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：
8479349
负责人：
Yinsheng Wang
金额：
$ 30.56万
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：
2009-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8479349
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 research study stable isotope

项目摘要

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.

抽象的人类基因组的完整性经常受到内源和外源的挑战代理。糖酵解或 N-亚硝基化合物中存在的内源性副产物胃肠道均可导致DNA中核碱基的羧烷基化。之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动物的细胞和组织中以显着水平存在。长期目标是该项目旨在从分子水平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方案提出实验目标：目标 1，评估重氮乙酸诱导的 DNA 修饰的化学性质，重氮乙酸是一种反应性物质。由内源诱导的 N-亚硝基化合物产生的中间体。目标 2，量化使用 LC-MS/MS 检测培养的人类细胞和糖尿病动物模型中的羧烷基化 DNA 加合物采用标准同位素稀释法。目标3，合成含有a的寡脱氧核糖核苷酸特定部位的羧烷基化病变。目标 4，采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，各种跨损伤合成 DNA 聚合酶将通过使用聚合酶缺陷细菌来描绘菌株，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。拟议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诱变特性的知识。拟议的研究还将揭示高血糖和接触 N-亚硝基化合物对人体的影响糖尿病并发症和胃肠道肿瘤的病因学。此外，拟议的研究可能会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。