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.

获得荧光显微镜用于DNA损伤的遗传询问 响应 项目概要/摘要： 端粒的替代性延长（ALT）是一种知之甚少的同源定向修复 在约15%的人类癌症中，端粒的维持机制。我们有 开发的系统，使定量，实时可视化的每个步骤在ALT。我们 来自该R 01的已发表的研究表明，ALT是由DNA损伤依赖性聚类引发的 端粒进入核体，作为长束同源定向修复的枢纽 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，我们 将同步端粒DNA双链断裂诱导与端粒纯化偶联， 鉴定端粒DNA损伤反应蛋白质组。这揭示了一种混合的损害反应 由同源性指导的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， 染色质以实现长束同源定向修复合成。我们的初步数据显示 能够促进或抑制ALT。为了系统地解决这一问题， 我们必须进行功能丧失实验，使我们能够定量评估 ALT期间每个因素的重要性。本提案的总体目标是执行阵列化 CRISPR筛选针对端粒损伤反应中的每个因素。我们将可视化 通过定量EdU在G2中端粒处共定位作为重组标记物对ALT的影响 依赖性端粒维持这是ALT的一个已知标志。我们将使用Cas9系统 允许引导RNA靶向每个基因中的功能域。屏幕将被执行 在未受干扰的ALT细胞中，在TRF 1-FokI诱导后，TRF 1-FokI是一种端粒特异性核酸酶 在端粒处产生DNA双链断裂，促进ALT。成像将是 以阵列形式在Nikon Ti 2 E电动倒置显微镜上进行， 具有在96孔格式中用40倍物镜对活细胞或固定细胞成像的能力。这些 基础研究将使我们能够确定端粒DNA损伤的关键因素， 以及它如何允许端粒延长，以在依赖于端粒的细胞中持续增殖。 备选.