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来源的挑战 探员们。内源性糖酵解副产物或N-亚硝基化合物存在于 胃肠道均可导致DNA中核苷酸碱基的羧烷基化。以前的研究 在这个和其他实验室中进行的研究表明，一些羧烷基化的核苷是 在人类和实验动物的细胞和组织中存在显著水平。的长期目标是 这个项目是为了在分子水平上了解羧烷基化的生物后果。 DNA加合物。在本申请中，我们根据以下四个具体情况提出实验 目的：目的1，评价重氮乙酸酯引起的DNA修饰的化学性质。 由内源诱导的N-亚硝基化合物产生的中间体。目标2，量化 用LC-MS/MS研究培养的人细胞和糖尿病动物模型中的羧烷基化DNA加合物 用标准同位素稀释法测定。目的3，合成含有A蛋白的寡脱氧核苷酸 特定部位的羧烷基化病变。目标4，使用穿梭载体技术并研究如何 羧烷基化的DNA损伤在大肠杆菌和人类细胞中复制。在这方面， 利用聚合酶缺陷细菌描述各种跨损伤合成dna聚合酶。 菌株，或者，对于使用哺乳动物细胞的复制研究，通过降低这些基因的表达 使用siRNA技术的聚合酶。拟议研究的结果将提供重要的新的 了解这组DNA加合物的细胞毒性和致突变特性。建议数 研究还将揭示高血糖和暴露于N-亚硝基化合物的影响 糖尿病并发症和胃肠道肿瘤的病因学。此外，拟议的研究可能导致 发现新的分子生物标记物和发生这些病理情况的危险因素。

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.