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

项目摘要： 线粒体是细胞内的细胞器，其具有多种对细胞生长至关重要的功能。 保持整体细胞健康。这种细胞器的独特之处之一是，它拥有 自己的DNA。线粒体DNA的损伤与广泛的生物学和疾病有关。 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，我们对线粒体修复的基本机制的了解 对DNA的损伤是有限的。目前所知的大部分都局限于单一类型的DNA损伤， 氧化损伤线粒体DNA修复领域缺乏进展主要是由于 缺乏鉴定和表征线粒体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 特别是线粒体基因组。这种方法是基于重新定位的一些 已知的和充分表征的DNA损伤剂特异性地针对线粒体DNA。中的每 这些重定向的化合物以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），使我们能够扩大我们对个体 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，我们将能够开发一个全面的线粒体DNA修复和阐明新的 线粒体核酸内不同病变类型的反应。在目标1中，我们将研究 已知的核DNA修复因子在响应许多不同类型的DNA中的潜在作用 线粒体DNA内的损伤。在目标2中，我们将扩大搜索范围，在全基因组范围内寻找 鉴定与维持无关的新的线粒体DNA损伤修复蛋白， 核DNA修复在目标3中，我们将描述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损伤社区与一个示范的效用，靶向的 DNA损伤剂作为线粒体生物学的功能探针， 功能筛选数据可用于激发线粒体DNA新的令人兴奋的方向 损害研究。这项研究将是第一个使用高通量筛选方法的研究。 再加上高度特异性的化学探针来研究线粒体生物学。我们认为这 这些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。

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.