Cross-talking pre-incision events of eukaryotic NER

描述(由申请人提供)： 真核生物基因组稳定性的维持受到DNA损伤的无情挑战。为了理解损伤耐受和避免有害影响的基础，重要的是不仅要掌握这种DNA损伤的性质及其在细胞中的处理途径，而且要清楚地了解细胞如何在高度致密和严格调控的真核染色质环境中管理损伤修复。这一建议是基于这样一个前提，即特定的生化途径依赖于途径组件之间广泛的分子间串扰，在大分子拥挤的条件下以更高的效率运行，并顺序地招募特定的因子来访问S以及修复哺乳动物基因组中的遗传毒性损伤。该建议建立在我们最近的观察结果的基础上，这些观察结果支持DNA修复与P53介导的和其他相关调控途径之间的强烈联系。整个实验旨在解决特定的假设，即细胞DNA损伤识别和转录因子聚集在DNA损伤部位，通过组蛋白乙酰化打开染色质，启动核苷酸切除修复(NER)的关键切开前事件。拟议的工作将利用PI在相关、细胞、分子、生化和免疫学技术方面的成熟专业知识来实现以下具体目标。(1)通过对已确定蛋白质状态的人细胞中NER亚通路的定量评估，评估受损DNA结合蛋白DDB在NER中的贡献性质和程度。(2)通过空间和时间的原位共定位，论证DDB在初始识别和下游核心NER因子的顺序招募中的作用。(3)通过定性蛋白质分类、靶向招募和相互作用因子的物理关联，确定组蛋白乙酰转移酶在损伤识别和切除中的作用。(4)通过将乙酰化事件仅放置在体内和体外的损伤部位，确定组蛋白乙酰化是DNA损伤的直接和关键结果。这些研究将通过识别串话事件和影响维持基因组稳定性的关键因素来提供关于DNA损伤识别和处理的动力学的重要见解，这些因素对于克服遗传毒性环境暴露的有害健康影响是至关重要的。

DESCRIPTION (provided by applicant): The maintenance of genomic stability in eukaryotes is relentlessly challenged by DNA damage. In order to understand the basis of damage tolerance and avoidance of deleterious effects, it is important to grasp not only the nature of such DNA damage and its processing pathways in cells, but also to have a clear understanding of how cells manage the lesion repair in the highly compact and tightly regulated chromatin milieu of the eukaryotic nucleus. This proposal is based on the premise that the specific biochemical pathways, relying on wide-ranging inter-molecular cross-talk between the pathway components, operate at a higher efficiency under conditions of macromolecular crowding and sequentially recruit specific factors for access a s well as repair of genotoxic lesions within mammalian genome. The proposal builds on our recent observations that support a strong link between DNA repair and p53 mediated and other related regulatory pathways. The overall experiments are designed to address the specific hypothesis that cellular DNA damage recognition and transcription factors, congregate at DNA lesions sites, open the chromatin via histone acetylation and initiate the key pre-incision events of nucleotide excision repair (NER). The proposed work will utilize the PI's established expertise in relevant, cellular, molecular, biochemical, and immunological technologies to undertake the following specific aims. (1) To assess the nature and extent of contribution of damaged DNA binding protein, DDB, in NER through quantitative assessment of NER sub-pathways in human cells of defined protein status. (2) To demonstrate the role of DDB for sequential recruitment of initial recognition and downstream core NER factors through spatial and temporal co-localization in situ. (3) To determine the participation of histone acetyl transferases in damage recognition and excision via qualitative protein cataloging and targeted recruitment and physical association of interacting factors. (4) To establish the histones acetylation as a direct and key consequence of DNA damage by placement of acetylation events exclusively at the lesion sites in vivo and in vitro. These studies will provide important insights regarding the dynamics of DNA damage recognition and processing through identification of cross-talking events and critical factors that influence the maintenance of genomic stability so important to overcome the hazardous health effects of genotoxic environmental exposures.