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: 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
• 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

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.

化石燃料的燃烧产生无处不在的多环芳烃（PAH） 以及潜在的致癌环境污染物。在有毒废物垃圾场发现了PAH， 超级基金地点，空气中的颗粒物，我们的食物和水以及烟草中的烟雾。 PAH化合物 据信据信有助于较高的肺部和其他已记录的癌症发生率 被污染的城市地区和吸烟者的居民。 PAH在生物学上是非活性的，但在代谢上是 被激活至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，前的 诱变DNA加合物。这种形式的DNA损伤积聚在暴露于PAH-的人的组织中 因此，受污染的环境有呼吸道疾病和癌症的风险。 并非所有DNA加合物都同样威胁到人类健康。其中许多可以通过 人DNA修复机制称为核苷酸切除修复（NER）。但是，NER的活动 系统是可变的：一些DNA病变缓慢修复，而另一些DNA病变对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 哮喘，肺癌和其他疾病。确切的结构特征呈现某些PAH-DNA加合物 耐Ner的理解很差。在识别并与NER结合的第一个哺乳动物NER因素中 底物是异二聚体XPC-RAD23B蛋白。病变的识别发生在两个步骤中 （两分）：（1）XPC-RAD23B识别，（2）随后的验证步骤，涉及 XPD在TFIIH中的解旋酶活性，TFIIH是一种与XPC-DNA复合物结合的多蛋白质因子。这 该项目的可行性基于先前开发的，广泛的不同PAH-DNA加合物的库 从完全抵抗力到完全易感性的NER活性展示了全部范围。目标是 阐明消除或促进其识别和修复的DNA病变的结构特征。 AIM 1专注于开发表面等离子体共振和其他方法（足迹，凝胶 电泳）用于研究XPC和TFIIH蛋白-DNA相互作用，利用一组良好的表征 苯并[a] pyrene -Diol环氧化物鸟嘌呤病变（BP -G）在两个不同的碱基序列上下文中。在其中之一中， BP-G病变要么是中等到良好的NER底物。在另一个序列中，单个核苷酸 在BP-G对面，缺少病变，相同的BP-G双链体对人类细胞中的NER具有完全抗性 提取物。在AIM 2中，这些方法将用于研究最初XPC-的机制 RAD23B和TFIIH-DNA结合现象，利用不同的笨重和小型NER-NER-NER- 抗性的PAH-瓜氨酸和 - 腺嘌呤DNA加合物。机械洞察力将通过探索本地来获得 使用NMR方法和分子建模引起的病变引起的DNA的热力学不稳定 和计算技术。