Novel Method For Quantifying Radiation-Induced Single-Strand-Break Yields In Plasmid DNA Highlights Tenfold Discrepancy

摘要

The widely used agarose gel electrophoresis method for assessing radiation-induced single-strand-break (SSB) yield in plasmid DNA involves measurement of the fraction of relaxed-circular (C) form that migrates independently from the intact supercoiled (SC) form. We rationalized that this method may underestimate the SSB yield since the position of the relaxed-circular form is not altered when the number of SSB per DNA molecule is >1. To overcome this limitation, we have developed a novel method that directly probes and quantifies SSBs. Supercoiled ³H-pUC19 plasmid samples were irradiated with γ-rays, alkali denatured, dephosphorylated, kinated with γ–[³²P-ATP], and the DNA-incorporated ³²P activities used to quantify the SSB yields per DNA molecule employing a standard curve generated using DNA molecules containing known number of SSBs. The same irradiated samples were analyzed by agarose gel and SSB yields were determined by conventional method. Comparison of the data demonstrated that the mean SSB yield per plasmid DNA molecule of [21.2 ± 0.59] × 10⁻² Gy⁻¹ as measured by direct probing, is ~10-fold higher than that obtained from the conventional gel-based method. These findings imply that the SSB yields inferred from agarose-gels need re-evaluation, especially when they were utilized in the determination of radiation risk.