Recognition of Environmental Carcinogen-DNA lesions by NER Proteins

NER 蛋白识别环境致癌物 DNA 损伤

基本信息

批准号：
8673463
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
项目类别：
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/8673463
关键词：
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide 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 DNA-Protein Interaction Disease Environment Environmental Carcinogens Environmental Pollution Epoxy Compounds Etiology Excision Exhibits Exposure to Food Fossil Fuels 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 public health relevance repaired research study superfund site transcription factor TFIIH urban area wasting

项目摘要

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 objectives 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.

化石燃料的燃烧会产生普遍存在的多环芳烃 (PAH) 以及潜在致癌的环境污染物。 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、我们的食物和水以及烟草烟雾中。多环芳烃化合物据信，这会导致肺癌和其他癌症的发病率升高，这些癌症已被记录在案污染城市地区的居民和吸烟者。 PAH 不具有生物活性，但具有代谢活性激活反应性二醇环氧化物，与 DNA 中的鸟嘌呤和腺嘌呤共价结合，形成稳定的预- 诱变 DNA 加合物。这种形式的 DNA 损伤在接触 PAH 的人的组织中积累受污染的环境，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。其中许多可以通过人类DNA修复机制称为核苷酸切除修复（NER）。然而，NER 的活动系统是可变的：一些DNA损伤被缓慢修复，而另一些则对NER有抵抗力。这这种持续性 DNA 损伤的积累会增加患肺部疾病的风险，例如哮喘、肺癌和其他疾病。产生某些 PAH-DNA 加合物的确切结构特征 NER 抵抗，人们知之甚少。第一个识别并结合 NER 的哺乳动物 NER 因子底物是异二聚体 XPC-RAD23B 蛋白。病变的识别分两步进行（双方方式）：（1）由 XPC-RAD23B 识别，以及（2）后续验证步骤，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。这该项目的可行性基于先前开发的不同 PAH-DNA 加合物的广泛库表现出全方位的 NER 活性，从完全耐药到完全敏感。目标是阐明 DNA 损伤的结构特征，从而消除或促进其识别和修复。目标 1 专注于开发表面等离子共振和其他方法（足迹法、凝胶法）电泳）用于利用一组充分表征的方法研究 XPC 和 TFIIH 蛋白质-DNA 相互作用两种不同碱基序列背景下的苯并[a]芘-二醇环氧化物鸟嘌呤损伤（BP-G）。其中之一， BP-G 病变是中等至良好的 NER 底物；在另一个序列中，单个核苷酸与 BP-G 相反，病变缺失，并且相同的 BP-G 双链体在人体细胞中完全抵抗 NER 提取物。在目标 2 中，这些方法将用于研究初始 XPC- RAD23B 和 TFIIH-DNA 结合现象利用不同的大和小 NER 熟练和 NER- 抗性 PAH-鸟嘌呤和腺嘌呤 DNA 加合物。通过探索当地的情况将获得机制见解使用 NMR 方法和分子建模对损伤引起的 DNA 热力学不稳定进行分析和计算技术。