Repair of Environmentally and Endogenously Induced Mitochondrial-DNA Damage

描述（由申请人提供） 环境因素，如紫外线、电离辐射、空气污染、化疗 药物和香烟烟雾中发现的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对细胞DNA造成损伤，如果不能正确修复，可能引发基因组不稳定和神经退行性疾病、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）途径去除ROS产生的DNA损伤，由于其靠近ROS产生的位点，线粒体DNA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的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，在启动BER中发挥关键作用。 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mtDNA修复中的作用。目的1将集中在理解的NEIL酶的表达的情况下，使用小鼠胚胎成纤维细胞线粒体DNA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货车Houten实验室（匹兹堡大学）进行。第二个目标，也开始在指导阶段，将集中在关键蛋白质的研究：蛋白质之间的相互作用NEIL酶和线粒体蛋白参与线粒体基因组的维护。这项工作将使用免疫共沉淀、酵母双杂交分析和纯化蛋白的组合进行，以验证所得结果。 R 00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。一个多学科的方法，使用小角X射线散射来表征NEIL酶和线粒体蛋白质和X射线晶体学之间形成的复合物，以确定复合物的晶体结构将进行。突变分析将用于检测这些相互作用的功能相关性。拟议的研究支持NIEHS的使命，通过研究环境和其他因素对与mtDNA损伤相关的疾病进展的影响。拟议研究的长期目标是设计阻碍关键蛋白质：蛋白质相互作用的方法，这可以抑制线粒体BER并防止异常细胞生长。

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.