Cross-talking pre-incision events of eukaryotic NER

描述（由申请人提供）：不同的外源性环境暴露在活细胞中引入有害的应激。DNA损伤反应（DDR）抵消了细胞内外无处不在的基因毒性损伤的影响。越来越多的因素被吸收到受损的基因组位点，不仅是错综复杂和不可分割的联系，而且它们的相互作用决定了DDR的性质和过程。由于DDR组分之间存在广泛的分子间串扰，该持续拨款将重点研究NER和检查点信号通路相关重叠因子的相互作用和影响。该建议是基于这样一个前提，即紫外线损伤同时激活各种影响损伤和修复的事件，以及表观遗传完整染色质和正常细胞周期的恢复。该研究的具体假设是，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与NER相关的DDR的所有关键方面。拟议的工作将利用大量相关的最新技术来实现以下相互关联的具体目标：(1)验证DDB和XPC在检查点激活中的作用；(2)了解组蛋白泛素化和乙酰化在检查点信号传递中的作用；(3)确定染色质重塑剂INO80在检查点维持中的作用；(4)确定组蛋白伴侣ASF1和NASP在细胞周期检查点恢复中的作用。各种缺乏单个蛋白因子的人类细胞系，无论是组成性的还是通过siRNA/shRNA介导的基因沉默，将在细胞周期的选择阶段被利用来分析对检查点蛋白标记的影响，并通过FACS分析、ChIP、共免疫沉淀和/或共定位分析揭示它们的功能相互作用。ATR/ATM底物的生化特性将通过SQ/TQ底物基序的突变改变以及它们的功能分析来实现。选择组蛋白修饰将在特定受损细胞中进行评估，以揭示调节NER和细胞周期进展的改变。最后，纯化的重组组蛋白和伴侣蛋白将在体外进行测试，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。这些系统的研究将为哺乳动物细胞外源暴露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。

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. PUBLIC HEALTH RELEVANCE: DNA damage from exposure to environmental agents provokes highly conserved cellular responses essential for maintaining genetic and epigenetic hallmarks of the human genome. The signals emanating from introduction of genomic damage activate checkpoints for arresting cells cycle, successful completion of DNA repair or elimination of irreparably injured cells through apoptosis. The recruitment of factors mediating these events at or near the damage site is not only intricately and inextricably linked but their interplay dictates the nature as well as course of DNA damage response. The proposed work, on the theme of deciphering the inter-molecular cross-talk between DNA repair and signaling, has important implications to human health risk assessment and management.