Synthesis and characterization of oligonucleotides containing a 4′-keto abasic site
Synthesis and characterization of oligonucleotides containing a 4′-keto abasic site
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DOI：
10.1021/bi0495376
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发表时间：
2004-05-11
期刊：
影响因子：
2.9
通讯作者：
Stubbe, J
中科院分区：
文献类型：
--
作者：
Chen, JY;Stubbe, J
DNA链断裂可作为对核酸碱基和/或脱氧核糖糖的氧化损伤的直接或间接后果而导致。电离辐射和抗肿瘤剂博来霉素（BLM）和烯二炔共同具有在从脱氧核糖部分夺取C4'氢原子后间接引起DNA链断裂的能力。在广泛研究的BLM的情况下，C4'自由基产生的Linder厌氧条件导致在C4'氧化成阳离子和H2O添加后产生4 '-酮脱碱基位点。为了研究这种病变的结构、稳定性和修复，报告了在任何序列背景下均质制备的一般方法。使用引物、在其3 ′-末端含有限制酶（NgoM IV）切割位点的模板和HIV-1逆转录酶将4 ′-叠氮基-2 ′-脱氧尿苷-5 ′-三磷酸掺入双链体DNA中。双链体的两条链在用限制酶切割后基于大小分离。当用尿嘧啶-DNA糖基化酶处理含有4 ′-叠氮基-2 ′-脱氧尿苷的单链（ss）DNA时，导致尿嘧啶、叠氮化物的定量释放，并产生含有4 ′-酮基脱碱基位点的ss-DNA。该损伤的特征直接通过MALDI-TOF MS和间接通过随后的还原、酶消化和GC/MS来表征。在生理条件下报道了含有4 '-酮基脱碱基位点相对于相同序列背景中的脱碱基位点的双链体DNA的稳定性。
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 Linder anaerobic conditions results in production of a 4'-keto abasic site after C4' oxidation to a cation and H2O 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 enzyrrie. 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.