Stover James S, Chowdhury Goutam, Zang Hong, Guengerich F Peter, Rizzo Carmelo J
Department of Chemistry, Center in Molecular Toxicology, and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235-1822, USA.
Chem Res Toxicol. 2006 Nov;19(11):1506-17. doi: 10.1021/tx0601455.
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is found in cooked meats and forms DNA adducts at the C8- and N2-positions of dGuo after appropriate activation. IQ is a potent inducer of frameshift mutations in bacteria and is carcinogenic in laboratory animals. We have incorporated both IQ-adducts into the G1- and G3-positions of the NarI recognition sequence (5'-G1G2CG3CC-3'), which is a hotspot for arylamine modification. The in vitro replication of the oligonucleotides was examined with Escherichia coli pol I Klenow fragment exo-, E. coli pol II exo-, and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and the extension products were sequenced by tandem mass spectrometry. Replication of the C8-adduct at the G3-position resulted in two-base deletions with all three polymerases, whereas error-free bypass and extension was observed at the G1-position. The N2-adduct was bypassed and extended by all three polymerases when positioned at the G1-position, and the error-free product was observed. The N2-adduct at the G3-position was more blocking and was bypassed and extended only by Dpo4 to produce an error-free product. These results indicate that the replication of the IQ-adducts of dGuo is strongly influenced by the local sequence and the regioisomer of the adduct. These results also suggest a possible role for pol II and IV in the error-prone bypass of the C8-IQ-adduct leading to frameshift mutations in reiterated sequences, whereas noniterated sequences result in error-free bypass.
2-氨基-3-甲基咪唑并[4,5-f]喹啉（IQ）存在于熟肉中，经过适当活化后会在脱氧鸟苷（dGuo）的C8和N2位置形成DNA加合物。IQ是细菌中移码突变的强效诱导剂，在实验动物中具有致癌性。我们已将两种IQ加合物分别引入NarI识别序列（5'-G1G2CG3CC-3'）的G1和G3位置，该序列是芳胺修饰的热点区域。使用大肠杆菌DNA聚合酶I Klenow片段（外切酶缺陷型）、大肠杆菌DNA聚合酶II（外切酶缺陷型）和嗜热栖热菌P2 DNA聚合酶IV（Dpo4）检测寡核苷酸的体外复制情况，并通过串联质谱对延伸产物进行测序。G3位置的C8加合物在所有三种聚合酶作用下的复制均导致两碱基缺失，而G1位置则观察到无错误的跨越和延伸。当位于G1位置时，所有三种聚合酶都能绕过并延伸N2加合物，且观察到无错误产物。G3位置的N2加合物具有更强的阻断作用，仅Dpo4能绕过并延伸该加合物以产生无错误产物。这些结果表明，dGuo的IQ加合物的复制受到局部序列和加合物区域异构体的强烈影响。这些结果还表明，DNA聚合酶II和IV可能在导致重复序列移码突变的C8-IQ加合物的易错跨越中发挥作用，而单拷贝序列则导致无错误跨越。
