Chemistry and Biology of DNA Carboxyalkylation

描述（由申请方提供）：人类基因组的完整性经常受到内源性和外源性因子的挑战。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中核碱基的羧烷基化。在这个实验室和其他实验室进行的先前研究显示，一些羧烷基化核苷在人类和实验室动物的细胞和组织中以显着水平存在。该项目的长期目标是在分子水平上了解羧烷基化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标提出实验：目标1，评估重氮乙酸盐诱导的DNA修饰的化学，重氮乙酸盐是由内源诱导的N-亚硝基化合物产生的反应性中间体。目的2：应用同位素稀释法建立LC-MS/MS定量检测人细胞和糖尿病动物模型中羧烷基化DNA加合物的方法。目的3，合成羧基化修饰的寡核苷酸。目的4，利用穿梭载体技术研究羧烷基化DNA损伤在大肠杆菌中的复制。大肠杆菌和人类细胞。在这方面，各种translesion合成DNA聚合酶的作用将通过使用聚合酶缺陷的细菌菌株来描绘，或者对于使用哺乳动物细胞的复制研究，通过用siRNA技术敲低这些聚合酶的表达来描绘。拟议的研究结果将提供有关这组DNA加合物的细胞毒性和致突变性的重要新知识。这项研究还将揭示高血糖和接触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，拟议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风险因素。公共卫生相关性：人类暴露于胃肠道中糖酵解的副产物和N-亚硝基化合物，这导致DNA中核碱基的羧烷基化。这项拟议的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风险因素

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