Chemistry and Biology of DNA Carboxyalkylation

DNA 羧烷基化的化学和生物学

基本信息

批准号：
8007480
负责人：
Yinsheng Wang
金额：
$ 9.95万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-31 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8007480
关键词：
2&apos -deoxyadenosine Animal Model Azaserine Biological Biological Markers Biology Bypass Cells Chemistry Complications of Diabetes Mellitus DNA DNA Adduction DNA Adducts DNA Modification Process DNA biosynthesis DNA lesion DNA-Directed DNA Polymerase Deoxyguanosine Escherichia coli Etiology Exposure to Gastrointestinal Neoplasms Gastrointestinal tract structure Glycolysis Goals Human Human Genome Hyperglycemia Individual Knowledge Laboratories Laboratory Animals Lead Lesion Light Malignant neoplasm of gastrointestinal tract Mammalian Cell Methods Molecular Mutation NMR Spectroscopy Nitroso Compounds Nucleosides Oligodeoxyribonucleotides Outcome Polymerase Property Radioisotope Dilution Technique Research Risk Factors Role Shuttle Vectors Site Small Interfering RNA Source Structure Techniques Technology animal tissue calf thymus DNA carboxymethylation chemical synthesis cytotoxic diabetic human DNA damage human tissue in vivo knock-down novel nucleobase public health relevance research study stable isotope

项目摘要

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

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试剂的挑战。胃肠道中存在的糖酵解或 N-亚硝基化合物的内源性副产物均可导致 DNA 中核碱基的羧烷基化。该实验室和其他实验室之前进行的研究表明，一些羧烷基化核苷在人类和实验动物的细胞和组织中含量很高。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，评估重氮乙酸诱导的DNA修饰的化学性质，重氮乙酸是由内源诱导的N-亚硝基化合物产生的反应中间体。目标 2，使用 LC-MS/MS 和标准同位素稀释方法对培养的人类细胞和糖尿病动物模型中的羧烷基化 DNA 加合物进行定量。目标 3，合成在特定位点带有羧烷基化损伤的寡脱氧核糖核苷酸。目标 4，采用穿梭载体技术并研究羧烷基化 DNA 损伤如何在大肠杆菌和人类细胞中复制。在这方面，各种跨损伤合成DNA聚合酶的作用将通过使用聚合酶缺陷的细菌菌株来描述，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述。拟议研究的结果将为这组 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影响。此外，拟议的研究可能会发现新的分子生物标志物和发展这些病理状况的危险因素。公共卫生相关性：人类在胃肠道中接触糖酵解副产物和 N-亚硝基化合物，导致 DNA 中核碱基的羧烷基化。拟议的研究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影响，并导致发现新的分子生物标志物和发生这些病理状况的危险因素