Ziegel Rebecca, Shallop Anthony, Upadhyaya Pramod, Jones Roger, Tretyakova Natalia
Department of Medicinal Chemistry, University of Minnesota School of Pharmacy, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2004 Jan 20;43(2):540-9. doi: 10.1021/bi035259j.
All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously methylated CG sites within the p53 tumor suppressor gene.
p53肿瘤抑制基因第5至8外显子上的所有CG二核苷酸均含有内源性5-甲基胞嘧啶（MeC）。这些相同的位点（如密码子157、158、245、248和273）是吸烟诱导的肺癌中的突变热点。几个研究小组使用UvrABC核酸内切酶切割试验证明，p53基因的甲基化CG二核苷酸是湾区多环芳烃（PAH）二醇环氧化物的优先结合位点。相比之下，内源性胞嘧啶甲基化对烟草特异性亚硝胺（如4-（甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮（NNK））诱导的DNA损伤分布的影响尚未阐明。在本文介绍的工作中，采用了一种稳定同位素标记的HPLC-ESI-MS/MS方法，来分析半甲基化和完全甲基化CG二核苷酸中鸟嘌呤的N7和O6位置对NNK衍生的甲基化和吡啶氧丁基化物种的反应活性。将15N3标记的鸟嘌呤碱基置于代表内源性甲基化p53密码子154、157和248的合成DNA序列中，然后用NNK重氮氢氧化物的乙酰化前体进行处理。HPLC-ESI-MS/MS分析用于确定15N3标记位置处N7-和O6-鸟嘌呤加合物的相对产率。在所有情况下，MeC的存在抑制了相邻G处N7-甲基鸟嘌呤、O6-甲基鸟嘌呤和O6-吡啶氧丁基鸟嘌呤的形成，在完全甲基化的二核苷酸和MeC之前的鸟嘌呤处观察到最大程度的减少。此外，在5'-相邻MeC存在的情况下，O6-Me-dG/N7-Me-G摩尔比降低，这表明观察到的O6-烷基鸟嘌呤加合物产率下降至少部分是甲基化CG二核苷酸中反应活性模式改变的结果。这些结果表明，与PAH二醇环氧化物的N2-鸟嘌呤加合物不同，NNK诱导的N7-和O6-烷基鸟嘌呤加合物并非优先在p53肿瘤抑制基因内源性甲基化的CG位点形成。
