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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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要：线粒体是细胞内的细胞器，具有维持生命活动所必需的多种功能维持整体细胞健康。该细胞器的独特特征之一是它具有自己的DNA。线粒体 DNA 损伤与多种生物和疾病有关过程包括衰老、神经功能障碍和癌症进展。尽管批评线粒体 DNA 的性质，我们对线粒体修复基本机制的了解他们的 DNA 受到的损害是有限的。大部分已知的知识仅限于单一类型的 DNA 损伤，氧化损伤。线粒体 DNA 修复领域缺乏进展主要是由于缺乏识别和表征线粒体 DNA 修复蛋白的工具和技术。最近，我们的实验室开发了一套化学探针，可以诱导一系列DNA损伤特别是在线粒体基因组中。该方法基于多个目标的重定向已知且已充分表征的 DNA 损伤剂，专门针对线粒体 DNA。每一个这些重新定位的化合物以独特的方式影响线粒体 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我们将能够开发 mtDNA 修复的全面概况并阐明新的工具箱对线粒体核酸内不同损伤类型的反应。在目标 1 中，我们将调查已知核 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在基因组范围内寻找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 损伤群落，展示了线粒体靶向的效用 DNA 损伤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 的新的、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与高度特异性的化学探针相结合来研究线粒体生物学。我们相信，这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以及调节细胞死亡至关重要。