Oxidative damage to a supercoiled DNA by water soluble peroxyl radicals characterized with DNA repair enzymes.

Earlier work (Paul, T., et al. (2000) Biochemistry 39, 4129-4135) has demonstrated that the water soluble positively charged peroxyl radical, (H(2)N)(2)(+)CC(CH(3))(2)OO(*) ((+)AOO(*)), caused direct strand scission of the Escherichia coli plasmid supercoiled DNA, pBR 322, with ca. 50% scission occurring at a (+)AOO(*)/base pair (bp) ratio of 0.2. There was no measurable direct scission with a negatively charged peroxyl ((-)BOO(*)) at (-)BOO(*)/bp = 24, nor with a neutral peroxyl (COO(*)) at COO(*)/bp = 5. Base modification (BM) of the same DNA by the same peroxyls has now been investigated using four base excision repair (BER) glycosylases. At (+)AOO(*)/bp = 0.04, there is 10% direct strand scission, and the Fpg protein recognized an additional 25% BM, while endonuclease (Endo) IV recognized an additional 20% BM and the other two BER enzymes did not give statistically significant BMs. None of the BER enzymes showed BMs in the DNA treated with -BOO(*). However, Fpg and Endo IV showed that at COO(*)/bp = 3.4 there was a BM comparable to that observed at (+)AOO(*)/bp = 0.04. Thus, COO(*) radicals are only ca. 1.2% as reactive toward the DNA's bases as (+)AOO(*). These results underline the importance of Coulombic forces in DNA reactions. It is also proposed that (+)AOO(*) has a higher intrinsic reactivity in H-atom abstractions and electron transfer processes than (-)BOO(*) or COO(*) radicals.

Cleavage of supercoiled DNA by horseradish peroxidase plus tert-butyl hydroperoxide is not due to tert-butylperoxyl radicals.

Two supercoiled (SC), double-stranded DNAs, pBR 322 and pUC 19, have been subjected to oxidative stress using horseradish peroxidase (HRP) and HRP + tert-butyl hydroperoxide (BOOH). HRP alone causes single-strand cleavage of these SC DNAs. Strand cleavage is enhanced substantially in the presence of commercial BOOH (which contains H(2)O(2)) but is, at best, only very slightly enhanced in the presence of pure BOOH. In the HRP/pure BOOH system, the DNA single-strand-scission which does occur appears to be due to a direct action of oxidized HRP. It is not due to tert-butylperoxyl radicals because strand-scision is not even retarded by 10 mM Trolox, an outstanding water-soluble trap for peroxyl radicals. The present results are congruent with our earlier conclusion [Paul, T., et al. (2000) Biochemistry 39, 4129] that neutral alkylperoxyl radicals produce little or no direct single-strand-scission in SC DNAs.