Sequence Distribution of Tobacco Carcinogen-DNA Adducts

描述(申请人提供)：这个项目集中在吸烟导致肺癌的两个基因中特定的烟草致癌物-DNA加合物的分布：p53肿瘤抑制基因和K-ras原癌基因。吸烟者的肺肿瘤通常包含K-ras密码子12和P53密码子157、158、248和273内的点突变的特征“热点”。这些突变被认为是DNA聚合酶错误的结果，DNA聚合酶在复制被代谢激活的烟草致癌物化学修饰的DNA时发生错误。K-ras和p53基因特定位点的大量突变可能源于它们对烟草致癌物的高反应性、DNA损伤修复不足或特定序列背景下病变错配率的增加。虽然通过定点突变可以准确地确定在特定序列环境中引入的DNA损伤的错配率，但缺乏能够绘制基因序列中特定DNA损伤的形成和修复的分析方法。本研究将使用本实验室最近开发的基于质谱学的方法来量化在p53和K-ras衍生的DNA序列中特定位置的DNA加合物的形成。这项研究将集中在两种主要的烟草致癌物质苯并[a]芘(B[a]P)和4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)对DNA的损伤。用B[a]P和NNK的反应性代谢物处理代表p53和K-ras基因突变易发区的双链合成寡核苷酸，并结合稳定同位素标记，用液相色谱-电喷雾电离串联质谱仪测定每个位置上加合物的形成程度。同样的方法将被用来分析内源性胞嘧啶甲基化对烟草致癌物诱导的鸟嘌呤损伤形成的影响，并确定K-ras和p53序列背景对NNK诱导的O6-烷基鸟嘌呤加合物修复的影响。这些研究将：1)确定含已知突变热点的K-ras和p53衍生的DNA序列中B[a]P二环氧化物诱导的核碱基的分布。2)沿着K-ras和P53衍生的DNA序列定位NNK诱导的甲基化和吡氧丁基化损伤的形成。3)分析内源性胞嘧啶甲基化对邻鸟嘌呤上B[a]P和NNK加合物形成的影响。4)检测K-ras序列上下文对NNK诱导的O6-烷基鸟嘌呤损伤修复的影响。这项工作的结果将对吸烟诱发肺癌中观察到的遗传变化的分子基础提供新的见解，并将有助于为高危个体开发合理的预防策略和基于机制的生物标志物。这项研究也将为未来在单核苷酸分辨率下进行致癌物修饰的碱基的体内研究奠定基础。

DESCRIPTION (provided by applicant): This project focuses on the distribution of specific tobacco carcinogen-DNA adducts within the two genes frequently targeted in smoking induced lung cancer: the p53 tumor suppressor gene and the K-ras proto-oncogene. Lung tumors of smokers often contain characteristic "hotspots" for point mutations within K-ras codon12 and p53 codons 157,158, 248, and 273. These mutations are believed to be a result of DNA polymerase errors during the replication of DNA chemically modified by metabolically activated tobacco carcinogens. The large numbers of mutations at specific sites of the K-ras and p53 genes may originate from their high reactivity towards tobacco carcinogens, deficient repair of DNA lesions, or elevated mispairing rates for the lesions within specific sequence context. While the mispairing rates of DNA lesions introduced in a defined sequence environment can be accurately established by site specific mutagenesis, analytical methods capable of mapping the formation and repair of specific DNA lesions within gene sequences are lacking. The present study will use a mass spectrometry based approach recently developed in this laboratory to quantify the formation of DNA adducts at specific positions within p53 and K-ras-derived DNA sequences. The research will focus on DNA damage induced by two prominent tobacco carcinogens, benzo[a]pyrene(B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Double-stranded synthetic oligodeoxynucleotides representing mutation-prone regions of the p53 and K-ras genes will be treated with reactive metabolites of B[a]P and NNK, and the extent of adduct formation at each position will be determined by liquid chromatography-electrospray ionization tandem mass spectrometry in combination with stable isotope labeling. The same approach will be used to analyze the effect of endogenous cytosine methylation on the formation of tobacco carcinogen-induced guanine lesions and to determine the effect of K-ras and p53 sequence context on the repair of NNK-induced O6-alkylguanine adducts. These studies will 1) Establish the distribution of B[a]P diolepoxide-induced nucleobasel esions within K-ras and p53-derived DNA sequences containing known mutational hotspots. 2) Map the formation of NNK-induced methylated and pyridyloxobutylated lesions along K-ras and p53-derived DNA sequences. 3) Analyze the effects of endogenous cytosine methylation on the formation of B[a]P and NNK adducts at neighboring guanines. 4) Examine the effects of K-ras sequence context on O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-alkylguanine lesions. The results of this work will afford new insights into the molecular basis of genetic changes observed in smoking-induced lung cancer and will aid in the development of rational prevention strategies and mechanism-based biomarkers for individuals at risk. This research will also lay a foundation for future in vivo studies of carcinogen-modified nucleobases at single nucleotide resolution.