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

基本信息

批准号：
9250131
负责人：
Aishwarya Prakash
金额：
$ 24.9万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9250131
关键词：
Address Affect Aging Air Pollution Base Excision Repairs Binding Binding Proteins Binding Sites Biology Cell Nucleus Cell Respiration Cell Survival Cells Cellular biology Chemicals Clinical Co-Immunoprecipitations Complex Crystallization 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 Mutation Analysis National Institute of Environmental Health Sciences Nerve Degeneration Neurodegenerative Disorders Nuclear Nucleosomes Oxidative Phosphorylation Oxidative Stress Oxides 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 experimental study flexibility interdisciplinary approach light scattering member mitochondrial genome oxidative DNA damage oxidative damage preference prevent protein complex protein protein interaction public health relevance repaired structural biology three dimensional structure time use tissue culture tool tumor yeast two hybrid system

项目摘要

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）环境药物，例如紫外线，电离辐射，空气污染，化学治疗在正常细胞代谢过程中产生的内部因素在香烟烟雾中发现的药物和化学物质在细胞中产生活性氧（ROS）。 ROS会损害细胞DNA，如果修复得不正确，可能会触发基因组不稳定性以及神经退行性疾病，衰老和癌症的进展。该建议旨在研究线粒体DNA（mtDNA）中的基本切除修复（BER）途径对ROS生成的DNA损伤的去除，这比其核对应物更容易受到氧化应激，因为它与ROS发电的位点接近。 DNA糖基酶在初始化BER中通过切除受损的碱并通过必需蛋白质相互作用介导修复过程的其他方面起着至关重要的作用。该提案的指导K99阶段旨在描述两个在MTDNA修复中的两个Nei样（Neil）DNA糖基酶，Neil 1和Neil 2的作用。 AIM 1将专注于在没有小鼠胚胎成纤维细胞表达尼尔酶的情况下，了解对mtDNA损伤的影响。使用Seahorse技术和QPCR技术在实时实时确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，它将集中于临界蛋白质的研究：尼尔酶与参与线粒体基因组维持的线粒体蛋白之间的蛋白质相互作用。这项工作将使用共免疫沉淀，酵母两种杂交分析和纯化蛋白质的组合来验证所获得的结果。 R00独立阶段将包括结构功能研究，以研究蛋白质的分子基础：由尼尔酶介导的蛋白质相互作用。使用小角度X射线散射的多学科方法，以表征尼尔酶和线粒体蛋白和X射线晶体学之间形成的络合物，以确定络合物的晶体结构。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药物对与mtDNA损伤相关的疾病进展的影响来支持NIEH的任务。拟议的研究的长期目标是设计阻碍关键蛋白质的方法：蛋白质相互作用，可以抑制线粒体BER并防止异常的细胞生长。