Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals wi th human fibroblast DNA. DNA damage consisted of both strand breaks and bas e modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitat ion of fragment size distributions using denaturing glyoxal-agarose gel ele ctrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative bas e modifications were observed to occur to a greater extent than strand brea ks at every concentration measured. The sequence-specific distribution of p eroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine po sitions in the examined sequences was found to be significantly oxidized. T he order of reactivity of DNA bases toward oxidation by peroxyl radicals wa s found to be G much greater than C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by per oxyl radicals depends on the exact specification of 5' and 3' flanking base s in a polarity dependent manner. Every guanine in the 5'XGC3' motif was fo und to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' puri ne flanks drastically reduced the extent of peroxyl radical G oxidation. Th e pattern of base modification and the influence of nearest neighbors diffe rs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequenc es.