Recognition of Environmental Carcinogen-DNA lesions by NER Proteins

描述（由申请人提供）：化石燃料的燃烧产生多环芳烃（PAH），这是普遍存在的潜在致癌环境污染物。PAH存在于有毒废物倾倒场和超级基金场地，空气中的颗粒物，我们的食物和水以及烟草烟雾中。PAH化合物被认为是导致肺癌和其他癌症的高发病率的原因，这些癌症在受污染的城市地区的居民和吸烟者中有记录。PAH是无生物活性的，但被代谢活化为反应性二醇环氧化物，其共价结合DNA中的鸟嘌呤和腺嘌呤以形成稳定的预诱变DNA加合物。这种形式的DNA损伤在暴露于PAH污染环境的人的组织中积累，因此这些人有患呼吸道疾病和癌症的风险。 并不是所有的DNA加合物都对人类健康构成同样的威胁。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损伤被缓慢修复，而另一些则对NER具有抗性。这种持续性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加合物耐NER的确切结构特征知之甚少。在第一个识别和结合NER底物的哺乳动物NER因子中，是异二聚体XPC-RAD 23 B蛋白。病变的鉴定分两步进行（二分方式）：（1）通过XPC-RAD 23 B识别，以及（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质NER因子。该项目的可行性是基于先前开发的，广泛的不同PAH-DNA加合物的库，这些加合物表现出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，消除或促进其识别和修复。 目的1是专注于开发表面等离子体共振和其他方法（足迹法，凝胶电泳），用于研究XPC和TFIIH蛋白质-DNA相互作用，利用一组充分表征的苯并[a]芘-二醇环氧化物鸟嘌呤损伤（BP-G）在两个不同的碱基序列背景。在其中一个序列中，BP-G病变是中度至良好的NER底物;在另一个序列中，与BP-G病变相对的单个核苷酸缺失，并且相同的BP-G双链体对人类细胞提取物中的NER完全具有抗性。在目的2中，这些方法将被应用于研究初始XPC-RAD 23 B和TFIIH-DNA结合现象的机制，其利用不同的大体积和小的NER-熟练的和NER-抗性的PAH-鸟嘌呤和-腺嘌呤DNA加合物。机制的见解将获得通过探索使用NMR方法的病变所造成的DNA的局部热力学不稳定，并通过分子建模和计算技术。

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.