Cao Huachuan, Jiang Yong, Wang Yinsheng
Department of Chemistry, University of California, Riverside, California 92521-0403, USA.
J Am Chem Soc. 2007 Oct 10;129(40):12123-30. doi: 10.1021/ja072130e. Epub 2007 Sep 18.
Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.
甲基乙二醛是一种高反应性的α-酮醛，它在体内产生，存在于环境和食物中。它可修饰DNA和蛋白质以形成晚期糖基化终产物（AGEs）。新出现的证据表明，N2-(1-羧乙基)-2'-脱氧鸟苷（N2-CEdG）是AGE相关DNA加合物的主要标志物。在此，我们首次报道了通过寡聚后合成方法制备含有N2-CEdG单个非对映异构体的寡脱氧核糖核苷酸（ODN），这为研究该损伤的复制和修复提供了可靠的底物。此外，从解链温度数据得出的热力学参数表明，N2-CEdG的两种非对映异构体显著破坏了双螺旋结构，在25℃下形成双链体的吉布斯自由能增加4千卡/摩尔。引物延伸试验结果表明，N2-CEdG的两种非对映异构体均可显著阻断由大肠杆菌DNA聚合酶I的无外切核酸酶活性的Klenow片段介导的复制合成。引人注目的是，与正确的核苷酸dCMP相比，聚合酶更优先地在损伤位点掺入错误的核苷酸dGMP和dAMP。
