Nucleosome Dynamics and the Repair of DNA Damage

描述（由申请人提供）：修复DNA损伤对于保护基因组的完整性和防止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性和结构性染色质结构域，它们在组蛋白修饰模式、染色质结合蛋白和核小体包装密度方面存在差异。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然不清楚。我们的初步数据表明一种组蛋白变体，组蛋白H2A。在DNA双链断裂（DSBs）中迅速交换到核小体上。H2A的交换。dsb上的Z改变核小体动力学，驱动断裂处开放、灵活的染色质结构域的形成。更进一步，这个H2A。Z交换促进组蛋白修饰的特定模式，对控制断裂位点DNA的切除和加工至关重要。中心假设是H2A。Z交换驱动dsb染色质的重塑，并控制组蛋白修饰和DNA的末端加工。锌指核酸酶（ZFNs）文库将用于在活性转录基因和紧凑的基因间区域中创建序列特异性的dsb。我们将决定H2A如何。Z交换重塑了基因和基因间区域的染色质结构，并确定了这些不同染色质结构域之间修复机制的关键差异。我们将决定H2A如何。Z交换影响核小体在dsb的定位，并决定核小体在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上的关键域。Z改变核小体动力学，促进开放染色质结构在dsb的形成。另外，我们将如何确定H2A的存在。DSB上的z核小体影响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产生DSB，我们可以确定DSB在不同功能域的修复过程，并揭示H2A的重要性。Z在改变dsb局部染色质结构中的作用。此外，由于许多肿瘤细胞既改变了染色质组织，又增加了组蛋白H2A的水平。Z，这项工作将为染色质结构和H2A的关系提供新的见解。Z影响与癌变有关的过程，肿瘤进展和肿瘤对放疗和化疗的敏感性。

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.