Repair of Environmentally and Endogenously Induced Mitochondrial-DNA Damage

环境和内源性诱导的线粒体 DNA 损伤的修复

• 批准号: 8762816
• 负责人: Aishwarya Prakash
• 金额: $ 8.57万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8762816
• 关键词: Address; Affect; Aging; Air Pollution; Base Excision Repairs; Binding; Binding Proteins; Biology; Cell Nucleus; Cell Respiration; Cell Survival; Cells; Cellular biology; Chemicals; Clinical; Co-Immunoprecipitations; Complex; DNA; DNA Damage; DNA Repair; DNA glycosylase; DNA lesion; DNA-Directed DNA Polymerase; DNA-Protein Interaction; Disease; Disease Progression; Drug Design; Embryo; Environment; Environmental Risk Factor; Enzyme Interaction; Enzymes; Excision; Family; Fibroblasts; Free Radicals; Generations; Genes; Genomic Instability; Genus Hippocampus; Goals; Health; Human; Hydrogen Peroxide; Individual; Ionizing radiation; Laboratories; Learning; Left; Lesion; Maintenance; Malignant Neoplasms; Measures; Mediating; Membrane; Mentors; Metabolism; Methods; Mission; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Molecular; Molecular Biology; Mus; Mutation; National Institute of Environmental Health Sciences; Nerve Degeneration; Neurodegenerative Disorders; Nuclear; Nucleosomes; Oxidative Phosphorylation; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Phase; Play; Polymerase; Process; Production; Proteins; Reactive Oxygen Species; Research; Research Support; Roentgen Rays; Role; Single Nucleotide Polymorphism; Site; Stress; Structure; Techniques; Technology; Testing; Two-Hybrid System Techniques; Ultraviolet Rays; Universities; Variant; Vermont; Visit; Work; X-Ray Crystallography; Yeasts; base; cell growth; cigarette smoking; design; endonuclease VIII; environmental agent; flexibility; light scattering; member; mitochondrial genome; oxidative DNA damage; oxidative damage; preference; prevent; protein complex; protein protein interaction; public health relevance; repaired; research study; structural biology; three dimensional structure; time use; tissue culture; tool; tumor; yeast two hybrid system

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.

描述（由申请人提供） 环境因素，例如紫外线、电离辐射、空气污染、化疗 香烟烟雾中的药物和化学物质与正常细胞代谢过程中产生的内部因素相结合，在细胞中产生活性氧（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。该提案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，由于线粒体 DNA 靠近 ROS 生成位点，因此比其核对应物更容易受到氧化应激的影响。 DNA 糖基化酶通过切除受损碱基并通过必需的蛋白质：蛋白质相互作用介导修复过程的其他方面，在初始化 BER 中发挥着关键作用。 该提案的指导 K99 阶段旨在描述两种 Nei 样 (NEIL) DNA 糖基化酶 NEIL 1 和 NEIL 2 在线粒体 DNA 修复中的作用。目标 1 将侧重于利用小鼠胚胎成纤维细胞了解在 NEIL 酶不表达的情况下对 mtDNA 损伤的影响。涉及使用 Seahorse 技术和 qPCR 技术实时测定线粒体功能以测量 DNA 损伤程度的实验将在 Van Houten 实验室（匹兹堡大学）进行。第二个目标也是在指导阶段启动的，将重点研究关键蛋白质：参与线粒体基因组维护的 NEIL 酶和线粒体蛋白质之间的蛋白质相互作用。这项工作将结合免疫共沉淀、酵母二杂交分析和纯化蛋白质来验证所获得的结果。 R00 独立阶段将包括结构功能研究，以研究 NEIL 酶介导的蛋白质：蛋白质相互作用的分子基础。将采用多学科方法，使用小角 X 射线散射来表征 NEIL 酶和线粒体蛋白之间形成的复合物，并使用 X 射线晶体学来确定复合物的晶体结构。突变分析将用于测试这些相互作用的功能相关性。拟议的研究通过研究环境和其他因素对 mtDNA 损伤相关疾病进展的影响来支持 NIEHS 的使命。该研究的长期目标是设计阻碍关键蛋白质与蛋白质相互作用的方法，从而抑制线粒体 BER 并防止异常细胞生长。