| Direct-type damage is the composite of the damage arising from direct ionization of DNA or from the transfer of dry electrons and holes to the DNA from its surrounding hydration waters. Here we test the hypothesis that the majority of DNA strand breaks produced by direct-type effects are due to sugar free radical precursors. The free radical dose response for crystalline DNA samples irradiated at 4 K was determined using electron paramagnetic resonance (EPR) spectroscopy. It was found that the dose response for sugar radicals correlates well with that of strand break products, supporting the hypothesis.; In X-irradiated plasmid DNA, free radical yields were measured by EPR at 4 K, and single strand breaks (ssb) and double strand breaks (dsb) were measured by gel electrophoresis. We determined the yields of (i) total DNA radicals, G(Sigmafr), (ii) sugar trapped radicals, Gsugar (fr), (iii) prompt single strand breaks, Gprompt(ssb), (iv) total single strand breaks, Gtotal(ssb), and (v) double strand breaks, G(dsb). The most provocative finding is that the yield of total single strand breaks exceeds the yield of trapped sugar radicals, i.e. G total(ssb) > Gsugar(fr). A significant fraction of strand breaks, therefore, is derived from precursors other than trapped DNA radicals. A novel mechanism is proposed whereby strand breaks are produced by two one-electron oxidations at a single site; the first oxidation is by direct ionization and the second is by a mobile guanine radical cation.; The role of DNA hydration (Gamma) in the induction of strand breaks and base lesions in pUC18 plasmid was investigated. The G(Sigmafr) and G total(ssb), are found to increase in tandem from Gamma = 2.5 to Gamma = 22.5 waters/nucleotide. The ratio Gtotal(ssb)/Gsugar(fr) however, is seen to decrease with increasing hydration. These findings argue in favor of the double oxidation at one site mechanism. The yield of specific types of base lesions was measured using base-excision repair endonucleases, Fpg and Nth. Base damage and strand break yields were found to be comparable. A working model that explains end product formation by direct effects in DNA is presented. |
