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pubmed:abstractText |
DNA strand breaks can result as a direct or indirect consequence of oxidative damage to the nucleic acid bases and/or deoxyribose sugars. Ionizing radiation and the antitumor agents, the bleomycins (BLMs) and enediynes, share in common the ability to indirectly cause DNA strand scission after C4' hydrogen atom abstraction from the deoxyribose moiety. In the case of extensively studied BLMs, the C4' radical generated under anaerobic conditions results in production of a 4'-keto abasic site after C4' oxidation to a cation and H(2)O addition. To study the structure, stability, and repair of this lesion, a general method is reported for its homogeneous preparation in any sequence context. 4'-Azido-2'-deoxyuridine-5'-triphosphate is incorporated into duplex DNA using a primer, a template containing a restriction enzyme (NgoM IV) cleavage site at its 3'-end, and HIV-1 reverse transcriptase. The two strands of the duplex are separated based on size after cleavage with the restriction enzyme. The single-stranded (ss) DNA containing 4'-azido-2'-deoxyuridine, when treated with uracil-DNA glycosylase, results in quantitative release of uracil, azide, and generation of a ss-DNA containing the 4'-keto abasic site. This lesion is characterized directly by MALDI-TOF MS and indirectly by subsequent reduction, enzymatic digestion, and GC/MS. The stability of duplex DNA containing a 4'-keto abasic site relative to an abasic site in the same sequence context is reported under physiological conditions.
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