increase in dsb yield caused by ethanol. It is proposed that the effects of elevated pH and of ethanol revealed the presence in intracellularly irradiated DNA of previously unidentified sites where both strands of the DNA were damaged as a result of single radiation events. The possible nature of the proposed sites and the relevance of these findings to the "neutral" elution technique are discussed.