Using an electrophoresis assay system developed in our laboratory, we have simultaneously measured single- and double-strand DNA breaks (SSBs and DSBs) induced by γ radiation in small SV40 viral DNA molecules, under conditions of greatly varying radical scavenger concentration and DNA configuration. In our experiments with aqueous solutions of SV40 DNA, we observe that SSB induction is linear with dose (one-hit response), over the entire hydroxyl scavenger efficiency range examined, from approximately 0 to$5\times 10^{9}\ {\rm s}^{-1}$, while DSB induction shifts from having a major quadratic component (two-hit response) at very low scavenger efficiencies to nearly pure linear for efficiencies >$10^{7}\ {\rm s}^{-1}$. The mean ratio of SSBs to one-hit DSBs remains relatively constant with increasing scavenger efficiency, decreasing from about 100:1 to 40:1 as the scavenger efficiency increases from$2\times 10^{5}\ {\rm s}^{-1}$ to$5\times 10^{9}\ {\rm s}^{-1}$, and the absolute induction efficiencies for breaks decrease by three orders of magnitude. This decrease takes place primarily at scavenger efficiencies above$1\times 10^{8}\ {\rm s}^{-1}$. Irradiation of intranuclear SV40 minichromosomes induces SSBs and DSBs at nearly the same efficiencies as does irradiation of free DNA at the highest scavenger concentrations examined, and at only about twice the efficiencies observed at -75°C, where direct effects are believed to predominate. Our observations that the linear-quadratic mix of the dose-response curve for DSBs depends critically on scavenger efficiency may help to clarify the considerable confusion in the literature on the shape of such curves. Our observations of a relatively constant ratio between one-hit SSBs and DSBs at low and moderate scavenger efficiencies are in agreement with the recent hypothesis of Siddiqi and Bothe (Radiat. Res. 112, 449-463 (1987)) that, contrary to widely and long-held beliefs, the formation by indirect effects of a one-hit DSB in DNA occurs under these conditions predominantly by a mechanism involving a single OH radical, with a presumed radical transfer between complementary DNA strands. In contrast, our results for strongly protective conditions are not consistent with this hypothesis, but are consistent with the predictions of Ward's hypothesis (Radiat. Res. 86, 185-195, (1981)) that one-hit DSBs from indirect effects are produced predominantly by local clusters of OH radicals from single energy deposition events (locally multiply damaged sites) rather than by single OH radicals. Finally, comparison of our intranuclear observations with our observations at -75°C suggests, but does not establish conclusively, that in the living cell all indirect effects are responsible for only about half of observed radiation-induced DSBs, with the other half resulting from direct effects.

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