Recognition of Environmental Carcinogen-DNA lesions by NER Proteins

化石燃料燃烧会产生普遍存在的多环芳烃(PAH) 以及可能致癌的环境污染物。多环芳烃在有毒废物堆放场和 超级基金网站，空气中的颗粒物，我们的食物和水，以及烟草烟雾。多环芳烃化合物 被认为是导致肺癌和其他癌症发病率较高的原因，这些癌症已在 污染城区的居民和吸烟者。多环芳烃在生物上是不活跃的，但在代谢上 活化到活性的二元醇环氧化物，它与DNA中的鸟嘌呤和腺嘌呤共价结合，形成稳定的，预 致突变的DNA加合物。这种形式的DNA损伤会在接触多环芳烃的人的组织中积累- 受污染的环境，他们因此有患呼吸道疾病和癌症的风险。 并不是所有的DNA加合物对人类健康都有同样的威胁。它们中的许多可以通过 人类DNA修复机制称为核苷酸切除修复(NER)。然而，非政府组织的活动 系统是可变的：一些DNA损伤被缓慢修复，而另一些对NER具有抵抗力。这个 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。导致某些PAH-DNA加合物的确切结构特征 耐NER，人们对此知之甚少。在第一批识别和结合NER的哺乳动物NER因子中 底物为异二聚体XPC-RAD23B蛋白。损伤的识别分两步进行 (两部分方式)：(1)由XPC-RAD23B识别，以及(2)后续验证步骤，涉及 XPD在TFIIH中的解旋酶活性，TFIIH是一种与XPC-DNA复合体结合的多蛋白NER因子。这个 该项目的可行性基于先前开发的不同多环芳烃-DNA加合物的广泛文库 它们表现出从完全抵抗到完全敏感的全谱NER活性。目标是 阐明DNA损伤的结构特征，以消除或促进其识别和修复。 目标1专注于开发表面等离子体共振和其他方法(足迹、凝胶 凝胶电泳法)研究XPC和TFIIH蛋白-DNA相互作用 苯并[a]芘二醇环氧鸟嘌呤损伤(BP-G)在两种不同的碱基序列背景下。在其中的一个中， BP-G损伤要么是中到好的NER底物；在另一个序列中，单核苷酸 与BP-G相反的是，病变缺失，相同的BP-G双链在人类细胞中对NER完全耐药 萃取物。在目标2中，这些方法将用于研究初始XPC的机制-- RAD23B和TFIIH-DNA结合现象利用不同的大小NER-熟练和NER- 抗性多环芳烃-鸟嘌呤和-腺嘌呤DNA加合物。机械的洞察将通过探索当地 用核磁共振方法和分子模拟研究损伤引起的DNA的热力学失稳 和计算技术。

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.