Excision of Carcinogen-DNA Adducts in Nucleosomes

描述（由申请人提供）：多环芳烃（PAH）是化石燃料燃烧的副产品，存在于我们的空气、食物和水中;这些遗传毒性环境致癌物在我们的环境中的存在继续对人类健康构成危害。PAH的结构特征和生物学影响，区分高活性的诱变剂和致瘤剂与结构相关的活性较低或无活性的化合物，长期以来一直是了解暴露人群中人类癌症病因的兴趣。PAH有两个重要的亚类，它们由不同的拓扑特征区分：（i）空间位阻的“峡湾”区域，其导致峡湾PAH的芳环系统中的显著非平面性，和（ii）空间位阻和平面的“海湾”区域PAH（例如，众所周知的环境致癌物苯并[a]芘）。两种类型的PAH都被代谢活化为反应性二醇环氧化物中间体，其主要与细胞DNA中的鸟嘌呤和腺嘌呤反应，在哺乳动物细胞和组织中形成预诱变共价加合物，如果不修复，最终导致人类癌症的病因学峡湾PAH引起了化学致癌界的极大关注，因为它们高达~ 100-比海湾地区的原型苯并[a]芘的致瘤性高出一倍。 本实验室以前的研究表明，在全细胞提取物中，人核苷酸切除修复（NER）系统对不同的立体化学定义良好的PAH-DNA加合物的相对切除效率存在显着差异。特别令人感兴趣的是观察到PAH-腺嘌呤DNA加合物来自高度致瘤性的峡湾PAH对NER有很强的抗性。然而，这些结论是建立在NER实验，进行了游离DNA在水环境中，它是不知道是否在生物学上更相关的蛋白质环境的核小体，在细胞中的DNA包装的基本亚单位，类似的层次结构的NER将观察到与DNA基板含有相同的单一海湾和峡湾PAH-DNA加合物。本项目的目标是评估核小体DNA中的NER效率。具体目标是：（1）研究DNA定位序列、热力学稳定性、核小体动力学以及核小体超螺旋不同位点上DNA加合物的可及性对NER效率的影响：（2）评价H3、H4、H2 A和H2 B组蛋白的共价修饰对相同核小体性质和NER的影响;（3）确定核小体中结构上不同的峡湾和海湾地区PAH-DNA加合物如何被NER系统与核小体中的人类细胞提取物差异切除，并阐明与未复合的DNA相比，组蛋白环境如何影响切除效率。这项研究的结果有望为未来研究大体积致癌物-DNA损伤是在体内加工的，以及核小体重塑因子如何增强和促进细胞环境中大体积DNA损伤的NER。

DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAH) are byproducts of fossil fuel combustion and are present in our air, food and water; the presence of these genotoxic environmental carcinogens in our environment continues to be a hazard to human health. The structural features and biological impact of PAH that distinguish highly active mutagens and tumorigens from structurally related less active, or inactive compounds, have long been of interest for understanding the etiology of human cancers in exposed populations. There are two important sub-classes of PAH that are distinguished by different topological features: (i) the sterically hindered 'fjord' region that causes significant non-planarity in the aromatic ring system of fjord PAH, and (ii) 'bay' region PAH that are sterically unhindered and planar (e.g., the well known environmental carcinogen benzo[a]pyrene). Both types of PAH are metabolically activated to reactive diol epoxide intermediates that react predominantly with guanine and adenine in cellular DNA to form pre-mutagenic covalent adducts in mammalian cells and tissues that can, if not repaired, ultimately contribute to the etiology of human cancers The fjord PAH have attracted significant attention by the chemical carcinogenesis community because they are up to ~ 100-fold more tumorigenic than the bay region prototype benzo[a]pyrene. Previous research from this laboratory has shown that there are remarkable differences in the relative excision efficiencies of different, stereochemically well defined PAH-DNA adducts by the human nucleotide excision repair (NER) system in whole cell extracts. Of particular interest are the observations that PAH- adenine DNA adducts derived from highly tumorigenic fjord PAH are strongly resistant to NER. However, these conclusions are built on NER experiments that were conducted with free DNA in aqueous environments; it is not known whether in biologically more relevant protein environments of nucleosomes, the fundamental sub-units of DNA packaging in the cell, similar hierarchies of NER will be observed with DNA substrates containing the same single bay and fjord PAH-DNA adducts. The objectives of this project are to evaluate NER efficiencies in nucleosomal DNA. The specific aims are: (1) to investigate the role of DNA positioning sequence, thermodynamic stability, nucleosome dynamics, and accessibility of DNA adducts at different sites of the nucleosomal superhelix on NER efficiencies; (2) Evaluate the effects of covalent modification of H3, H4, H2A and H2B histones on the same nucleosome properties and NER; (3) Determine how structurally distinct fjord and bay region PAH-DNA adducts in nucleosomes are differentially excised by the NER system with human cell extracts in nucleosomes, and elucidate how the excision efficiencies are impacted by the histone environment compared with uncomplexed DNA The outcome of this study is expected to provide a molecular basis for future investigations of how bulky carcinogen-DNA lesions are processed in vivo and how nucleosome remodeling factors enhance and facilitate the NER of bulky DNA lesions in cellular environments.