Chemical and Molecular Mechanisms of Mitochondrial DNA Degradation

项目摘要/摘要 线粒体是亚细胞隔间，对能量产生、细胞信号传递和 高等真核细胞中蛋白质辅因子的生物合成。线粒体DNA(MtDNA)基因组是 是线粒体功能不可缺少的，因为它编码电子传递链的蛋白质亚单位和 一套完整的转移和核糖体RNA。线粒体DNA降解是线粒体的一种重要机制 基因组维护。此外，线粒体DNA降解是应对的一项重要质量控制措施 线粒体DNA损伤源于内源性和环境化学物质。它的作用机制 人们对线粒体DNA降解及其相关因素知之甚少，这是一个重大的知识鸿沟。 这些知识是理解线粒体基因组维持和病理学的基础， 因为线粒体DNA降解可能导致线粒体DNA耗竭综合征的病因，并导致 通过循环线粒体DNA激活先天免疫系统。本项目的目标是定义 线粒体DNA损伤降解的化学和分子基础以及阐明其在转录中的重要作用 因子和DNA包装蛋白TFAM(线粒体转录因子A)在DNA降解和修复中的作用。 解决这一关键的知识差距将有助于国际和平研究所的长期目标，即解开 线粒体DNA周转及其在线粒体病理生物学中的作用这个项目关注的是一个无处不在的 DNA损伤和中央DNA修复中间体，即基本(AP)位点。这一点的中心假设是 应用是TFAM调节AP病变的稳定性，并介导AP-DNA的降解。这 假设建立在强大的初步数据和经验证据的基础上。初步结果将是 通过使用定量生化、计算和细胞方法的组合来进一步评估。 具体地说，这项研究计划将描绘TFAM介导的AP-DNA的化学和动力学基础 不稳定，描述TFAM在人类细胞AP-DNA降解中的作用，并阐明 TFAM在线粒体DNA修复中的调节作用。预期的结果是，该项目将填补一个关键的知识 线粒体DNA降解的化学和分子机制及新的蛋白质因子研究进展 参与了这一过程。这一应用建立在PI在DNA和蛋白质生物化学方面的强大背景之上， 机械酶学，并进行定量分析，加速了令人振奋的、多产的进步 线粒体生物学的研究领域。这个项目的意义在于，它将第一次定义 线粒体DNA降解途径的化学和分子基础及TFAM在线粒体DNA中的作用 退化和修复。考虑到AP位点是线粒体DNA修复的关键中间体，我们对AP的见解- DNA降解将对理解线粒体基因组的维持和 不稳定。这项研究获得的新知识将深刻地推动线粒体DNA维护领域的发展 并有可能为开发治疗线粒体疾病的新疗法提供参考。

PROJECT SUMMARY/ABSTRACT Mitochondria are subcellular compartments that are critical for energy production, cell signaling, and the biosynthesis of protein cofactors in higher eukaryotic cells. The mitochondrial DNA (mtDNA) genome is indispensible for mitochondrial function because it encodes protein subunits of the electron transport chain and a full set of transfer and ribosomal RNAs. MtDNA degradation is an essential mechanism in mitochondrial genomic maintenance. In addition, mtDNA degradation is an important quality control measure to cope with mitochondrial DNA damage sourced from endogenous and environmental chemicals. The mechanism of mtDNA degradation and factors involved are poorly understood, which represents a significant knowledge gap. Such knowledge is fundamental to the understanding of mitochondrial genomic maintenance and pathology, because mtDNA degradation may contribute to the etiology of mtDNA depletion syndromes and to the activation of the innate immune system by circulating mtDNA. The objective of this project is to define the chemical and molecular basis of damaged mtDNA degradation and to clarify the role of a major transcription factor and DNA packaging protein TFAM (mitochondrial transcription factor A) in DNA degradation and repair. Addressing this critical knowledge gap will facilitate the PI's long-term goal of unraveling the basis of mitochondrial DNA turnover and its role in mitochondrial pathobiology. This project focuses on a ubiquitous DNA lesion and central DNA repair intermediate, i.e. abasic (AP) sites. The central hypothesis of this application is that TFAM modulates the stability of AP lesions and mediates AP-DNA degradation. This hypothesis is grounded in both strong preliminary data and empirical evidence. Preliminary results will be further evaluated by using a combination of quantitative biochemical, computational, and cellular approaches. Specifically, this research program will delineate the chemical and kinetic basis of TFAM-mediated AP-DNA destabilization, describe the involvement of TFAM in AP-DNA degradation in human cells, and clarify the regulatory role of TFAM in mtDNA repair. The expected outcome is that the project will fill a critical knowledge gap concerning the chemical and molecular mechanisms of mtDNA degradation and novel protein factors involved in the process. This application builds on the PI's strong background in DNA and protein biochemistry, mechanistic enzymology, and quantitative analysis, and accelerates the progress in an exciting, productive area of research into mitochondrial biology. The significance of this project is that it will, for the first time, define the chemical and molecular basis of an mtDNA-degradation pathway and the role of TFAM in mtDNA degradation and repair. Considering that AP sites are key intermediates in mtDNA repair, our insights into AP- DNA degradation will have broad implications for understanding mitochondrial genomic maintenance and instability. New knowledge gained from this research will profoundly advance the field of mtDNA maintenance and potentially inform the development novel therapeutics for mitochondrial diseases.