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；在我们的环境中存在这些基因毒性环境致癌物继续对人类健康构成危害。多环芳烃的结构特征和生物学影响区分了高活性诱变剂和致瘤剂与结构相关的低活性或无活性化合物，长期以来一直是了解暴露人群中人类癌症病因学的兴趣所在。多环芳烃有两个重要的亚类，它们以不同的拓扑特征来区分：(i)空间受阻的“峡湾”区，在峡湾多环芳烃的芳香环系统中导致显著的非平面性；（ii）空间不受阻的“海湾”区多环芳烃（例如，众所周知的环境致癌物苯并[a]芘）。这两种类型的多环芳烃被代谢激活为活性环氧二醇中间体，这些中间体主要与细胞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致突变前的共价加合物，如果不进行修复，最终会导致人类癌症的病因。峡湾多环芳烃引起了化学致癌界的极大关注，因为它们的致癌性比海湾地区的原型苯并[a]芘高100倍。该实验室先前的研究表明，在全细胞提取物中，人类核苷酸切除修复（NER）系统对不同的、立体化学上定义良好的PAH-DNA加合物的相对切除效率存在显著差异。特别有趣的是观察到多环芳烃-腺嘌呤DNA加合物来源于高致瘤性的峡湾多环芳烃对NER有很强的抗性。然而，这些结论是建立在用游离DNA在水环境中进行的NER实验之上的；目前尚不清楚是否在核小体（细胞中DNA包装的基本亚基）的生物学上更相关的蛋白质环境中，含有相同单一海湾和峡湾PAH-DNA加合物的DNA底物是否会观察到类似的NER层次。本项目的目的是评估核小体DNA中的NER效率。具体目的是：(1)研究DNA定位序列、热力学稳定性、核小体动力学和核小体超螺旋不同位点DNA加合物的可及性对NER效率的影响；(2)评价H3、H4、H2A和H2B组蛋白共价修饰对相同核小体性质和NER的影响；(3)确定NER系统与核小体中的人类细胞提取物如何在结构上不同的峡湾和海湾地区切除核小体中的PAH-DNA加合物；并阐明组蛋白环境对切除效率的影响。本研究的结果有望为未来研究大块致癌物质-DNA病变在体内是如何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块DNA病变的NER提供分子基础。

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.