Cellular Responses to DNA-Protein Crosslinks

描述(申请人提供)：暴露于来自内源和外源的各种化学制剂，以及暴露在电离和紫外线辐射下，除了更广泛地表征单个DNA或蛋白质加合物外，还会在DNA和蛋白质分子之间产生交联链。所有这些物质要么是已知的，要么是可疑的致癌物，因此，对暴露于此的人群构成重大的健康风险。尽管这些DNA-蛋白质交联物(DPC)普遍存在，并可能在一些癌症、老年性黄斑变性和一些神经退行性疾病(如帕金森病)中起致病作用，但关于这些损伤的生物处理方面的数据很少，因为直到最近，还没有可用的机制来在DNA中创建位置特异性、蛋白质特异性损伤。为了建立对细胞对DPC存在的反应的基本了解，本文首次开发了创建位于DNA的主要和次要沟槽以及沿糖-磷酸骨架的位点特异性DPC的方法。含有位点特异性和蛋白质特异性DPC的DNA的可获得性将使关于这些损伤如何在哺乳动物细胞中复制和修复的假说得到检验。具体目标(1)为含有各种DPC损伤的已定义DNA的构建和物理特征奠定基础；开发了在DNA中创建各种大小的DPC(600-120,000 Da)的策略，并通过分析弯曲角度、足迹和热失稳来表征它们对DNA结构的调制。特殊目的(2)将使用原核生物和哺乳动物的NER分析、DNA复制旁路分析和DNA解旋酶研究来表征包含这些DPC损伤的DNA的体外复制和修复。特殊目的(3)通过将这些损伤工程成单链和双链载体，并通过修复熟练和缺陷的细胞复制它们，来评估DPC的突变潜力和体内修复途径；将分析这些特定部位损伤的突变谱。

DESCRIPTION (provided by applicant): Exposure to a variety of chemical agents that arise from both endogenous and exogenous sources, as well as exposure to ionizing and ultraviolet radiation, produces crosslinks between DNA and protein molecules, in addition to the more extensively characterized individual DNA or protein adducts. All of these agents are either known or suspected carcinogens and as such, pose significant health risks to exposed human populations. Despite the pervasiveness of these DNA-protein crosslinks (DPCs) and their probable causative role in some cancers, age-related macular degeneration and some neurodegenerative diseases such as Parkinson's, there exists a paucity of data concerning the biological processing of these lesions, because until recently, there have been no mechanisms available to create site-specific, protein-specific lesions in DNA. In order to establish a fundamental understanding of cellular responses to the presence of DPCs, herein for the first time, methodologies are developed that create site-specific DPCs that are located in the major and minor grooves of DNA and along the sugar-phosphate backbone. The availability of DNAs containing site-specific and protein-specific DPCs will enable the testing of hypotheses on how these lesions are replicated and repaired in mammalian cells. Specific Aim (1) lays the foundation for the construction and physical characterization of defined DNAs containing a variety of DPC lesions; strategies are developed to create various sized DPCs (600 - 120,000 Da) in DNAs and characterize their modulation of the DNA structure by analyses of bend angles, footprint, and thermal destabilization. Specific Aim (2) will characterize in vitro replication and repair of DNAs containing these DPC lesions, using both prokaryotic and mammalian NER assays, DNA replication bypass analyses, and DNA helicase unwinding studies. Specific Aim (3) evaluates the mutagenic potential and in vivo repair pathways for DPCs by engineering these lesions into single- and double-stranded vectors and replicating them through repair proficient and deficient cells; mutational spectra will be analyzed for these site-specific lesions.