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.

项目摘要/摘要 DNA破坏不同起源的药物不断挑战生物体的基因组和影响其正常功能。真核生物通过部署有效的DNA损伤响应（DDR）来抵消迅速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募集和及时清除多个交叉说话因素。在上一个项目期间，我们已经确定了ATM检查点激酶（主单元格循环检查点控制器）和CRL4DDB2泛素连接酶复合物（螺旋延伸DNA病变传感器），作为DNA修复依赖性染色质组装的两个关键调节剂。此的基本前提更新提案是DNA损伤激活了撞击（i）逮捕细胞以允许的平行事件获取DNA损伤和修复机械，以及（ii）恢复表观遗传完整的染色质和正常细胞循环的恢复。因此，研究旨在专注于（1）揭露 ATM定向的H3K56AC驱动的维修依赖性染色质组件的分子基础和（2）在组蛋白的集成事件中阐明CRL4DDB2泛素连接酶复合物的功能和机制乙酰化，泛素化和染色质沉积。结果将证明相关假设（a）ATM激酶通过新型的磷酸化信号传导促进依赖修复的染色质组装级联和（b）CRL4DDB2泛素连接酶指导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相互连接的特定目标：（1）确定ATM介导的DNA损伤信号的作用调节依赖修复的染色质组装，（2）以阐明CRL4DDB2 UB - 牵引酶在 H3K56乙酰化和（3）建立CRL4DDB2 UB-ligase介导的调节后的机理修复染色质恢复中的组蛋白沉积。正常或具体的人类细胞系种类缺乏组成型或siRNA/shRNA介导的基因沉默的单个蛋白质因子将是在细胞周期的精选阶段使用来分析对检查点蛋白质因子的影响，并揭示其通过FACS分析，芯片，共免疫沉淀和/或通过共定位的功能相互作用测定。将在特异性折衷的细胞中评估一系列组蛋白修饰，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，乙酰基转移酶和CRL4DDB2将在体外测试，以描绘其在体内的特定生化作用。这些DDR的系统机械研究不仅可以提供与癌症相关的知识病因，以及管理人类健康风险的新工具，目标和策略。