Nucleosome Dynamics and the Repair of DNA Damage

描述（由申请人提供）：DNA 损伤的修复对于保护基因组的完整性和预防可能导致癌症的基因毒性事件至关重要。然而，哺乳动物细胞含有多种功能和结构染色质结构域，其在组蛋白修饰模式、染色质结合蛋白和核小体堆积密度方面有所不同。 因此，损伤部位染色质结构的重塑对于 DNA 损伤的检测和修复至关重要。然而，驱动 DSB 核小体结构变化的潜在机制仍不清楚。我们的初步数据表明，组蛋白变体，组蛋白 H2A.Z，在 DNA 双链断裂 (DSB) 处快速交换到核小体上。 DSB 处 H2A.Z 的交换改变了核小体动力学，驱动断裂处开放、灵活的染色质结构域的形成。此外，这种 H2A.Z 交换促进了组蛋白修饰的特定模式，对于控制断裂位点 DNA 的切除和加工至关重要。中心假设是 H2A.Z 交换驱动 DSB 处染色质的重塑，并控制组蛋白修饰和 DNA 末端加工。锌指核酸酶 (ZFN) 文库将用于在活跃转录的基因和紧凑的基因间区域中创建序列特异性 DSB。我们将确定 H2A.Z 交换如何重塑基因和基因间区域中的染色质结构，并确定这些不同染色质结构域之间修复机制的关键差异。我们将确定 H2A.Z 交换如何影响核小体在 DSB 的定位，并确定核小体在 DSB 的定位如何影响组蛋白的后续修饰和末端切除的程度。此外，我们将确定 H2A.Z 上改变核小体动力学并促进 DSB 开放染色质结构形成的关键结构域。此外，我们将确定 DSB 上 H2A.Z 核小体的存在如何影响 DSB 修复的机制和保真度。通过使用锌指核酸酶在基因和基因间区域中创建 DSB，我们可以确定 DSB 修复如何在不同的功能域中进行，并揭示 H2A.Z 在改变 DSB 局部染色质结构中的重要性。此外，由于许多肿瘤细胞的染色质组织发生了改变，组蛋白 H2A.Z 水平也有所增加，这项工作将为染色质结构和 H2A.Z 如何影响与癌发生、肿瘤进展以及肿瘤对放疗和化疗敏感性相关的过程提供新的见解。

DESCRIPTION (provided by applicant): The repair of DNA damage is critical to protect the integrity of the genome and prevent genotoxic events which can lead to cancer. However, mammalian cells contain a diverse array of functional and structural chromatin domains, which differ in the pattern of histone modifications, chromatin binding proteins and in the density of nucleosome packing. Consequently, remodeling of the chromatin structure at sites of damage is critical for the detection and repair of DNA damage. However, the underlying mechanism driving changes in nucleosome structure at DSBs remains poorly defined. Our preliminary data demonstrates that a histone variant, histone H2A.Z, is rapidly exchanged onto nucleosomes at DNA double-strand breaks (DSBs). The exchange of H2A.Z at DSBs alters nucleosomes dynamics, driving the formation of open, flexible chromatin domains at the break. Further, this H2A.Z exchange promotes specific patterns of histone modification and is critical for controlling resection and processing of the DNA at the break site. The central hypothesis is that H2A.Z exchange drives remodeling of the chromatin at DSBs and controls both histone modification and end processing of the DNA. A library of Zinc Finger Nucleases (ZFNs) will be used to create sequence-specific DSBs in actively transcribed genes and compact, intergenic regions. We will determine how H2A.Z exchange remodels chromatin structure in genes and intergenic regions and identify key differences in the mechanism of repair between these distinct chromatin domains. We will determine how H2A.Z exchange impacts the positioning of nucleosomes at DSBs and determine how nucleosome positioning at DSBs impacts the subsequent modification of histones and the extent of end resection. Further, we will identify key domains on H2A.Z which alter nucleosome dynamics and promote the formation of open chromatin structures at DSBs. In addition, we will determine how the presence of H2A.Z-nucleosomes at DSBs impacts the mechanism and fidelity of DSB repair. By using Zinc Finger Nucleases to create DSBs in genes and intergenic regions, we can determine how DSB repair proceeds in distinct functional domains and unravel the importance of H2A.Z in altering the local chromatin architecture at DSBs. Further, because many tumor cells have both altered chromatin organization and increased levels of histone H2A.Z, this work will provide new insights into how chromatin structure and H2A.Z impacts processes related to carcinogenesis, tumor progression and the sensitivity of tumors to both radiation therapy and chemotherapy.