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(PP087) Novel methods for quantifying ionizing radiation-induced deoxyribose oxidation in DNA.
Zhou, Xinfeng*,1, Chen, Bingzi1, Yi, Huilan1, Jiang, Tao1, Dedon, Peter1, 1 Biological Engineering Division, Cambridge, MA, USA
ABSTRACT- Deoxyribose oxidation in DNA plays an important role in the cellular response to oxidative stress induced by ionizing radiation and free radical agents. We have therefore developed a variety of sensitive analytical methods to quantify different deoxyribose oxidation products and their secondary derivatives formed under biological conditions. We now report a rapid and highly sensitive (1/106nt using 1 g DNA) 14C labeling/accelerator mass spectrometry (AMS) method to quantify strand breaks and abasic sites, as well as its extension to quantify base lesions. This method offers an index for total DNA deoxyribose oxidation. We also report gas chromatography-mass spectrometry (GC/MS) methods to identify and quantify products of two different deoxyribose 4'-oxidation pathways in DNA: 3'-phosphoglycolate and 4'-keto-1'-aldehyde abasic site (with a limit of detection of 100 fmol using 170 g of DNA.). These methods were applied to DNA oxidized by a variety of agents, including ionizing radiation, to assess the basis for different partitioning of 4'-chemistry of deoxyribose oxidation. Combined with methods to quantify 3'- and 5'-oxidation products, these results also offer information about the partitioning of oxidation at different deoxyribose carbon positions. Finally, we will report a LC/MS method to study N-formylation of histone lysine residues by 3'-formylphosphate, a product of 5'-oxidation of deoxyribose in DNA. These methods provide new insights into the chemistry of deoxyribose oxidation in DNA in vitro and in cells.
Key words: oxidative DNA damage, deoxyribose oxidation, abasic site, DNA damage chemistry
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