High-resolution analyses of chromatin responses to DNA damage

DNA中的遗传信息以染色质的形式包装，受到多条DNA修复途径的保护，了解DNA修复的这一关键任务是如何执行的，这是本研究的长期目标。该项目将以前所未有的空间和时间分辨率检测单个活细胞中因DNA损伤而发生的染色质实时变化，从而确定DNA修复过程中染色质重组的意义。除了提高DNA损伤反应和修复机制的知识外，该项目还将开发和应用几项创新技术，这些技术可能会引起其他研究人员的极大兴趣。PI和共同PI致力于在研究界广泛传播这些技术。该项目的多学科性质将为研究生和博士后研究员以及本科生和高中生提供一个独特而宝贵的培训机会。他们有望学习与研究哺乳动物细胞DNA修复机制相关的基础和尖端技术，包括高分辨率成像。染色质对DNA损伤的反应是高度动态的，必须受到调控，以维持适当的结构组织，促进高保真的DNA修复并保持基因组的完整性。这些染色质结构动力学如何与DNA修复相协调还不是很清楚。PI将开发包括对相关光谱(PCF)和纳米成像跟踪技术在内的多重新型荧光相关方法，以分析DNA损伤如何影响染色质动力学，进而决定DNA修复途径的选择。这些现象将被研究以响应在不同细胞环境中诱导的相对简单的DNA断裂或复杂的DNA损伤。目标1包括监测损伤部位染色质通路的变化，询问与DNA修复途径选择的关系，并确定责任因素。目的2包括对特定DNA双链断裂位点的染色质动力学进行高分辨率多路分析，并研究DNA修复和转录之间的相互作用。一个总的目标也是开发新的方法，当DNA损伤时，产生单个哺乳动物细胞染色质动态的4D和纳米尺度的视图，以便定量地了解细胞机制如何评估损伤并决定如何修复其基因组。

The genetic information in DNA, which is packaged in the form of chromatin, is protected from damage by multiple DNA repair pathways, and understanding how this critical task of DNA repair is performed is the long-term goal of this research. The project will examine real-time changes in chromatin in response to DNA damage in individual living cells with unprecedented spatial and temporal resolution, and thereby determine the significance of chromatin restructuring during DNA repair. In addition to advancing knowledge of DNA damage response and repair mechanisms, the project will develop and apply several innovative technologies that will likely be of great interest to other researchers. The PI and co-PI are committed to disseminating these techniques broadly in the research community. The multidisciplinary nature of this project will provide a unique and valuable training opportunity for graduate students and postdoctoral fellows as well as undergraduate and high school students. They are expected to learn fundamental and cutting-edge techniques relevant to studying DNA repair mechanisms in mammalian cells, including high-resolution imaging. The chromatin response to DNA damage is highly dynamic and must be regulated to maintain the appropriate architectural organization that promotes high-fidelity DNA repair and preserves genomic integrity. How these chromatin structural dynamics are coordinated with DNA repair is not well understood. The PIs will develop multiplexing novel fluorescence correlation methods including pair correlation spectroscopy (pCF) and nanoimaging tracking techniques to analyze how DNA damage affects chromatin dynamics and how that in turn dictates DNA repair pathway choice. These phenomena will be investigated in response to relatively simple DNA breaks or complex DNA damage lesions induced in different cellular contexts. Aim 1 includes monitoring changes in chromatin access at the damage site, interrogating the relationship to DNA repair pathway choice, and determining responsible factors. Aim 2 involves high-resolution multiplexed analyses of chromatin dynamics at defined DNA double-strand break sites, and investigating the interplay between DNA repair and transcription. An overall goal is also to develop new methods that yield 4D and nanoscale views of chromatin dynamics in single mammalian cells when DNA is damaged, for a quantitative understanding of how the cellular machinery assesses the damage and decides how to repair its genome.