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.