Chemistry and Biology of DNA Carboxyalkylation

摘要 人类基因组的完整性经常受到内源性和外源性基因的挑战。 剂.糖酵解的内源性副产物或存在于糖酵解产物中的N-亚硝基化合物 胃肠道的反应都可以导致DNA中核碱基的羧烷基化。以前的研究 在这个实验室和其他实验室进行的研究表明，一些羧烷基化核苷是 在人类和实验室动物的细胞和组织中以显著水平存在。的长期目标 这个项目是为了在分子水平上了解羧烷基化的生物学后果。 DNA加合物。在本申请中，我们根据以下四个具体实施方案提出实验。 目的：目的1，评估重氮乙酸诱导的DNA修饰的化学，重氮乙酸是一种反应性的 由内源诱导的N-亚硝基化合物产生的中间体。目标2：量化 使用LC-MS/MS在培养的人细胞和糖尿病动物模型中检测羧烷基化DNA加合物 用标准同位素稀释法目的3，合成含a 在特定部位的羧烷基化病变。目的4，利用穿梭载体技术， 羧烷基化DNA损伤在E.大肠杆菌和人类细胞。在这方面， 各种跨损伤合成DNA聚合酶将通过使用聚合酶缺陷的细菌 或者，对于使用哺乳动物细胞的复制研究，通过敲低这些菌株的表达， 聚合酶与siRNA技术。这项研究的结果将提供重要的新成果。 关于这组DNA加合物的细胞毒性和致突变性质的知识。拟议 研究还将揭示高血糖症和暴露于N-亚硝基化合物对糖尿病的影响。 糖尿病并发症和胃肠道肿瘤的病因。此外，拟议的研究可能导致 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。

ABSTRACT 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.