Cross-talking pre-incision events of eukaryotic NER

描述（由申请方提供）：不同的外源性环境暴露会在活细胞中引入有害应激。DNA损伤反应（DDR）是细胞内外基因毒性损伤的一种免疫反应。招募一个不断增加的名单的因素受损的基因组位点不仅是错综复杂的和不可分割的联系，但它们的相互作用决定了DDR的性质以及过程。由于DDR组件之间广泛的分子间串扰，该继续资助将专注于研究NER和检查点信号通路的相关重叠因素的相互作用和影响。该提议基于这样的前提，即UV损伤同时激活不同的事件，这些事件影响损伤和修复的获得以及表观遗传学上完整的染色质和正常细胞周期的恢复。具体假设的基础上提出的工作是，初始传感器的紫外线损伤，DDB和XPC复合物，是密切相关的信号激酶，ATR和ATM，和它们的相互作用，结合染色质重塑因子，组蛋白伴侣和组蛋白修饰蛋白，确定所有关键方面的DDR相关NER。拟议的工作将利用相关的大量最先进的技术来解决以下相互关联的具体目标：（1）证明DDB和XPC在检查点激活中的功能，（2）了解组蛋白泛素化和乙酰化在检查点信号传导中的作用，（3）确定染色质重塑剂INO 80在检查点维持中的影响，和（4）确定组蛋白伴侣ASF 1和NASP参与细胞周期检查点恢复。将在细胞周期的选定阶段使用组成型或通过siRNA/shRNA介导的基因沉默缺乏单个蛋白因子的各种人类细胞系，以分析对检查点蛋白标志物的影响，并通过FACS分析、ChIP、免疫共沉淀和/或通过共定位测定揭示其功能相互作用。ATR/ATM底物的生物化学表征将通过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价选择的组蛋白修饰，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蛋白将在体外进行测试，以描绘其NER，检查点和细胞周期特异性的生物化学作用在体内。这些系统的研究将提供关键的见解，对哺乳动物细胞的外源性暴露后启动的关键事件，人类健康风险评估和管理的最终目标。

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.