Functional genetic screening to elucidate novel mitochondrial DNA repair factors using organelle-targeted chemical probes

使用细胞器靶向化学探针进行功能性遗传筛查以阐明新型线粒体 DNA 修复因子

• 批准号: 9174919
• 负责人: Shana O Kelley
• 金额: $ 18.36万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9174919
• 关键词: Affect; Aging; Aging-Related Process; Alkylation; Biochemical; Biochemical Pathway; Biological; Biological Phenomena; Biology; CRISPR/Cas technology; Cell Death; Cell Fractionation; Cell model; Cell physiology; Cells; Cessation of life; Chemical Agents; Chemicals; Communities; Complement; Complex; Coupled; Cultured Cells; DNA; DNA Alkylation; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA lesion; Data; Disease; Eukaryotic Cell; Family; Fluorescence Microscopy; Genes; Genetic Materials; Genetic Screening; Genome; Genomic DNA; Health; Human; Individual; Knock-out; Knowledge; Lesion; Life; Link; Maintenance; Mammalian Cell; Mitochondria; Mitochondrial DNA; Molecular; Nature; Neoplasm Metastasis; Neurodegenerative Disorders; Neurologic Dysfunctions; Nuclear; Nucleic Acids; Organelles; Oxidative Phosphorylation; Pathway interactions; Peptides; Process; Production; Proteins; Proteome; Protocols documentation; RNA Interference; Research; Resources; Role; Series; Set protein; Small Interfering RNA; Source; System; Techniques; Time; Translations; Work; base; drug development; genome-wide; genome-wide analysis; high throughput screening; interest; knock-down; knockout gene; malignant neurologic neoplasms; mammalian genome; mitochondrial genome; new therapeutic target; novel; protein function; repaired; response; screening; small molecule; tool; tumor progression

Project Summary: Mitochondria are organelles within the cell that have a variety of functions essential to maintaining overall cellular health. One of the unique features of this organelle is that it possesses its own DNA. Damage to mitochondrial DNA is linked to a broad range of biological and disease processes including aging, neurological dysfunction and cancer progression. Despite the critical nature of mitochondrial DNA, our knowledge of the basic mechanisms by which mitochondria repair damage to their DNA is limited. Much of what is known is restricted to a single type of DNA damage, oxidative lesions. This lack of progress in the mitochondrial DNA repair field results primarily from a lack of tools and techniques for identifying and characterizing mitochondrial DNA repair proteins. Recently, our lab developed a set of chemical probes that can induce a range of DNA lesions specifically in the mitochondrial genome. This approach is based on the retargeting of a number of known, and well-characterized, DNA-damaging agents specifically to mitochondrial DNA. Each of these retargeted compounds affect the mitochondrial DNA in a distinct manner (oxidative lesions, alkylation lesions, and double stranded breaks) allowing us to expand our understanding of individual cellular responses to a single type of DNA lesion. We hypothesize that by using this molecular toolbox we will be able to develop a comprehensive profile of mtDNA repair and elucidate novel responses for distinct lesion types within mitochondrial nucleic acids. In Aim 1 we will investigate the potential role of known nuclear DNA repair factors in response to a number of distinct types of DNA lesions within mitochondrial DNA. In Aim 2 we will broaden the search and look genome-wide to identify novel mitochondrial DNA damage repair proteins that are not linked to maintenance and repair of nuclear DNA. In Aim 3, we will characterize the biochemical roles of these proteins within the context of mitochondrial DNA repair. At the conclusion of these studies, we will have elucidated novel aspects of mitochondrial DNA damage repair and response. This work will provide the mitochondrial DNA damage community with a demonstration of the utility of mitochondria-targeted DNA damaging agents as functional probes of mitochondrial biology and generate a rich resource of functional screening data that can be used to spark new and exciting directions in mitochondrial DNA damage research. The proposed study will be the first to use high-throughput screening approaches coupled with highly specific chemical probes to study mitochondrial biology. We believe that this combination of approaches will provide new, important information about the function of an organelle that is critical for the supporting cellular life as well as regulating cellular death.

项目总结： 线粒体是细胞内的细胞器，具有多种必需的功能 维持整体细胞健康。这种细胞器的一个独特特征是它拥有自己的 拥有自己的DNA。线粒体DNA的损伤与广泛的生物学和疾病有关 这些过程包括衰老、神经功能障碍和癌症进展。尽管关键的是 线粒体DNA的性质，我们对线粒体修复基本机制的了解 对它们DNA的损害是有限的。已知的很多东西仅限于一种类型的DNA损伤， 氧化损伤。线粒体DNA修复领域缺乏进展的主要原因是 缺乏识别和表征线粒体DNA修复蛋白的工具和技术。 最近，我们实验室开发了一套化学探针，可以引发一系列DNA损伤 特别是在线粒体基因组中。该方法基于对多个 已知的、特征明确的DNA损伤剂，专门针对线粒体DNA。每一个 这些重定向化合物以一种不同的方式影响线粒体DNA(氧化损伤， 烷基化损伤和双链断裂)使我们能够扩大对个体的理解 细胞对单一类型DNA损伤的反应。我们假设通过使用这种分子 工具箱我们将能够开发线粒体DNA修复的全面概况并阐明新的 对线粒体核酸中不同损伤类型的反应。在目标1中，我们将调查 已知的核DNA修复因子对多种不同类型DNA的潜在作用 线粒体DNA内的损伤。在目标2中，我们将扩大搜索范围，并着眼于全基因组 鉴定新的线粒体DNA损伤修复蛋白，这些蛋白与维持和修复无关 核DNA的修复。在目标3中，我们将描述这些蛋白质在体内的生化作用。 线粒体DNA修复的背景。在这些研究的结论中，我们将阐明 线粒体DNA损伤修复和反应的新方面。这项工作将提供 线粒体DNA损伤群落与线粒体靶向功能的论证 DNA损伤剂作为线粒体生物学的功能探针并产生丰富的DNA资源 可用于激发线粒体DNA新的和令人兴奋的方向的功能筛选数据 损害研究。这项拟议的研究将是第一次使用高通量筛查方法 再加上高度特异的化学探针来研究线粒体生物学。我们相信这一点 各种方法的结合将提供有关细胞器功能的新的重要信息 这对于支持细胞生命和调节细胞死亡至关重要。