Recognition of Environmental Carcinogen-DNA lesions by NER Proteins

描述（由申请人提供）：化石燃料的燃烧会产生多环芳烃（PAH），这是一种普遍存在的潜在致癌环境污染物。多环芳烃存在于有毒废物堆和超级基金场所、空气中的微粒、我们的食物和水以及烟草烟雾中。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，这在污染城市地区的居民和吸烟者中已经有记录。多环芳烃在生物学上是无活性的，但在代谢过程中被激活为活性环氧二醇，与DNA中的鸟嘌呤和腺嘌呤共价结合，形成稳定的、致突变前的DNA加合物。这种形式的DNA损伤在暴露于多环芳烃污染环境的人的组织中积累，因此他们有患呼吸系统疾病和癌症的风险。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。确切的结构特征，使某些多环芳烃dna加合物耐ner，是知之甚少。在哺乳动物中最早识别并结合内源性内源性蛋白的因子是异二聚体XPC-RAD23B蛋白。病变的识别分两步进行：(1)由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是一种结合XPC-DNA复合物的多蛋白NER因子。该项目的可行性基于先前开发的广泛的不同多环芳烃- dna加合物文库，这些加合物展示了从完全抗性到完全易感的全谱NER活性。目的是阐明妨碍或促进其识别和修复的DNA损伤的结构特征。目标1的重点是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，利用一组具有良好特征的苯并[a]芘-环氧二醇鸟嘌呤病变（BP-G）在两种不同的碱基序列背景下。在其中一种情况下，BP-G病变要么是中度至良好的NER底物；在另一个序列中，BP-G对面的单个核苷酸缺失，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，这些方法将应用于研究XPC- RAD23B和TFIIH-DNA结合现象的初始机制，利用不同的大体积和小体积的NER-精通和NER-耐药的pah -鸟嘌呤和腺嘌呤DNA加合物。通过使用核磁共振方法、分子建模和计算技术，探索由病变引起的DNA局部热力学不稳定，将获得机制见解。

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.