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A high throughput assay to identify novel chromatin factors that regulate DNA repair

鉴定调节 DNA 修复的新型染色质因子的高通量测定

基本信息

批准号：
9767137
负责人：
Raul Mostoslavsky
金额：
$ 21.06万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767137
关键词：
Amplifiers Antibodies Binding Biochemical Biological Assay Biological Process Cell division Cell physiology Cells Chromatin Chromatin Remodeling Factor DNA DNA Binding DNA Binding Domain DNA Damage DNA Double Strand Break DNA Repair DNA Repair Pathway DNA biosynthesis DNA lesion DNA-Binding Proteins Disease Eukaryotic Cell Exclusion Exposure to Gene Mutation Generations Genetic Genetic Materials Genetic Transcription Genome Grant Histone Acetylation Histones Image Immunologics Kinetics Lasers Lead Libraries Life Malignant Neoplasms Measures Methodology Microscope Monitor Nerve Degeneration Nucleosomes Organism Peptides Play Process Proteins Reader Recruitment Activity Reporter Resolution Role Secure Signal Pathway Site Structure Syndrome System Testing Transference Validation Variant Work base cDNA Library chromatin remodeling experience genetic information genotoxicity high throughput screening high throughput technology histone modification imaging approach new technology novel quantitative imaging recruit repaired response screening sensor

项目摘要

Damage to our DNA poses one of the most direct threats to both the ability of our cells to function normally, as well as our ability to faithfully transmit genetic information to our progeny. Our cells experience a significant amount of spontaneous DNA lesions. Life, as we know it, is possible thanks to specific DNA repair mechanisms that eukaryotic cells have evolved. Such mechanisms involved the highly coordinated action of sensors, amplifiers and effectors that work in concert with signaling pathways to allow efficient DNA repair, in what is known as the DNA Damage Response (DDR). Our DNA is densely packed in chromatin, a barrier that needs to be overcome for proper recognition and repair of DNA lesions. Indeed, chromatin dynamics has emerged as an important module in the DDR, however the specific chromatin factors that are directly involved in the repair process remain poorly known. In this context, advances in the field have been hampered by limited availability of high-throughput technologies. In this exploratory grant, we will attempt to tackle these limitations by developing both a library of chromatin factors and a microscope based-high throughput assay to follow kinetics and recruitment of chromatin factors to DNA breaks. Specifically, in Aim 1 we will develop a library (ChromORFeome) of chromatin factors and use a high-throughput laser breaks assay to identify novel chromatin factors recruited to sites of DNA breaks. Aim 2 will provide proof of principle validation of these novel identified chromatin factors. Results from this exploratory grant will establish advanced new technologies to study the process of DNA repair and identify novel chromatin factors that play key roles in this process.

对我们DNA的损伤是对我们细胞功能的最直接威胁之一，正常情况下，以及我们忠实地将遗传信息传递给后代的能力。我们的细胞会出现大量的自发性DNA损伤生命，正如我们所知，是可能的，真核细胞进化出的特定DNA修复机制。这些机制涉及与信号协同工作的传感器、放大器和效应器的高度协调的动作这是一种允许有效DNA修复的途径，即所谓的DNA损伤反应（DDR）。我们 DNA密集地堆积在染色质中，这是一个需要克服的障碍，以进行正确的识别，修复DNA损伤。事实上，染色质动力学已经成为一个重要的模块，然而，直接参与修复过程的特定染色质因子仍然存在知之甚少。在这方面，由于可获得的资源有限，高通量技术。在这个探索性的补助金，我们将试图解决这些限制，开发染色质因子文库和基于显微镜的高通量测定，遵循染色质因子对DNA断裂的动力学和募集。具体而言，在目标1中，我们将开发染色质因子库（ChromORFeome），并使用高通量激光断裂检测以鉴定募集到DNA断裂位点的新型染色质因子。Aim 2将提供以下证据：这些新鉴定的染色质因子的原理验证。这项探索性赠款的结果将建立先进的新技术，研究DNA修复过程，在这个过程中起关键作用的染色质因子。