Evidence for Induction of DNA Double-Strand Breaks at Paired Radical Sites

Isolated plasmid pBR322 DNA was irradiated in the gas explosion apparatus in the presence of 10 mmol${\rm dm}^{-3}$ GSH. By varying the time of the oxygen shot relative to the 5-ns pulse of electrons, the chemical repair kinetics of the oxygen-dependent free-radical precursors of DNA single- and double-strand breaks (SSBs and DSBs) can be determined. The first-order repair rate of the SSB precursors was$1370\ {\rm s}^{-1}$ in comparison to$2900\ {\rm s}^{-1}$ for DSB precursors. Under these conditions the oxygen enhancement ratio for SSBs was 3.0 in comparison to 7.5 for DSBs. This twofold difference in chemical repair rate may be interpreted on the basis of the free-radical precursor of a DSB consisting of two radicals, one on either strand of the DNA. With the chemical repair of one or other of these radicals by hydrogen atom donation from GSH, a DSB is not produced. This process will occur at twice the rate of the chemical repair of an SSB precursor consisting of a single radical. These data are consistent with the concept that DSBs are formed at the sites of clustered energy depositions with the production of a paired radical.