Recognition of Environmental Carcinogen-DNA lesions by NER Proteins

NER 蛋白识别环境致癌物 DNA 损伤

• 批准号: 9057542
• 负责人: Nicholas E Geacintov
• 金额: $ 35.34万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9057542
• 关键词: Adenine; Affect; Affinity; Amaze; Aromatic Polycyclic Hydrocarbons; Asthma; Base Sequence; Benzo(a)pyrene; Binding; Biochemical; Biological Markers; Cancer Etiology; Carcinogens; Cell Extracts; Characteristics; Chemical Structure; Chicago; Cisplatin; Collaborations; Complex; Computational Technique; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Maintenance; DNA Repair; DNA lesion; Disease; Environment; Environmental Carcinogens; Environmental Pollution; Epoxy Compounds; Etiology; Excision; Exhibits; Exposure to; Food; Fossil Fuels; GTF2H1 gene; Glycols; Guanine; Health; Human; Illinois; Individual; Laboratories; Lesion; Libraries; Lung; Lung diseases; Malignant Neoplasms; Malignant neoplasm of lung; Methodology; Methods; Molecular Conformation; Molecular Models; Nucleotide Excision Repair; Nucleotides; Oxidation-Reduction; Oxidoreductase; Particulate; Process; Property; Proteins; Quinones; Reactive Oxygen Species; Resistance; Risk; Roentgen Rays; Shapes; Site; Smoker; Specificity; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Surgical incisions; System; Techniques; Testing; Thermodynamics; Tissues; Tobacco smoke; Water; Workplace; adduct; base; crosslink; exposed human population; gel electrophoresis; helicase; human DNA; human disease; improved; insight; molecular modeling; prototype; repaired; research study; superfund site; transcription factor TFIIH; urban area; wasting

DESCRIPTION (provided by applicant): The combustion of fossil fuels generates polycyclic aromatic hydrocarbons (PAH) that are ubiquitous and potentially cancer-causing environmental contaminants. PAH are found at toxic waste dumps and superfund sites, in airborne particulates, in our food and water, as well as in tobacco smoke. PAH compounds are believed to contribute to the higher rates of lung and other cancers that have been documented in residents of polluted urban areas and smokers. The PAH are biologically inactive but are metabolically activated to reactive diol epoxides that covalently bind to guanine and adenine in DNA to form stable, pre- mutagenic DNA adducts. Such forms of DNA damage accumulate in tissues of people exposed to PAH- contaminated environments, who are thus at risk of developing respiratory diseases and cancer. Not all DNA adducts are equally threatening to human health. Many of them can be removed by the human DNA repair mechanism called nucleotide excision repair (NER). However, the activity of the NER system is variable: some DNA lesions are slowly repaired, while some others are resistant to NER. The accumulation of such persistent DNA damage enhances the risk of developing diseases of the lung such as asthma, lung cancer, and other disorders. The exact structural features that render certain PAH-DNA adducts NER-resistant, are poorly understood. Among the first mammalian NER factors that recognize and bind to NER substrates is the heterodimeric XPC-RAD23B protein. The identification of the lesions occurs in a two-step (bipartite manner): (1) recognition by XPC-RAD23B, and (2) a subsequent verification step that involves the helicase activity of XPD in TFIIH, a multi-protein NER factor that binds to the XPC-DNA complex. The feasibility of this project is based on a previously developed, extensive library of different PAH-DNA adducts that exhibit the full spectrum of NER activities, from fully resistant to fully susceptible. The objecties are to elucidate the structural features of DNA lesions that abrogate or promote their recognition and repair. Aim 1 is focused on developing surface plasmon resonance and other methods (footprinting, gel electrophoresis) for studying XPC and TFIIH protein-DNA interactions utilizing a set of well characterized benzo[a]pyrene - diol epoxide guanine lesions (BP-G) in two different base sequence contexts. In one of these, the BP-G lesions are either moderate-to-good NER substrates; in the other sequence, a single nucleotide opposite the BP-G, lesion is missing, and the same BP-G duplexes are fully resistant to NER in human cell extracts. In Aim 2, these methodologies will be applied to investigate the mechanisms of the initial XPC- RAD23B and TFIIH-DNA binding phenomena utilizing different bulky and small NER-proficient and NER- resistant PAH-guanine and -adenine DNA adducts. Mechanistic insights will be gained by exploring the local thermodynamic destabilization of DNA caused by the lesions using NMR methods, and by molecular modeling and computational techniques.

描述（由申请人提供）：化石燃料的燃烧会产生多环芳烃（PAH），它们是普遍存在且可能致癌的环境污染物。 PAH 存在于有毒废物堆放场和超级基金场所、空气中的颗粒物、我们的食物和水中以及烟草烟雾中。多环芳烃化合物被认为会导致污染城市地区居民和吸烟者患肺癌和其他癌症的发病率升高。 PAH 没有生物活性，但在代谢上被激活为活性二醇环氧化物，该二醇环氧化物与 DNA 中的鸟嘌呤和腺嘌呤共价结合，形成稳定的、诱变前的 DNA 加合物。这种形式的 DNA 损伤会在暴露于 PAH 污染环境的人的组织中积累，因此面临患呼吸道疾病和癌症的风险。 并非所有 DNA 加合物都同样对人类健康构成威胁。其中许多可以通过称为核苷酸切除修复 (NER) 的人类 DNA 修复机制去除。然而，NER 系统的活性是可变的：一些 DNA 损伤会缓慢修复，而另一些则对 NER 具有抵抗力。这种持续性 DNA 损伤的积累会增加患哮喘、肺癌和其他疾病等肺部疾病的风险。人们对某些 PAH-DNA 加合物具有 NER 抗性的确切结构特征知之甚少。第一个识别并结合 NER 底物的哺乳动物 NER 因子是异二聚体 XPC-RAD23B 蛋白。病变的识别分两步（二分方式）进行：(1) XPC-RAD23B 识别，(2) 随后的验证步骤，涉及 TFIIH 中 XPD 的解旋酶活性，TFIIH 是一种与 XPC-DNA 复合物结合的多蛋白 NER 因子。该项目的可行性基于先前开发的不同 PAH-DNA 加合物的广泛库，该库表现出从完全耐药到完全敏感的全谱 NER 活性。目的是阐明 DNA 损伤的结构特征，从而消除或促进其识别和修复。 目标 1 的重点是开发表面等离子体共振和其他方法（足迹法、凝胶电泳），利用一组在两种不同碱基序列背景下充分表征的苯并[a]芘 - 二醇环氧化物鸟嘌呤损伤 (BP-G)，研究 XPC 和 TFIIH 蛋白质-DNA 相互作用。在其中之一中，BP-G 病变要么是中等至良好的 NER 底物，要么是中等至良好的 NER 底物。在另一个序列中，与 BP-G 相对的单个核苷酸缺失，病变缺失，并且相同的 BP-G 双链体在人类细胞提取物中完全抵抗 NER。在目标 2 中，这些方法将用于利用不同的大体积和小 NER 熟练和 NER 抗性 PAH-鸟嘌呤和腺嘌呤 DNA 加合物来研究初始 XPC-RAD23B 和 TFIIH-DNA 结合现象的机制。通过使用 NMR 方法以及分子建模和计算技术探索由损伤引起的 DNA 局部热力学不稳定，将获得机理见解。