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: 9521821
• 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/9521821
• 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 screen; genome-wide; 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.

项目摘要： 线粒体是细胞内的细胞器，具有多种功能 保持整体细胞健康。该细胞器的独特特征之一是它具有 自己的DNA。线粒体DNA的损害与广泛的生物学和疾病有关 包括衰老，神经功能障碍和癌症进展的过程。尽管很关键 线粒体DNA的性质，我们对线粒体修复的基本机制的了解 损害其DNA是有限的。已知的大部分内容仅限于单一类型的DNA损伤， 氧化病变。线粒体DNA修复场缺乏进展，主要是由于 缺乏用于识别和表征线粒体DNA修复蛋白的工具和技术。 最近，我们的实验室开发了一组可以诱导一系列DNA病变的化学探针 特别是在线粒体基因组中。这种方法基于许多重新定位 已知且特征良好的DNA损害剂专门针对线粒体DNA。每个 这些重新定位的化合物以不同的方式影响线粒体DNA（氧化病变， 烷基化病变和双链休息）使我们能够扩大对个人的理解 细胞对单一类型的DNA病变的反应。我们通过使用此分子来假设 工具箱我们将能够开发出mtDNA修复的全面概况并阐明新颖 线粒体核酸内不同病变类型的反应。在AIM 1中，我们将调查 已知的核DNA修复因子对多种不同类型的DNA的潜在作用 线粒体DNA中的病变。在AIM 2中，我们将拓宽搜索，并将范围全基因组看起来 识别与维护无关的新型线粒体DNA损伤修复蛋白 修复核DNA。在AIM 3中，我们将表征这些蛋白质的生化作用 线粒体DNA修复的背景。在这些研究的结束时，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损伤社区，展示了线粒体靶向的实用性 DNA破坏剂作为线粒体生物学的功能探针，并产生丰富的资源 功能筛选数据可用于激发线粒体DNA中的新和令人兴奋的方向 损害研究。拟议的研究将是第一个使用高通量筛选方法的研究 再加上高度特定的化学探针来研究线粒体生物学。我们相信这个 方法的组合将提供有关细胞器功能的新的重要信息 这对于支持细胞寿命以及调节细胞死亡至关重要。