Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.
Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.
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DOI：
10.1021/jacs.5b00669
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发表时间：
2015-03-25
影响因子：
15
通讯作者：
Gates KS
中科院分区：
文献类型：
--
作者：
Catalano MJ;Liu S;Andersen N;Yang Z;Johnson KM;Price NE;Wang Y;Gates KS
最近发现了一种新型的链间交联，这种交联是由 DNA 脱碱基位点与双螺旋相对链上的鸟嘌呤残基反应产生的，但该交联的化学连接性尚未严格建立。这里描述的工作旨在表征双链 DNA 中生成的 dG-AP 交联的化学结构和特性。该方法涉及对含有 dG-AP 交联的 DNA 双链体进行酶消化所释放的核苷交联“残余物”的表征。我们首先对由连接到 dG 环外 N2-氨基的 2-脱氧核糖加合物组成的假定交联残余物 9b 进行了化学合成和完整的光谱结构测定。还制备了交联残余物的还原类似物(11b)。液相色谱-串联质谱 (LC-MS/MS) 分析表明，合成标准品 9b 和 11b 的保留时间和质谱特性分别与酶消化含有天然和还原 dG-AP 交联的双链体释放的真实交联残留物相匹配。这些结果建立了双链 DNA 释放的 dG-AP 交联的化学连通性，并为检测生物样品中的这种病变提供了基础。双链 DNA 中的 dG-AP 交联非常稳定，在 pH 7 和 23 °C 下分解的半衰期为 22 天。 dG-AP 交联的内在化学稳定性表明，双链 DNA 中的这种损伤可能能够阻断参与转录和复制的 DNA 加工酶。
A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG–AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link “remnant” released by enzymatic digestion of DNA duplexes containing the dG–AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N2-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography–tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG–AP cross-link, respectively. These results establish the chemical connectivity of the dG–AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG–AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG–AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.