Recognition of Environmental Carcinogen-DNA lesions by NER Proteins

描述(申请人提供)：化石燃料燃烧产生的多环芳烃(PAH)是普遍存在的潜在致癌环境污染物。在有毒废物堆放场和超级基金地点、空气颗粒物、我们的食物和水以及烟草烟雾中都发现了多环芳烃。多环芳烃化合物被认为是造成污染城市地区居民和吸烟者肺癌和其他癌症发病率较高的原因。多环芳烃在生物上是不活性的，但在代谢过程中被激活为活性二元醇环氧化物，这些环氧化物与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损伤会在暴露于多环芳烃污染环境中的人的组织中积累，因此他们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。其中许多可以被称为核苷酸切除修复(NER)的人类DNA修复机制移除。然而，NER系统的活性是可变的：一些DNA损伤被缓慢修复，而另一些对NER具有抵抗力。这种持续的DNA损伤的积累增加了发生肺部疾病的风险，如哮喘、肺癌和其他疾病。使某些PAH-DNA加合物具有NER抗性的确切结构特征，目前还知之甚少。在哺乳动物中识别和结合NER底物的第一个NER因子是异二聚体XPC-RAD23B蛋白。病变的识别分两步进行(两部分)：(1)XPC-RAD23B的识别；(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复合体结合的多蛋白NER因子。该项目的可行性是基于之前开发的不同多环芳烃-DNA加合物的广泛文库，这些加合物展示了从完全抗性到完全敏感的全谱NER活性。目的是阐明DNA损伤的结构特征，以消除或促进其识别和修复。目的1利用一组在两种不同碱基序列背景下表征良好的苯并[a]芘二醇环氧化物鸟嘌呤损伤(BP-G)，建立表面等离子体共振和其他方法(足迹法、凝胶电泳法)来研究XPC和TFIIH蛋白与DNA的相互作用。在其中一个序列中，BP-G损伤是中到好的NER底物；在另一个序列中，与BP-G损伤相反的单核苷酸缺失，并且相同的BP-G双链在人类细胞提取物中对NER完全耐药。在目标2中，这些方法将被应用于研究最初的XPC-RAD23B和TFIIH-DNA结合现象的机制，使用不同体积和小的NER熟练和NER抗性的PAH-鸟嘌呤和腺嘌呤DNA加合物。通过使用核磁共振方法探索损伤引起的DNA局部热力学不稳定，以及通过分子建模和计算技术，将获得机理上的见解。

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.