Cross-talking pre-incision events of eukaryotic NER

真核 NER 的串扰切前事件

基本信息

批准号：
8462251
负责人：
ALTAF A WANI
金额：
$ 43.62万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8462251
关键词：
ATM activation Ablation Acetylation Address Apoptosis BRCA1 gene Binding Biochemical Biological Assay Cell Cycle Cell Cycle Arrest Cell Cycle Checkpoint Cell Cycle Progression Cell Cycle Stage Cell Proliferation Cell physiology Cells Chromatin Chromatin Remodeling Factor Co-Immunoprecipitations Complex DNA Damage DNA Repair EP300 gene Environmental Exposure Epigenetic Process Event Exposure to Gene Silencing Genetic Genome Genomics Goals Grant Health Histones Human Human Cell Line Human Genome Individual Laboratories Life Link Maintenance Mammalian Cell Mediating Molecular Molecular Chaperones Nature Normal Cell Nucleotide Excision Repair Pathway interactions Phosphotransferases Proteins Recombinants Recovery Regulation Risk Assessment Risk Management Role Signal Pathway Signal Transduction Site Small Interfering RNA Stress Surgical incisions Technology UV induced DNA damage Ubiquitination Work Xenobiotics base chromatin remodeling environmental agent histone modification in vitro testing in vivo injured insight public health relevance repaired response restoration sensor small hairpin RNA ultraviolet damage ultraviolet irradiation

项目摘要

DESCRIPTION (provided by applicant): Diverse xenobiotic environmental exposures introduce deleterious stress in living cells. DNA damage response (DDR) counteracts the effects of omnipresent genotoxic insult from within and outside cell. The recruitment of an ever-increasing list of factors to damaged genomic sites is not only intricately and inextricably linked but their interplay dictates the nature as well as course of DDR. Due to a wide-ranging inter-molecular crosstalk between DDR components this continuation grant will focus on studying the interaction and influence of relevant overlapping factors of NER and checkpoint signaling pathways. The proposal is based on the premise that UV damage simultaneously activates diverse events impinging on access to damage and repair as well as restoration of epigenetically intact chromatin and normal cell cycling. Specific hypothesis underlying the proposed work is that initial sensors of UV damage, DDB and XPC complexes, are intimately associated with signaling kinases, ATR and ATM, and their interaction, in conjunction with chromatin remodeling factors, histone chaperons and histone modifying proteins, determines all key aspects of DDR related to NER. The proposed work will utilize a relevant plethora of state-of-the art technologies to address following inter-related specific objectives: (1) to demonstrate the function of DDB and XPC in checkpoint activation, (2) to understand the roles of histone ubiquitination and acetylation in checkpoint signaling, (3) to ascertain the influence of chromatin remodeler, INO80, in checkpoint maintenance, and (4) to establish the participation of histone chaperons, ASF1 and NASP, in cell cycle checkpoint recovery. Variety of human cell lines lacking individual protein factors, either constitutively or by siRNA/shRNA mediated gene silencing, will be utilized at select stages of cell cycle to analyze the effects on checkpoint protein markers and reveal their functional interactions through FACS analysis, ChIP, co-immunoprecipitation and/or by co-localization assays. Biochemical characterization of ATR/ATM substrates will be achieved by mutational alterations of SQ/TQ substrate motifs followed by their functional analysis. Select histone modifications will be evaluated in specifically compromised cells to reveal alterations regulating NER and cell cycle progression. Lastly, purified recombinant histones and chaperons will be tested in vitro to delineate their NER, checkpoint and cell cycle specific biochemical roles in vivo. These systematic studies will furnish crucial insights regarding the key events initiated upon xenobiotic exposures of mammalian cells with the ultimate goal of human health risk assessment and management.

描述（由申请人提供）：不同的异生素环境暴露会给活细胞带来有害的应激。 DNA 损伤反应 (DDR) 可以抵消细胞内外普遍存在的基因毒性损伤的影响。越来越多的因素被招募到受损的基因组位点，这不仅错综复杂、密不可分，而且它们的相互作用决定了 DDR 的性质和进程。由于 DDR 组件之间存在广泛的分子间串扰，这项继续拨款将重点研究 NER 和检查点信号通路相关重叠因素的相互作用和影响。该提议的前提是，紫外线损伤会同时激活多种事件，影响损伤和修复以及表观遗传完整染色质和正常细胞周期的恢复。这项工作的具体假设是，紫外线损伤的初始传感器 DDB 和 XPC 复合物与信号激酶、ATR 和 ATM 密切相关，它们与染色质重塑因子、组蛋白伴侣和组蛋白修饰蛋白的相互作用决定了所有DDR 与 NER 相关的关键方面。拟议的工作将利用大量相关的最先进技术来解决以下相互关联的具体目标：（1）展示 DDB 和 XPC 在检查点激活中的功能，（2）了解组蛋白泛素化的作用检查点信号传导中的乙酰化和乙酰化，(3) 确定染色质重塑剂 INO80 在检查点维持中的影响，以及 (4) 确定组蛋白伴侣 ASF1 和NASP，细胞周期检查点恢复。将在细胞周期的选定阶段利用组成型或通过 siRNA/shRNA 介导的基因沉默缺乏单个蛋白质因子的各种人类细胞系来分析对检查点蛋白质标记物的影响，并通过 FACS 分析、ChIP、co 揭示其功能相互作用。 -免疫沉淀和/或通过共定位测定。 ATR/ATM 底物的生化表征将通过 SQ/TQ 底物基序的突变改变及其功能分析来实现。将在特定受损细胞中评估选定的组蛋白修饰，以揭示调节 NER 和细胞周期进程的改变。最后，纯化的重组组蛋白和伴侣将在体外进行测试，以描述它们在体内的 NER、检查点和细胞周期特异性生化作用。这些系统研究将为哺乳动物细胞接触异生物质时引发的关键事件提供重要见解，最终目标是人类健康风险评估和管理。