Cross-talking pre-incision events of eukaryotic NER

真核 NER 的串扰切前事件

• 批准号: 7072794
• 负责人: ALTAF A WANI
• 金额: $ 34.67万
• 依托单位: OHIO STATE 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 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072794
• 关键词: DNA binding protein; DNA damage; DNA repair; acetylation; acyltransferase; binding sites; biological signal transduction; cell line; cell nucleus; chromatin; chromatin immunoprecipitation; enzyme activity; eukaryote; genome; histones; nucleotides; p53 gene /protein; polymerase chain reaction; protein localization; protein protein interaction; protein structure function; transcription factor; western blottings

DESCRIPTION (provided by applicant): The maintenance of genomic stability in eukaryotes is relentlessly challenged by DNA damage. In order to understand the basis of damage tolerance and avoidance of deleterious effects, it is important to grasp not only the nature of such DNA damage and its processing pathways in cells, but also to have a clear understanding of how cells manage the lesion repair in the highly compact and tightly regulated chromatin milieu of the eukaryotic nucleus. This proposal is based on the premise that the specific biochemical pathways, relying on wide-ranging inter-molecular cross-talk between the pathway components, operate at a higher efficiency under conditions of macromolecular crowding and sequentially recruit specific factors for access a s well as repair of genotoxic lesions within mammalian genome. The proposal builds on our recent observations that support a strong link between DNA repair and p53 mediated and other related regulatory pathways. The overall experiments are designed to address the specific hypothesis that cellular DNA damage recognition and transcription factors, congregate at DNA lesions sites, open the chromatin via histone acetylation and initiate the key pre-incision events of nucleotide excision repair (NER). The proposed work will utilize the PI's established expertise in relevant, cellular, molecular, biochemical, and immunological technologies to undertake the following specific aims. (1) To assess the nature and extent of contribution of damaged DNA binding protein, DDB, in NER through quantitative assessment of NER sub-pathways in human cells of defined protein status. (2) To demonstrate the role of DDB for sequential recruitment of initial recognition and downstream core NER factors through spatial and temporal co-localization in situ. (3) To determine the participation of histone acetyl transferases in damage recognition and excision via qualitative protein cataloging and targeted recruitment and physical association of interacting factors. (4) To establish the histones acetylation as a direct and key consequence of DNA damage by placement of acetylation events exclusively at the lesion sites in vivo and in vitro. These studies will provide important insights regarding the dynamics of DNA damage recognition and processing through identification of cross-talking events and critical factors that influence the maintenance of genomic stability so important to overcome the hazardous health effects of genotoxic environmental exposures.

描述（由申请人提供）： 真核生物基因组稳定性的维持不断受到 DNA 损伤的挑战。 为了理解损伤耐受和避免有害影响的基础，不仅要掌握这种DNA损伤的性质及其在细胞中的加工途径，而且要清楚地了解细胞如何管理损伤修复真核细胞核高度紧凑且严格调控的染色质环境。 该提议的前提是，特定的生化途径依赖于途径成分之间广泛的分子间串扰，在大分子拥挤的条件下以更高的效率运行，并依次招募特定的因子进行访问和修复哺乳动物基因组内的遗传毒性损伤。 该提案建立在我们最近的观察结果之上，这些观察结果支持 DNA 修复与 p53 介导的以及其他相关调控途径之间的密切联系。 整个实验旨在解决特定的假设，即细胞 DNA 损伤识别和转录因子聚集在 DNA 损伤位点，通过组蛋白乙酰化打开染色质并启动核苷酸切除修复 (NER) 的关键切口前事件。 拟议的工作将利用 PI 在相关细胞、分子、生化和免疫学技术方面的既定专业知识来实现​​以下具体目标。 (1) 通过定量评估特定蛋白质状态的人类细胞中的 NER 子通路，评估受损 DNA 结合蛋白 (DDB) 在 NER 中的性质和贡献程度。 (2) 证明 DDB 通过原位时空共定位顺序招募初始识别和下游核心 NER 因子的作用。 (3)通过定性蛋白质编目和相互作用因子的靶向招募和物理关联来确定组蛋白乙酰转移酶在损伤识别和切除中的参与。 (4) 通过将乙酰化事件仅放置在体内和体外的损伤位点，确定组蛋白乙酰化是 DNA 损伤的直接和关键后果。 这些研究将通过识别串扰事件和影响基因组稳定性维持的关键因素，提供有关 DNA 损伤识别和处理动态的重要见解，这对于克服基因毒性环境暴露对健康的危害非常重要。