Cross-talking pre-incision events of eukaryotic NER

描述(由申请人提供)：不同的外来环境暴露会在活细胞中引入有害的压力。DNA损伤反应(DDR)抵消了来自细胞内外的无处不在的遗传毒性侮辱的影响。越来越多的因素被招募到受损的基因组位置，不仅错综复杂和千丝万缕地联系在一起，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，这项延续拨款将侧重于研究NER和检查点信号通路的相关重叠因素的相互作用和影响。该建议的前提是紫外线损伤同时激活影响损伤和修复的各种事件，以及恢复表观遗传完整的染色质和正常的细胞周期。支持这项工作的具体假设是，紫外线损伤的初始传感器DDB和XPC复合体与信号激酶、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DDR与NER相关的所有关键方面。这项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)了解组蛋白泛素化和乙酰化在检查点信号中的作用，(3)确定染色质重构体INO80在检查点维持中的影响，以及(4)确定组蛋白伴侣ASF1和NASP在细胞周期检查点恢复中的参与。各种缺乏单个蛋白质因子的人类细胞系，无论是结构性的还是通过siRNA/shRNA介导的基因沉默，将在细胞周期的特定阶段被用于分析对检查点蛋白标记物的影响，并通过FACS分析、芯片、免疫共沉淀和/或通过共定位分析来揭示它们之间的功能相互作用。ATR/ATM底物的生化特性将通过SQ/TQ底物基序的突变和功能分析来实现。选择的组蛋白修饰将在特定受损的细胞中进行评估，以揭示调节NER和细胞周期进展的变化。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的生化作用。这些系统的研究将为人类健康风险评估和管理的最终目标提供关于哺乳动物细胞暴露于异种生物时引发的关键事件的关键见解。

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.