DNA double-strand break chromatin alterations and genome integrity

DNA 双链断裂染色质改变和基因组完整性

• 批准号: 10799132
• 负责人: Roger A Greenberg
• 金额: $ 14.04万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799132
• 关键词: Address; Affect; CRISPR screen; Cancer Etiology; Cells; Chromatin; Coupled; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; Data; Elements; Event; Genes; Genetic; Genetic Recombination; Genome; Goals; Guide RNA; Human; Hybrids; Image; Length; Maintenance; Malignant Neoplasms; Microscope; Microscopy; Molecular; Motor; Names; Nuclear; Proliferating; Proteome; Publishing; Research; System; TERF1 gene; Telomere Maintenance; Time; Visualization; cell fixing; examination questions; experimental study; fluorescence microscope; genome integrity; insight; loss of function; nuclease; programs; repaired; replication stress; response; telomere; treatment response

Acquisition of a fluorescent microscope for genetic interrogation of DNA damage responses Project Summary/Abstract: Alternative lengthening of telomeres (ALT) is poorly understood homology directed repair mechanism that is responsible for telomere maintenance in ~15% of human cancers. We have developed systems that enable quantitative, real-time visualization of each step during ALT. Our published studies from this R01 reveal that ALT is initiated by DNA damage dependent clustering of telomeres into nuclear bodies that serve as hubs for long tract homology directed repair synthesis. We named this mechanism Break Induced Telomere Synthesis and showed that it is critical for telomere lengthening in cells that utilize ALT. To examine the question of how DNA damage responses assemble on telomeric chromatin, we coupled synchronous telomere DNA double-strand break induction with telomere purification to identify the telomere DNA damage response proteome. This revealed a hybrid damage response that consists of both homology directed DNA repair and replication stress factors. We hypothesize that ALT occurs by assembling elements of several different DNA repair mechanisms on telomeric chromatin to achieve long tract homology directed repair synthesis. Our preliminary data reveals that responses that either promote or inhibit ALT. To systematically address this response, it is essential for us to perform loss of function experiments that allow us to quantitatively assess the importance of each factor during ALT. The overall goal of this proposal is to perform arrayed CRISPR screens that target each factor in the telomere damage response. We will visualize the effect on ALT by quantifying EdU colocalization at telomeres in G2 as a marker of recombination dependent telomere maintenance. This is a known hallmark of ALT. We will use the Cas9 system that allows guide RNAs targeted to functional domains in each gene. The screen will be performed in unperturbed ALT cells and following TRF1-FokI induction, which is a telomere specific nuclease that generates DNA double-strand breaks at telomeres that promote ALT. Imaging will be performed in an arrayed format on a Nikon Ti2E motorized inverted microscope that is equipped with the capacity to image either live or fixed cells with a 40X objective in a 96 well format. These fundamental studies will allow us to define the critical elements of the telomere DNA damage response and how it allows telomere lengthening for sustained proliferation in cells that rely on ALT.

获取用于 DNA 损伤基因检测的荧光显微镜 回应 项目摘要/摘要： 端粒替代延长 (ALT) 对同源定向修复知之甚少 负责维持约 15% 人类癌症端粒的机制。我们有 开发了能够定量、实时可视化 ALT 期间每个步骤的系统。我们的 R01 已发表的研究表明 ALT 是由 DNA 损伤依赖性聚类启动的 将端粒形成核体，作为长链同源定向修复的中心 合成。我们将这种机制命名为“断裂诱导端粒合成”，并表明它是 对于利用 ALT 的细胞中端粒延长至关重要。 为了研究 DNA 损伤反应如何在端粒染色质上组装的问题，我们 将同步端粒 DNA 双链断裂诱导与端粒纯化相结合 鉴定端粒 DNA 损伤反应蛋白质组。这揭示了混合损伤反应 它由同源定向 DNA 修复和复制应激因子组成。我们假设 ALT 是通过在端粒上组装几种不同 DNA 修复机制的元件而发生的 染色质实现长链同源定向修复合成。我们的初步数据显示 促进或抑制 ALT 的反应。为了系统地解决这个问题， 对于我们进行功能丧失实验至关重要，这些实验使我们能够定量评估 ALT 期间每个因素的重要性。该提案的总体目标是执行阵列 针对端粒损伤反应中每个因素的 CRISPR 筛选。我们将可视化 通过量化 G2 端粒上的 EdU 共定位作为重组标记对 ALT 的影响 依赖端粒维持。这是 ALT 的一个众所周知的标志。我们将使用Cas9系统 允许引导 RNA 靶向每个基因的功能域。将执行画面 在未受干扰的 ALT 细胞中并在 TRF1-FokI 诱导后（一种端粒特异性核酸酶） 在端粒处产生 DNA 双链断裂，从而促进 ALT。影像学将 在配备了尼康 Ti2E 电动倒置显微镜上以阵列形式进行 能够使用 96 孔格式的 40X 物镜对活细胞或固定细胞进行成像。这些 基础研究将使我们能够确定端粒 DNA 损伤的关键因素 反应以及它如何使端粒延长以实现依赖于细胞的持续增殖 ALT。