Environmental Carcinogen-DNA Adducts: NER Recognition

 描述(申请人提供)：人类基因组不断受到环境污染物、炎症反应期间在人体组织中分泌的内源性活性氧化物种以及阳光紫外线成分的攻击。在外源致癌环境污染物中，有多环芳香化合物，它们是在有毒垃圾场和超级基金地点、空气颗粒物以及我们的食物和水中发现的化石燃料燃烧的副产品。由多环芳香化合物、炎症相关的活性氧化物种和紫外线引起的DNA损伤会导致恶性突变的积累，从而导致各种人类癌症。然而，并不是所有的DNA损伤在促进人类疾病方面都同样有效：虽然损伤可以通过人类核苷酸切除修复(NER)机制来切除，但一些DNA损伤可以快速修复，一些修复缓慢，还有一些对NER完全抵抗，因此特别具有遗传毒性。在由多种NER基因突变引起的毁灭性的人类疾病着色性干皮病中，NER的重要作用得到了证明。然而，为什么某些DNA损伤是NER耐药的，而其他DNA损伤不是NER正常的，这一点还不清楚。本项目的目的是通过重点研究NER中损伤识别的关键步骤，从机制上深入了解DNA损伤修复的这种令人费解的变异性，以获得对NER抗性的分子理解。我们假设，损伤被识别的程度取决于它对DNA施加的不稳定或稳定的程度：稳定导致修复阻力，而不稳定促进修复。我们将剖析一组选定的DNA损伤的结构、动力学和热力学属性，这些属性决定了它们是否能被Rad4-RAD23识别，Rad4-RAD23是人类XPC-RAD23B损伤识别因子的酵母同源基因。在目标1中，我们将确定含有损伤的DNA的局部热力学稳定性调节其识别的程度。在目标2中，我们将确定Rad4-RAD23成功识别病变并正确招募后续NER因子的生产性结合的分子机制，以及结合途径和沿该途径的自由能如何依赖于病变结构。在目标3中，我们将研究核小体中的DNA与组蛋白的复合体，核小体是细胞中DNA的基本包装单位。我们将确定核小体中NER蛋白对DNA损伤的访问如何受损伤的结构和动态特性的影响，以促进或抑制修复。我们对NER的DNA损伤识别机制的新见解可能会导致开发更有效、对NER不敏感的化疗药物，因为现有药物的疗效会受到NER的影响。此外，这种了解将有助于在许多环境污染物中确定最具遗传毒性的致癌前体，从而能够制定更有针对性的减少政策和相关健康风险的生物监测方法。

 DESCRIPTION (provided by applicant): The human genome is under constant attack from environmental pollutants, endogenous reactive oxidizing species that are secreted in human tissues during the inflammatory response, and ultraviolet components of sunlight. Among the exogenous cancer-causing environmental contaminants are polycyclic aromatic compounds that are byproducts of fossil fuel combustion found at toxic waste dumps and superfund sites, in airborne particulates, and in our food and water. The DNA lesions derived from polycyclic aromatic compounds, inflammation-related reactive oxidizing species, and ultraviolet light result in the accumulation of malignant mutations that lead to a variety of human cancers. However, not all DNA lesions are equally effective in promoting human diseases: while lesions can be excised by the human nucleotide excision repair (NER) mechanism, some DNA lesions are rapidly repaired, some are repaired slowly, and some are entirely resistant to NER and are therefore particularly genotoxic. The vital importance of NER is demonstrated in the devastating human disorder xeroderma pigmentosum, caused by mutations in various NER genes. However, why certain DNA lesions are NER-resistant and others are not when NER is normal, is not understood. The objective of this project is to provide mechanistic insights into this puzzling variability of DNA lesion repair, by focusing on the key step of lesion recognition in NER, to yield a molecular understanding of NER resistance. We hypothesize that how well a lesion is recognized is determined by the extent of destabilization or stabilization that it impose on DNA: stabilization leads to repair resistance and destabilization facilitates repair. We will dissect the structural, dynamic and thermodynamic properties for a selected set of DNA lesions that govern whether they are recognized by Rad4-Rad23, the yeast ortholog of the human XPC-RAD23B lesion recognition factor. In Aim 1 we will determine the extent that local thermodynamic stability of lesion-containing DNA regulates their recognition. In Aim 2 we will determine the molecular mechanism for productive binding of Rad4-Rad23 that successfully recognizes the lesions and correctly recruits subsequent NER factors, and how the binding pathway and free energies along this pathway depend on lesion structures. In Aim 3 we will investigate DNA complexed with histone proteins in nucleosomes, the fundamental packaging unit of DNA in cells. We will determine how access of the NER proteins to DNA lesions in nucleosomes is governed by the lesion's structural and dynamic properties to promote or inhibit repair. The novel insights into the DNA lesion recognition mechanisms of NER that we will gain may lead to the development of more effective, less NER- susceptible chemotherapeutic agents, since the efficacy of current drugs is impaired by NER. Furthermore, such understanding will help to identify the most genotoxic cancer-causing precursors among the many environmental contaminants, thus allowing for the development of better targeted abatement policies and biomonitoring methods of the associated health risks.