Chemistry and Biology of DNA Carboxyalkylation

描述（由申请人提供）：人类基因组的完整性经常受到内源性和外源性病原体的挑战。内源性产生的糖酵解副产物或胃肠道中存在的n -亚硝基化合物都可以导致DNA核碱基的羧基烷基化。先前在本实验室和其他实验室进行的研究表明，在人类和实验动物的细胞和组织中，一些羧基烷基化核苷的含量很高。该项目的长期目标是在分子水平上了解羧基烷基化DNA加合物的生物学后果。在本申请中，我们根据以下四个具体目的提出实验：目的1，评估重氮乙酸盐诱导的DNA修饰的化学性质，重氮乙酸盐是内源性诱导的n -亚硝基化合物产生的反应性中间体。目的2：采用LC-MS/MS和标准同位素稀释法，定量培养的人细胞和糖尿病动物模型中羧基烷基化DNA加合物。目的3，合成在特定位点含有羧基烷基化损伤的寡脱氧核糖核苷酸。目的4：利用穿梭载体技术，研究羧基烷基化DNA损伤如何在大肠杆菌和人类细胞中复制。在这方面，各种翻译合成DNA聚合酶的作用将通过使用聚合酶缺乏的细菌菌株来描述，或者在哺乳动物细胞的复制研究中，通过使用siRNA技术敲低这些聚合酶的表达来描述。所提出的研究结果将为这组DNA加合物的细胞毒性和诱变特性提供重要的新知识。拟议的研究还将揭示高血糖和暴露于n -亚硝基化合物在糖尿病并发症和胃肠道肿瘤病因学中的意义。此外，提出的研究可能会导致发现新的分子生物标志物和发展这些病理条件的危险因素。公共卫生相关性：人类在胃肠道中暴露于糖酵解和n -亚硝基化合物的副产物，这导致DNA核碱基的羧基烷基化。该研究将揭示高血糖和暴露于n -亚硝基化合物在糖尿病并发症和胃肠道肿瘤病因学中的意义，并导致发现新的分子生物标志物和发展这些病理条件的危险因素

DESCRIPTION (provided by applicant): The integrity of human genome is frequently challenged by endogenous and exogenous sources of agents. Endogenously produced byproducts of glycolysis or N-nitroso compounds present in the gastrointestinal tract can both lead to the carboxyalkylation of nucleobases in DNA. Previous studies carried out in this and other laboratories revealed that some of the carboxyalkylated nucleosides are present at significant levels in cells and tissues of humans and laboratory animals. The long-term goal of this project is to understand, at the molecular level, the biological consequences of the carboxyalkylated DNA adducts. In the present application, we propose experiments according to the following four specific aims: Aim 1, to assess the chemistry of DNA modifications induced by diazoacetate, which is a reactive intermediate arising from endogenously induced N-nitroso compounds. Aim 2, to quantify the carboxyalkylated DNA adducts in cultured human cells and in diabetic animal models by using LC-MS/MS with the standard isotope dilution method. Aim 3, to synthesize oligodeoxyribonucleotides harboring a carboxyalkylated lesion at a specific site. Aim 4, to employ shuttle vector technology and investigate how the carboxyalkylated DNA lesions are replicated in E. coli and human cells. In this respect, the roles of various translesion synthesis DNA polymerases will be delineated by using polymerase-deficient bacterial strains or, for replication studies using mammalian cells, by knocking down the expression of these polymerases with the siRNA technique. The outcome of the proposed research will provide significant new knowledge about the cytotoxic and mutagenic properties of this group of DNA adducts. The proposed research will also reveal the implications of hyperglycemia and exposure to N-nitroso compounds in the etiology of diabetic complications and gastrointestinal tumors. Furthermore, the proposed study may lead to the discovery of novel molecular biomarkers and risk factors for developing these pathological conditions. PUBLIC HEALTH RELEVANCE: Humans are exposed to byproducts of glycolysis and N-nitroso compounds in the gastrointestinal tract, which results in the carboxyalkylation of nucleobases in DNA. The proposed research will reveal the implications of hyperglycemia and exposure to N-nitroso compounds in the etiology of diabetic complications and gastrointestinal tumors, and lead to the discovery of novel molecular biomarkers and risk factors for developing these pathological conditions