Cellular Responses to DNA-Protein Crosslinks

描述（由申请人提供）：暴露于内源性和外源性来源的各种化学试剂以及暴露于电离辐射和紫外线辐射，除了更广泛表征的单个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。所有这些物质都是已知或疑似致癌物，因此对接触人群构成重大健康风险。尽管这些DNA-蛋白质交联（DPC）的普遍性及其在某些癌症、年龄相关性黄斑变性和某些神经退行性疾病（如帕金森病）中的可能致病作用，但关于这些病变的生物学处理的数据仍然很少，因为直到最近，还没有机制可用于在DNA中产生位点特异性、蛋白质特异性病变。为了建立对DPC存在的细胞反应的基本理解，本文首次开发了产生位点特异性DPC的方法，所述DPC位于DNA的大沟和小沟中并且沿着糖-磷酸骨架。含有位点特异性和蛋白特异性DPC的DNA的可用性将使得能够测试关于这些损伤如何在哺乳动物细胞中复制和修复的假设。特定目的（1）为含有各种DPC损伤的定义DNA的构建和物理表征奠定了基础;开发了在DNA中产生各种大小的DPC（600 - 120，000 Da）的策略，并通过分析弯曲角度、足迹和热不稳定来表征其对DNA结构的调节。特定目的（2）将使用原核和哺乳动物NER测定、DNA复制旁路分析和DNA解旋酶解旋研究，表征含有这些DPC病变的DNA的体外复制和修复。特定目的（3）通过将这些病变工程化到单链和双链载体中，并通过修复熟练和缺陷细胞复制它们，评价DPC的致突变潜力和体内修复途径;将分析这些位点特异性病变的突变谱。

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.