Cross-talking events of eukaryotic DDR

真核DDR的串扰事件

基本信息

批准号：
9174683
负责人：
ALTAF A WANI
金额：
$ 45.23万
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 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9174683
关键词：
Ablation Acetylation Address Biochemical Biological Assay Cancer Etiology Cell Cycle Cell Cycle Checkpoint Cell Cycle Progression Cell Cycle Stage Cell Proliferation Cells Chromatin Chromatin Modeling Co-Immunoprecipitations Complex DNA Damage DNA Repair DNA Replication Damage DNA lesion Deposition Distal EP300 gene Environmental Exposure Eukaryota Eukaryotic Cell Event Gene Silencing Genome Genomic Instability Goals Health Histone Acetylation Histones Human Human Cell Line Image Imagery Imaging technology Immunofluorescence Immunologic Individual Knowledge Life Ligase Link Lysine Malignant Neoplasms Mediating Molecular Molecular Chaperones Mutation NBS1 gene Normal Cell Organism Pathway interactions Phosphorylation Phosphotransferases Play Proteins Recombinants Regulation Research Design Risk Role S-nitro-N-acetylpenicillamine Series Signal Transduction Small Interfering RNA Technology Time Transferase Ubiquitination Work abstracting ataxia telangiectasia mutated protein genetic approach genome integrity histone acetyltransferase histone modification in vitro testing in vivo insight novel repaired response restoration sensor small hairpin RNA tool ubiquitin ligase

项目摘要

PROJECT SUMMARY/ABSTRACT DNA damaging agents of diverse origins continuously challenge the genome of living organisms and impact its normal function. Eukaryotes counteract by deploying an effective DNA damage response (DDR) by promptly activating cell cycle checkpoints, initiating DNA repair and reassembling the intact chromatin. This intricate phenomenon depends on sequential recruitment and timely clearance of a number of cross- talking factors. During the previous project period, we have identified ATM checkpoint kinase (a master cell cycle checkpoint controller), and CRL4DDB2 ubiquitin ligase complex (a helix-distorting DNA lesion sensor), as the two key regulators of DNA repair-dependent chromatin assembly. The underlying premise of this renewal proposal is that DNA damage activates parallel events that impinge on (i) arresting cells to allow access to DNA damage and repair machinery and (ii) restoration of epigenetically intact chromatin and resumption of normal cell cycling. Accordingly the studies are designed to focus on (1) uncovering the molecular underpinning of ATM-directed H3K56ac-driven repair-dependent chromatin assembly, and (2) elucidate the function and mechanism of CRL4DDB2 ubiquitin ligase complex in integrated events of histone acetylation, ubiquitination and deposition in chromatin. The results would prove the related hypothesis that (a) ATM kinase facilitates repair-dependent chromatin assembly via a novel phosphorylation signaling cascade, and (b) CRL4DDB2 ubiquitin ligase directs the H3K56 acetylation and histone deposition during chromatin restoration. The proposed work will utilize a series of relevant technologies to address following inter-linked specific objectives: (1) to establish the role of ATM-mediated DNA damage signaling in regulating repair-dependent chromatin assembly, (2) to elucidate the function of CRL4DDB2 Ub-ligase in H3K56 acetylation, and (3) to establish the mechanism of CRL4DDB2 Ub-ligase-mediated regulation of post- repair histone deposition in chromatin restoration. Variety of human cell lines, normal or specifically 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 factors and reveal their functional interactions through FACS analysis, ChIP, co-immunoprecipitation and/or by co-localization assays. A battery of histone modifications will be evaluated in specifically compromised cells to reveal the alterations that regulate DNA repair and cell cycle progression. Lastly, purified recombinant histones, acetyltransferases, and CRL4DDB2 will be tested in vitro to delineate their specific biochemical roles in vivo. These systematic mechanistic studies of DDR would not only provide the knowledge relevant to cancer etiology, but also new amenable tools, targets and strategies for managing human health risk.

项目概要/摘要不同来源的 DNA 损伤剂不断挑战生物体的基因组并产生影响其正常功能。真核生物通过部署有效的 DNA 损伤反应 (DDR) 来抵消及时激活细胞周期检查点，启动 DNA 修复并重新组装完整的染色质。这种错综复杂的现象取决于顺序招募和及时清除许多交叉谈话因素。在之前的项目期间，我们已经鉴定了ATM检查点激酶（主细胞循环检查点控制器）和 CRL4DDB2 泛素连接酶复合物（螺旋扭曲 DNA 损伤传感器），作为 DNA 修复依赖性染色质组装的两个关键调节因子。这样做的基本前提是更新建议是，DNA 损伤会激活并行事件，影响 (i) 阻止细胞以允许获得 DNA 损伤和修复机制，以及 (ii) 恢复表观遗传完整的染色质和恢复正常的细胞循环。因此，研究的目的是集中于（1）揭示 ATM 引导的 H3K56ac 驱动的修复依赖性染色质组装的分子基础，以及 (2) 阐明CRL4DDB2泛素连接酶复合物在组蛋白整合事件中的功能和机制染色质中的乙酰化、泛素化和沉积。结果将证明相关假设 (a) ATM 激酶通过新型磷酸化信号促进修复依赖性染色质组装 (b) CRL4DDB2 泛素连接酶在级联过程中指导 H3K56 乙酰化和组蛋白沉积染色质修复。拟议的工作将利用一系列相关技术来解决以下问题相互关联的具体目标：(1) 确定 ATM 介导的 DNA 损伤信号传导在调节修复依赖性染色质组装，(2) 阐明 CRL4DDB2 Ub 连接酶在 H3K56 乙酰化，以及 (3) 建立 CRL4DDB2 Ub 连接酶介导的后调节机制修复染色质修复中的组蛋白沉积。各种人类细胞系，正常的或特殊的缺乏单个蛋白质因子，无论是组成型还是通过 siRNA/shRNA 介导的基因沉默，都会用于细胞周期的选定阶段，分析对检查点蛋白因子的影响并揭示其作用通过 FACS 分析、ChIP、免疫共沉淀和/或共定位进行功能相互作用化验。将在特定受损的细胞中评估一组组蛋白修饰，以揭示调节 DNA 修复和细胞周期进程的改变。最后，纯化的重组组蛋白，乙酰转移酶和 CRL4DDB2 将在体外进行测试，以描述它们在体内的特定生化作用。这些 DDR 的系统机制研究不仅可以提供与癌症相关的知识病因学，以及管理人类健康风险的新的合适工具、目标和策略。