Detect Multiply Damaged Sites and Determine Their Yields

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These experiments are designed to optimize the chances of measuring the yields of specific types of multiply damaged sites. They test, among other things, the hypothesis that some stable sites of reductive damage are spatially correlated with a nearby site of sugar oxidation.

The transition of plasmid DNA from a supercoiled to an open circle conformation, as detected by gel electrophoresis, affords an extraordinarily sensitive method for detecting single strand breaks (ssb), one measure of deoxyribose damage. In order to determine the yield of ssb, G(ssb), by this method it is commonly assumed that Poisson statistics apply such that, on average, one ssb occurs per supercoiled plasmid lost. For the direct effect, at a large enough plasmid size, this assumption may be invalid. In this report, the assumption that one ssb occurs per pUC18 plasmid (2686 bp) is tested by measuring freebase (fbr), which is also a measure of deoxyribose damage in films preparedunder controlled relative humidity so as to produce known levels of DNA hydration. The level of DNA hydration, Γ, is expressed in mol water/mol nucleotide. The yield of fbr, G(fbr), was measured by HPLC following exposure of the films to 70 kV X-rays and subsequent dissolution in water. It is well known that damage in deoxyribose leads to ssb and fbr.

Based on known mechanisms,there exists a close correspondence between fbr and ssb, i.e., G(fbr) ≅ G(ssb). Following this assumption, the ssb multiplicity, m(ssb), was determined, where m(ssb) was defined as the mean number of ssb per supercoiled plasmid lost. The yield of lost supercoil was determined previously (S. Purkayastha,, J. Phys. Chem. B 110, 26286-26291, 2006). We found that m(ssb) = 1.4 ±0.2 at Γ = 2.5 and m(ssb) = 2.8 ±0.5 to 3.1 ±0.5 at Γ = 22.5, indicating that the assumption of one ssb per lost supercoil is not likely to hold for a 2686 bp size plasmid exposed to the direct effect. In addition, an increase in G(fbr), upon steppingfrom Γ of 2.5 to 22.5, was paralleled by an increase in the yield of trapped deoxyribose radicals, GdRib(fr), also measured previously. As a consequence, the shortfall between ssb and trapped radicals, G(diff) = G(ssb) – GdRib(fr), remained relatively constant at 90-110 nmol/J. The lack of change between the two extremes of hydration is in keeping with the suggestion that non-radical species, such as doubly-oxidized deoxyribose, are responsible for the shortfall.

Ratio for yield of free base release

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