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(PP020) The effects of hydration on strand break formation due to direct ionization of plasmid DNA.
Purkayastha, Shubhadeep*,1, Milligan, Jamie2, Bernhard, William1, 1 Department of Biochemistry & Biophysics, Rochester, New York, United States2 Department of Radiology, La Jolla, California, United States
ABSTRACT- The purpose of the study was to determine how the free radical formation in X-irradiated DNA correlates with the induction of single strand break (ssb) and double strand break (dsb) as a function of hydration (). Solid state films of pUC18 (2686 bp) plasmids were hydrated to in the range 2.5 < < 22.5 mol water/mol nucleotide. 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 in the exact same samples of X-irradiated plasmid DNA. We determined the yields of (i) total DNA radicals, G(fr), (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). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(fr) is seen to increase from 0.28 ± 0.01 mol/J at = 2.5 to 0.63 ± 0.01 mol/J at = 22.5, respectively. Gtotal(ssb) is seen to increase in a comparable fashion from 0.10 ± 0.01 mol/J at = 2.5 to 0.15 ± 0.01 mol/J at = 22.5, respectively. The ratio Gtotal(ssb)/G(fr) is observed to decrease from 0.38 ± 0.05 at = 2.5 to 0.23 ± 0.03 at =22.5. The ratio of total ssb to dsb in pUC18 is found to vary between 18 ± 4 and 20 ± 4 over the range of . The most provocative finding is that in pUC18 films, irrespective of , the yield of total single strand breaks exceeds the yield of trapped sugar radicals, i.e. Gtotal(ssb)>Gsugar(fr). We suggest, therefore, that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. Alternate reaction pathways are considered. A novel mechanism, whereby strand breaks are produced by two one-electron oxidations at a single site, is proposed. The first oxidation is by direct ionization and the second is by a mobile guanine radical cation. Supported by NCI Grants 2-R01-CA32546 (WAB) and 2-R01-CA46295 (JRM).
Key words: direct effect, free radical yield, strand break yield, epr
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