Chemical and Molecular Mechanisms of Mitochondrial DNA Degradation

项目总结/摘要 线粒体是亚细胞区室，其对于能量产生、细胞信号传导和细胞增殖至关重要。 在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 它是线粒体功能不可或缺的，因为它编码电子传递链的蛋白质亚基， 一套完整的转移和核糖体RNA线粒体DNA降解是线粒体内的一种重要机制， 基因组维护此外，线粒体DNA降解是一个重要的质量控制措施，以科普 线粒体DNA损伤来源于内源性和环境化学物质。的机理 线粒体DNA降解和相关因素知之甚少，这代表了一个重大的知识差距。 这些知识对于理解线粒体基因组的维持和病理学是基础， 因为线粒体DNA降解可能导致线粒体DNA缺失综合征的病因学， 通过循环线粒体DNA激活先天免疫系统。本项目的目标是确定 受损mtDNA降解的化学和分子基础，并阐明一个主要转录因子的作用。 转录因子和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PI的长期目标，即揭开 线粒体DNA周转及其在线粒体病理生物学中的作用。这个项目的重点是一个无处不在的 DNA损伤和中央DNA修复中间体，即脱碱基（AP）位点。这个问题的核心假设是 TFAM调节AP损伤的稳定性并介导AP-DNA降解。这 这一假设是基于强有力的初步数据和经验证据。初步结果将是 通过使用定量生物化学、计算和细胞方法的组合进一步评估。 具体而言，本研究计划将描绘TFAM介导的AP-DNA的化学和动力学基础 不稳定，描述了TFAM参与人类细胞中的AP-DNA降解，并阐明了 TFAM在mtDNA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以及新的蛋白质因子方面的空白 参与了这个过程。这项申请建立在PI在DNA和蛋白质生物化学方面的强大背景之上， 机械酶学和定量分析，并加速了令人兴奋的，多产的进展， 线粒体生物学的研究领域。该项目的意义在于，它将首次定义 mtDNA降解途径的化学和分子基础以及TFAM在mtDNA中的作用 降解和修复。考虑到AP位点是线粒体DNA修复的关键中间体，我们对AP- DNA降解将对理解线粒体基因组的维持和 不稳定从这项研究中获得的新知识将深刻推动mtDNA维护领域的发展 并可能为线粒体疾病的新疗法的开发提供信息。

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.