Repair of Environmentally and Endogenously Induced Mitochondrial-DNA Damage

描述(由申请人提供) 紫外线、电离辐射、空气污染、化疗等环境物质 在香烟烟雾中发现的药物和化学物质，与正常细胞新陈代谢过程中产生的内部因素相结合，在细胞中产生活性氧物种(ROS)。ROS会对细胞DNA造成损害，如果修复不当，可能会引发基因组不稳定以及神经退行性疾病、衰老和癌症的进展。这一建议旨在研究通过线粒体DNA(MtDNA)中的碱基切除修复(BER)途径消除ROS产生的DNA损伤，线粒体DNA比核DNA更容易受到氧化应激的影响，因为它靠近ROS产生的部位。DNA糖基酶通过切除受损的碱基，并通过基本蛋白质：蛋白质相互作用来调节修复过程的其他方面，在启动BER中起着关键作用。 该提案的指导K99阶段试图描述两个Nei样(Neil)DNA糖基酶Neil 1和Neil 2在mtDNA修复中的作用。目的1将重点了解在缺乏Neil酶表达的情况下，小鼠胚胎成纤维细胞对mtDNA损伤的影响。将在Van Houten实验室(匹兹堡大学)进行使用SeaHorse技术和qPCR技术实时测定线粒体功能的实验，以测量DNA损伤的程度。第二个目标也是在指导阶段启动的，将专注于关键蛋白质的研究：Neil酶和参与线粒体基因组维护的线粒体蛋白质之间的蛋白质相互作用。这项工作将使用免疫共沉淀、酵母双杂交分析和纯化蛋白相结合的方法来验证所获得的结果。 R00独立阶段将包括结构-功能研究，以探索蛋白质的分子基础：Neil酶介导的蛋白质相互作用。将采用一种多学科的方法，使用小角X射线散射来表征Neil酶与线粒体蛋白质之间形成的复合体，并使用X射线结晶学来确定复合体的晶体结构。突变分析将用于测试这些相互作用的功能相关性。这项拟议的研究通过研究环境和其他因素对线粒体DNA损伤相关疾病进展的影响，支持了NIEHS的使命。这项拟议研究的长期目标是设计方法来阻止关键蛋白质：蛋白质相互作用，这种相互作用可以抑制线粒体BER，防止细胞异常生长。

DESCRIPTION (provided by applicant) Environmental agents such as ultraviolet light, ionizing radiation, air pollution, chemotherapeutic drugs, and chemicals found in cigarette smoke, combined with internal factors produced during processes of normal cellular metabolism generate reactive oxygen species (ROS) in cells. ROS cause damage to cellular DNA, which if not properly repaired, can trigger genome instability and the progression of neurodegenerative disorders, aging, and cancer. This proposal seeks to study the removal of ROS-generated DNA damage via the base excision repair (BER) pathway in mitochondrial DNA (mtDNA), which is more susceptible than its nuclear counterpart to oxidative stress owing to its proximity to sites of ROS generation. DNA glycosylases play a critical role in initializing BER by excising damaged base and mediating other aspects of the repair process via essential protein:protein interactions. The mentored K99 phase of the proposal seeks to delineate a role for two Nei-like (NEIL) DNA glycosylases, NEIL 1 and NEIL 2, in the repair of mtDNA. Aim 1 will focus on understanding effects on mtDNA damage in the absence of expression of the NEIL enzymes using mouse embryo fibroblasts. Experiments involving the determination of mitochondrial function in real-time using Seahorse technology and qPCR techniques to measure the extent of DNA damage will be performed in the Van Houten laboratory (University of Pittsburgh). The second aim, also initiated during the mentored phase, will focus on the study of critical protein:protein interactios between the NEIL enzymes and mitochondrial proteins involved in mitochondrial genome maintenance. This work will be performed using a combination of co-immunoprecipitation, yeast two- hybrid analysis, and purified proteins to validate the results obtained. The R00 independent phase will consist of structure-function studies to investigate the molecular basis of protein:protein interactions mediated by the NEIL enzymes. A multi-disciplinary approach using small-angle X-ray scattering to characterize the complexes formed between the NEIL enzymes and mitochondrial proteins and X-ray crystallography to determine the crystal structures of the complexes will be undertaken. Mutational analysis will be used to test the functional relevance of these interactions. The proposed research supports the mission of the NIEHS by studying the effects of environmental and other agents on the progression of diseases associated with mtDNA damage. The long-term goal of the proposed research is to design ways to hinder key protein:protein interactions, which could inhibit mitochondrial BER and prevent aberrant cell growth.