ATP-Dependent Chromatin Remodeling and Genomic Instability in Mammalian Cells

摘要 INO80染色质重塑复合体在骨肉瘤的修复中起重要作用 低等真核生物辐射诱导的DNA双链断裂。这一发现 提供了第一个强有力的证据，表明在高度 紧密的染色质结构是DNA损伤反应的关键因素。它变成了 越来越清楚的是，维持基因组的稳定依赖于高度协调 DNA损伤修复、细胞周期检查点和染色质重塑的作用 机械装置。虽然前两个机制一直是广泛讨论的主题 在过去几十年的研究中，染色质修饰和 DNA损伤反应的重塑在很大程度上仍不清楚，特别是在哺乳动物中 系统。我们提出的研究旨在描绘染色质重塑 支持去除DNA损伤和启动损伤诱导的细胞周期的活动 检查点信号。我们的重点将放在INO80依赖于ATP的两个关键亚单位上 染色质重塑复合体、INO80和Arp5。INO80是SNF2的唯一成员 被认为是协助DNA的特殊染色质重构体的超家族 修理。Arp5是一种肌动蛋白相关的核蛋白，是INO80的一个完整亚基 很复杂。芽殖酵母中任一基因的突变使细胞对 广谱的遗传毒性物质。在本应用程序中，我们试图了解如何 INO80调节DNA损伤反应。我们已经成功地通过 INO80和ARP5的同源靶向、功能丧失的人类细胞模型。 这些遗传模型系统将成为研究INO80功能的独特工具 和Arp5在细胞增殖、电离辐射修复和紫外线诱导的DNA损伤中的作用， 损伤诱导的细胞周期停滞和细胞凋亡。我们的结果有望进一步 阐明DNA损伤反应系统和分子生物学机制 基因组总体上不稳定的基础。我们的结果也应该有助于识别 新的治疗靶点，特别是辐射增敏的靶点。叙事 获得DNA损伤是许多细胞机制的关键先决条件，这些机制作用于 保护人类基因组的完整性。在本申请中提出的研究试图 了解染色质重塑复合体在建立这种通路中的作用 分子和遗传学方法。拟议工作的结果有可能 揭示遗传不稳定性的新机制并寻找癌症的新靶点 心理治疗。

ABSTRACT The Ino80 chromatin remodeling complex plays an important role in the repair of radiation-induced DNA double strand breaks in lower eukaryotes. This discovery provides the first strong evidence that accessibility to DNA in the context of highly compact chromatin structure is a critical factor in DNA damage response. It becomes increasingly clear that maintenance of genome stability depends on highly coordinated actions of DNA damage repair, cell cycle checkpoint, and chromatin remodeling mechanisms. While the first two mechanisms have been the subject of extensive investigations during the past decades, the role of chromatin modification and remodeling in DNA damage response remains largely unclear, particularly in mammalian systems. Our proposed studies are aimed at delineating how chromatin remodeling activities support removal of DNA lesions and initiation of damage-induced cell cycle checkpoint signals. Our focus will be on two key subunits of the Ino80 ATP-dependent chromatin remodeling complex, Ino80 and Arp5. Ino80 is a unique member of the SNF2 superfamily that is believed to be a specialized chromatin remodeler assisting in DNA repair. Arp5 is an actin-related nuclear protein and an integral subunit of the Ino80 complex. Mutations of either gene in budding yeast render cells hypersensitive to a broad spectrum of genotoxic agents. In this application, we seek to understand how Ino80 modulates DNA damage responses. We have successfully created, via homologous targeting, loss-of-function human cellular models for INO80 and ARP5. These genetic model systems will serve as unique tools to study the function of Ino80 and Arp5 in cell proliferation, repair of ionizing radiation and UV-induced DNA lesions, damage-induced cell cycle arrest, and apoptosis. Our results are expected to further elucidate the mechanisms of the DNA damage response system and the molecular basis of genomic instability at large. Our results should also be useful for identification of novel therapeutic targets, especially targets for radiation sensitization. NARRATIVE Access to DNA lesions is a key prerequisite for many cellular mechanisms that act to protect the integrity of the human genome. Studies proposed in this application seek to understand the role of chromatin remodeling complex in creating such access by using molecular and genetics approaches. Results from the proposed work have the potential to unveil novel mechanism of genetic instability and to identify novel targets for cancer therapy.