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）：多种武物环境暴露会在活细胞中引入有害压力。 DNA损伤反应（DDR）抵消了细胞内外无处不在的遗传毒性损伤的影响。招募不断受损的基因组部位因素的列表不仅复杂且密不可分地联系在一起，而且它们的相互作用决定了DDR的性质和过程。由于DDR组件之间的分子间串扰广泛，该延续赠款将着重于研究NER和检查点信号通路相关重叠因子的相互作用和影响。该提案基于这样的前提：紫外线损伤同时激活影响损害和修复的各种事件，以及恢复表观遗传上完整的染色质和正常细胞循环的恢复。所提出的工作的基本假设是，UV损伤，DDB和XPC复合物的初始传感器与信号激酶，ATR和ATM及其相互作用密切相关，及其相互作用与染色质重塑因子，组蛋白伴侣，组蛋白和组蛋白修饰的蛋白质相结合，确定与NER与NER相关的所有关键方面。 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组蛋白伴侣，ASF1和NASP参与细胞周期检查点恢复。在细胞周期的某些阶段，将使用缺乏单个蛋白质因子的人类细胞系的种类或通过siRNA/SHRNA介导的基因沉默，以分析对检查点蛋白标记物的影响，并通过FACS分析，CHIP，CoS-nip，co-noprecipitation和co-munumunoprication和/os/os/os/或通过共取代分析揭示其功能相互作用。 ATR/ATM底物的生化表征将通过SQ/TQ底物基序的突变改变，然后进行功能分析来实现。将在特定损害的细胞中评估选定的组蛋白修饰，以揭示调节NER和细胞周期进程的变化。最后，将在体外测试纯化的重组组蛋白和伴侣，以描绘其NER，检查点和细胞周期特定的生化作用。这些系统的研究将提供有关哺乳动物细胞异生物暴露的关键事件的关键见解，这是人类健康风险评估和管理的最终目标。