Site specific radical action in DNA oligonucleotides
thesisposted on 15.12.2014, 10:35 by Zara Anne. Doddridge
The interaction of ionising radiation and reactive oxygen species with DNA results in a plethora of radical intermediates and final products, making the determination of specific mechanisms problematic. To assist in the elucidation of individual mechanisms, oligonucleotides containing modified bases that are (i) photolabile precursors of defined single radical intermediates, or (ii) sites which are sensitive to oxidative effects, have been examined.;Towards (i), UV-induced strand breakage in BrdU-containing DNA oligonucleotides has been investigated. Aerobic photolysis proceeds with the specific loss of the nucleoside immediately 5' to BrdU and yields strand breaks possessing phosphate termini. This is consistent with a BrdU-derived base peroxyl radical-mediated abstraction of the C'1 H-atom from the neighbouring 5'deoxyribose. Studies of duplex systems revealed that cleavage occurs predominantly in the BrdU-containing strand, with the yields of breakage being dependent on both conformation and sequence. Studies using ionizing radiation revealed site-specific cleavage only when the oligonucleotides were irradiated in solution in the presence of scavenger; irradiation of the oligonucleotides as a 'dry-film' or in dilute aqueous solution failed to yield evidence of specific damage.;Towards (ii), the unique aspect of the key oxidative purine DNA base lesion, 7,8-dihydro-8-oxo-guanine, acting as a locus for further oxidative action has been investigated. The observations of site-specific damage at the 7,8-dihydro-8-oxo-guanine site resulting from radiogenic direct and indirect effects, and peroxyl radical action are interpreted in terms of the facile migration of charge along the DNA and the migration of oxidising radicals to sensitive sites on DNA, respectively. These observations suggest that redox-sensitive base lesions, that already exist or are formed in the genome, may act as sensitive sites ('hot' spots) for radiogenic and oxidative effects and furthermore, raise doubts about the reliability of using the 7,8-dihydro-8-oxo-guanine lesion as a sole marker of oxidative stress.