Cellular Responses to DNA-Protein Crosslinks

细胞对 DNA-蛋白质交联的反应

• 批准号: 6898814
• 负责人: R. Stephen Lloyd
• 金额: $ 27.86万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6898814
• 关键词: DNA; DNA damage; DNA footprinting; DNA repair; DNA replication; X ray crystallography; adduct; carcinogens; crosslink; fluorescence resonance energy transfer; helicase; ionizing radiation; mutagen testing; mutagens; nuclear magnetic resonance spectroscopy; nucleic acid structure; proteins; ultraviolet radiation

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.

描述（由申请人提供）：暴露于内源性和外源性来源产生的多种化学剂，以及暴露于电离和紫外线辐射中，除了具有更广泛特征的个体DNA或蛋白质加入器外，还会在DNA和蛋白质分子之间产生交联。所有这些药物都是已知的或疑似的致癌物，因此，暴露的人群构成了重大健康风险。尽管这些DNA-蛋白交联（DPC）及其在某些癌症中的可能致病作用，但与年龄有关的黄斑变性和某些神经退行性疾病（如帕金森氏病），但存在与这些病态的生物学处理相关的数据，因为直到最近，就可以使用机械源，但仍存在与帕金森的生物处理相关的数据。为了建立对DPC存在的细胞反应的基本理解，首次开发了一种方法，该方法论创建了位于DNA的主要和次要的dna且沿糖磷酸骨链的主要和小凹槽。含有位点特异性和蛋白质特异性DPC的DNA的可用性将对哺乳动物细胞中如何复制和修复这些病变的假设进行测试。具体目的（1）为定义的DNA的构建和物理表征奠定了基础，这些DNA包含各种DPC病变；开发了创建DNA中各种大小的DPC（600-120,000 DA）的策略，并通过弯曲角，足迹和热不稳定的分析来表征它们对DNA结构的调节。具体目的（2）将使用原核和哺乳动物NER分析，DNA复制旁路分析和DNA解旋酶解放研究来表征包含这些DPC病变的DNA的体外复制和修复。特定目的（3）通过将这些病变的工程化为单链和双链载体，评估DPC的诱变潜力和体内修复途径，并通过修复熟练且缺乏细胞来复制它们；将分析这些部位特异性病变的突变光谱。