Sequence Distribution of Tabacco Carcinogen-DNA Adducts

人类基因组中5‘-CG-3’位点的内源性胞嘧啶甲基化参与了许多 生理过程，包括基因表达、宿主防御、基因组印记和X染色体 失活。有趣的是，许多肿瘤类型，包括吸烟引起的肺癌，其特征是 异常的DMA甲基化模式。5-甲基胞嘧啶(MEC)的存在引起的影响很小，但值得注意 DNA结构和动力学的变化，导致DNA-蛋白质相互作用和染色质的改变 改建。此外，MEC能够提高MeCG二核苷酸中鸟嘌呤碱基的反应性 致癌物质。值得注意的是，大多数肺癌的突变“热点”观察到在P53 抑癌基因发现于内源性甲基化的MeCG二核苷酸，例如P53密码子157， 158、245、248和273。我们之前的调查显示，EC在这些地点的存在 调节邻近的鸟嘌呤碱基对烟草致癌物的反应性，导致靶向 苯并[a]芘二环氧化物(BPDE)与MeCG序列的结合。相比之下，MeCG的烷基化反应 烟草特有亚硝胺的反应性代谢物4-(甲基亚硝基)-1-(3-吡啶)-1- 丁酮(NNK)，被抑制。我们现在建议通过以下方式确定这些影响的结构基础 使用一系列MEC类似物进行稳定的同位素标记研究。我们还将检验这一假设 胞嘧啶甲基化状态和局部DMA序列上下文通过反应性影响鸟嘌呤加合物的形成 暴露在烟草烟雾中产生的氧物种。最后，MEC对O6的任何影响- 烷基鸟嘌呤DMA烷基转移酶介导的NNK诱导的O6-烷基鸟嘌呤加合物的修复也将 检查过了。这些研究将会： 1.确定5-甲基胞嘧啶(MEC)影响邻体反应性的机制 鸟嘌呤对烟草致癌物质具有碱基作用。 2.考察MEC及其结构类似物对N_2-鸟嘌呤加合物立体化学的影响 由苯并[a]P二环氧化物诱导。 3.定位氧化型DNA损伤在P53和K-ras衍生DNA序列中的分布。 4.检测O6-烷基鸟嘌呤DNA烷基转移酶对NNK诱导的O6-鸟嘌呤损伤的修复作用。 甲基化和未甲基化的CG二核苷酸。 这项研究的结果将确定内源性MEC调节反应性的机制 Cg位点对烟草致癌物的影响，提供了对遗传和表观遗传起源的洞察 在肺癌中观察到的变化。我们的方法是创新的，因为我们的实验室正在使用一种新颖的， 基于质谱学的方法来探测DNA双链中特定碱基的反应性 烷基化和氧化剂。这项拟议的研究具有重要意义，因为人类广泛存在 与烟草产品的接触以及烟草产品在人类肺癌发病中的中心作用。

Endogenous cytosine methylation at 5'-CG-3' sites within the human genome is involved in many physiological processes, including gene expression, host defense, genomic imprinting, and X chromosome inactivation. Interestingly, many tumor types, including smoking-induced lung cancer, are characterized by aberrant DMA methylation patterns. The presence of 5-methylcytosine (MeC) induces small, but noticeable changes in DMA structure and dynamics, leading to altered DNA-protein interactions and chromatin remodeling. Furthermore, MeC is capable of increasing the reactivity of guanine bases in MeCG dinucleotides towards carcinogens. Remarkably, the majority of lung cancer mutational "hot spots" observed within the p53 tumor suppressor gene are found at endogenously methylated MeCG dinucleotides, e.g. p53 codons 157, 158, 245, 248, and 273. Our previous investigations have revealed that the presence of eC at these sites modulates the reactivity of neighboring guanine bases towards tobacco carcinogens, leading to targeted binding of benzo[a]pyrene diolepoxides (BPDE) to MeCG sequences. In contrast, alkylation of MeCG dinucleotides by reactive metabolites of tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), is inhibited. We now propose to determine the structural basis for these effects by conducting stable isotope labeling studies with a series of MeC analogs. We will also test the hypothesis that cytosine methylation status and local DMA sequence context influence guanine adduct formation by reactive oxygen species generated as a result of exposure to tobacco smoke. Finally, any effects of MeC on O6- alkylguanine DMA alkyltransferase-mediated repair of NNK-induced O6-alkylguanine adducts will also be examined. These studies will: 1. Determine the mechanisms by which 5-methylcytosine (MeC) influences the reactivity of neighboring guanine bases towards tobacco carcinogens. 2. Examine the effects of MeC and its structural analogs on the stereochemistry of N2-guanine adducts induced by B[a]P diolepoxides. 3. Map the distribution of oxidative DMA lesions within p53 and K-ras derived DNA sequences. 4. Examine O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-guanine lesions at methylated and unmethylated CG dinucleotides. The results of this research will identify the mechanisms by which endogenous MeC modulates the reactivity of CG sites towards tobacco carcinogens, providing an insight into the origins of genetic and epigenetic changes observed in lung cancer. Our approach is innovative because our laboratory is employing a novel, mass spectrometry based approach to probe the reactivity of specific bases within DNA duplexes towards alkylating and oxidative agents. The proposed research is significant because of the widespread human exposure to tobacco products and because of their central role in the initiation of lung cancer in humans.