Oxidative pathways of guanine in DNA

DNA中鸟嘌呤的氧化途径

• 批准号: 8272592
• 负责人: VLADIMIR SHAFIROVICH
• 金额: $ 33.81万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8272592
• 关键词: 8-hydroxyguanosine; Abbreviations; Affect; Amino Acids; Attention; Base Excision Repairs; Base Pairing; Base Sequence; Biological Markers; Cations; Cell Extracts; Cell Line; Cells; Characteristics; Chemicals; Chromatin; Chronic; Clinical Treatment; DNA; DNA Damage; DNA Maintenance; DNA Repair; DNA lesion; Development; Disease; Disease Progression; Electron Transport; Electrons; Environment; Environmental Pollutants; Environmental Risk Factor; Equilibrium; Etiology; Excision; Exogenous Factors; Guanine; High Pressure Liquid Chromatography; Histones; Human; Hydration status; Hydroxyl Radical; Infection; Inflammation; Inflammatory; Inflammatory Response; Investigation; Ionizing radiation; Kinetics; Lasers; Lead; Lesion; Link; Malignant Neoplasms; Methods; Monitor; Nature; Nitrogen; Nitrogen Dioxide; Nucleic Acids; Nucleosome Core Particle; Nucleosomes; Nucleotide Excision Repair; Oxidants; Oxidative Stress; Oxygen; Pathway interactions; Peptides; Peroxonitrite; Play; Positioning Attribute; Predisposition; Prevention strategy; Production; Progress Reports; Proteins; Reaction; Reactive Oxygen Species; Relative (related person); Research; Resistance; Risk; Role; Simulate; Site; Solvents; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis; Superoxides; System; Thymine; Time; Tobacco smoke; Ultraviolet Rays; Virus Diseases; Water; absorption; adduct; base; cancer cell; cell transformation; chemical bond; chemical reaction; crosslink; deprotonation; design; innovation; novel; oxidation; oxidative DNA damage; peptide G; public health relevance; repaired; research study; response; spiroiminodihydantoin; tool

DESCRIPTION (provided by applicant): Chronic inflammation caused by diverse chemical, physical and infectious factors increases the risk of malignant cell transformations and the progression of human cancers. The enhanced production of reactive oxygen and nitrogen species (ROS and RNS, respectively) in inflammatory cells leads to oxidative stress that induces an imbalance between oxidative DNA damage and DNA repair. The accumulation of oxidatively generated DNA lesions enhances the mutagenic burden of the cells that can lead to cancer. A primary target of ROS and RNS overproduced at sites of inflammation is guanine, the most easily oxidizable natural nucleic acid base. The primary oxidation step generates guanine radicals that undergo a cascade of chemical reactions with cellular nucleophiles and other substances that lead to the formation of a variety of unstable intermediates and stable genotoxic guanine lesions in DNA. During the previous project period, we have developed new methods and approaches for investigating reaction pathways of guanine radicals, the formation of a variety of stable end-products, and the repair of these lesions by base excision repair (BER) and nucleotide excision repair (NER) mechanisms. The major tools include real time monitoring of the reactions of unstable guanine radical and other intermediates by kinetic laser transient absorption spectroscopy, the isolation and identification of the stable DNA lesions formed by HPLC, LC-MS/MS and MALDI-TOF/MS, and 1D and 2D NMR methods. Major findings are (1) that the one-electron oxidation of guanine is base-sequence dependent, (2) the discovery of a novel guanine-thymine (G*-T*) intrastrand cross-linked lesion that competes with the formation of 8-oxoG, spiroiminodihydantoins (Sp), and other lesions, and (3) that the G*-T* and Sp lesions are substrates for both BER and NER repair mechanisms. The new specific aims build on these preliminary findings with the objectives of clarifying the relationships between the competitive reaction pathways of guanine radicals, the resulting formation of stable guanine lesions, and the susceptibilities of the latter to repair by BER and NER mechanisms. The specific aims of this project are: 1) determine the effects of base sequence on the distributions of the guanine lesions generated by inflammatory ROS and RNS (peroxynitrite, nitrogen dioxide, superoxide radicals); 2) Compare the base sequence-dependent formation of these lesions in naked DNA and nucleosomal DNA; 3) Determine the susceptibilities to BER and NER mechanisms of repair using single oxidative guanine lesions in naked and nucleosomal DNA, and monitor oxidative guanine damage and repair in selected repair-proficient and deficient cell lines. A better understanding of DNA damage and repair under conditions simulating oxidative stress should provide a rational basis for discovering new strategies for the prevention and clinical treatments of disease. PUBLIC HEALTH RELEVANCE: Chronic inflammation developed in response to environmental pollutants, tobacco smoke, viral infections, ionizing and UV radiations, and other exogenous factors, is tightly implicated in the etiology of many human cancers. A better understanding of the DNA damage and DNA repair mechanisms under inflammatory conditions will provide a rational basis for discovering new specific biomarkers of oxidative stress, the development of new strategies for the prevention and clinical treatment of inflammatory diseases, and the progression of cancers under inflammatory conditions.

描述（由申请人提供）：由多种化学、物理和感染因素引起的慢性炎症会增加恶性细胞转化和人类癌症进展的风险。炎症细胞中活性氧和氮物质（分别为ROS和RNS）的产生增加导致氧化应激，其诱导氧化DNA损伤和DNA修复之间的不平衡。氧化产生的DNA损伤的积累增加了细胞的诱变负荷，可导致癌症。在炎症部位过度产生的ROS和RNS的主要靶标是鸟嘌呤，其是最容易氧化的天然核酸碱基。初级氧化步骤产生鸟嘌呤自由基，其与细胞亲核试剂和其他物质发生级联化学反应，导致在DNA中形成各种不稳定中间体和稳定的遗传毒性鸟嘌呤损伤。在上一个项目期间，我们已经开发了新的方法和途径，用于研究鸟嘌呤自由基的反应途径，各种稳定的终产物的形成，以及通过碱基切除修复（BER）和核苷酸切除修复（NER）机制修复这些病变。主要手段包括动态激光瞬态吸收光谱法对不稳定鸟嘌呤自由基和其他中间体反应的真实的实时监测，HPLC、LC-MS/MS和MALDI-TOF/MS对DNA稳定损伤的分离鉴定，以及一维和二维NMR方法。主要发现是（1）鸟嘌呤的单电子氧化是碱基序列依赖性的，（2）发现了一种新的鸟嘌呤-胸腺嘧啶（G*-T*）链内交联损伤，它与8-oxoG、螺亚氨基二乙内酰脲（Sp）和其他损伤的形成竞争，（3）G*-T* 和Sp损伤是BER和NER修复机制的底物。 新的具体目标建立在这些初步研究结果的基础上，其目的是澄清鸟嘌呤自由基的竞争性反应途径之间的关系，从而形成稳定的鸟嘌呤病变，以及后者通过BER和NER机制修复的能力。本研究的具体目的是：1）确定碱基序列对炎症性ROS和RNS引起的鸟嘌呤损伤分布的影响（过氧亚硝酸根、二氧化氮、超氧自由基）; 2）比较裸DNA和核小体DNA中这些损伤的碱基序列依赖性形成; 3）使用裸DNA和核小体DNA中的单个氧化鸟嘌呤损伤确定对BER和NER修复机制的敏感性，并监测氧化鸟嘌呤损伤和修复在选定的修复熟练和缺陷细胞系。 更好地了解在模拟氧化应激条件下的DNA损伤和修复，应该为发现疾病预防和临床治疗的新策略提供合理的基础。 公共卫生相关性：由于环境污染物、烟草烟雾、病毒感染、电离和紫外线辐射以及其他外源性因素而产生的慢性炎症与许多人类癌症的病因学密切相关。更好地理解炎症条件下DNA损伤和DNA修复机制将为发现新的特异性氧化应激生物标志物、开发预防和临床治疗炎症性疾病的新策略以及炎症条件下癌症的进展提供合理的基础。