Repair of Environmentally Induced Mitochondrial DNA Damage

项目总结/摘要 这一提议将代谢功能障碍与线粒体DNA修复后的调节联系起来， （mtDNA）的损伤。环境因素如电离辐射， 香烟烟雾、除草剂和重金属以及正常细胞代谢中发现的化学物质 这些过程在细胞中产生活性氧（ROS）。ROS会破坏细胞DNA， 适当修复，可以触发基因组不稳定性和代谢疾病的进展， 神经退行性疾病、衰老和癌症。线粒体DNA比它的核对应物更容易受到 氧化应激碱基切除修复（BER）途径修复细胞核和细胞核中受损的碱基， 线粒体区室称为DNA糖基化酶的特殊酶在初始化 BER通过切除受损的基地和调解其他方面的修复过程中，通过必要的 蛋白质：蛋白质相互作用。 我们将确定两种DNA糖基化酶NEIL 1和NEIL 2在修复中的作用和调节。 线粒体DNA我们假设NEIL酶与线粒体形成独特的复合物， 负责线粒体DNA复制和转录的蛋白质，包括线粒体单链 DNA结合蛋白（mtSSB）、转录因子A（TFAM）、聚合酶γ（Polγ）和闪烁解旋酶。 我们的中心假设是NEIL酶和线粒体蛋白之间的复合物形成驱动了 在复制/转录叉之前进行修复，并通过（去）乙酰化进行调节。为了解决这个 假设，我们将研究NEIL酶和命名的 线粒体蛋白质通过结构驱动的分析。实验使用蛋白质绘画，小角度X射线 散射和X射线晶体学将被用来确定之间形成的复合物的结构 NEIL 1、mtSSB、Polγ和Twinkle之间以及NEIL 2、TFAM和Polγ之间的差异。接下来，我们将测试 （de）乙酰化对NEIL功能的影响。乙酰辅酶A的高水平的驱动化学 我们的初步数据表明NEIL 2是以这种方式修饰的。脱乙酰 NEIL蛋白的NAD+依赖性沉默调节蛋白质的功能，并将探讨 这里.细胞代谢，线粒体功能障碍，以及环境毒物，导致增加 ROS水平对脱乙酰化所需的关键代谢物（NAD+）的生物利用度产生不利影响; 我们研究的一个方面。最后，我们将研究NEIL酶的定位条件下， 环境诱导的氧化应激及其对线粒体呼吸，膜电位， 和形态学。这将阐明氧化应激导致的重要的核-线粒体串扰， 应力 线粒体DNA修复是一个新兴领域，NEIL酶被置于修复过程的最前沿， 我们最近的发现通过解决NEIL复合物的形成，活性调节， 定位，拟议的研究将提供必要的分子洞察力，了解如何修复 由环境毒物引起的线粒体DNA损伤可以防止诱变， 线粒体代谢功能和线粒体DNA修复之间的联系。

Project Summary/ Abstract This proposal connects metabolic dysfunction to the regulation of repair following mitochondrial DNA (mtDNA) damage caused by environmental toxicants. Environmental agents such as ionizing radiation, chemicals found in cigarette smoke, herbicides, and heavy metals as well as normal cellular metabolic processes, 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 metabolic diseases including neurodegenerative disorders, aging, and cancer. MtDNA is more susceptible than its nuclear counterpart to oxidative stress. The base excision repair (BER) pathway mends damaged bases in both nuclear and mitochondrial compartments. Specialized enzymes called DNA glycosylases play a critical role in initializing BER by excising damaged bases and mediating other aspects of the repair process via essential protein:protein interactions. We will determine the role and regulation of two DNA glycosylases, NEIL1 and NEIL2, in the repair of mtDNA. We hypothesize that the NEIL enzymes form unique and distinct complexes with mitochondrial proteins that are responsible for mtDNA replication and transcription including mitochondrial single-stranded DNA binding protein (mtSSB), transcription factor A (TFAM), polymerase γ (Polγ), and the twinkle helicase. Our central hypothesis is that complex formation between the NEIL enzymes and mitochondrial proteins drives repair ahead of the replication/ transcription forks and is regulated via (de)acetylation. To address this hypothesis, we will examine the functional interactions between the NEIL enzymes and the named mitochondrial proteins via structure-driven analyses. Experiments using protein painting, small angle-X-ray scattering, and X-ray crystallography will be used to determine the structures of complexes formed between NEIL1, mtSSB, Polγ, and Twinkle as well as between NEIL2, TFAM, and Polγ. Next, we will test the impact of (de)acetylation on NEIL function. High levels of acetyl-coenzyme A in the mitochondrion drives chemical acetylation of proteins and our preliminary data suggests that NEIL2 is modified in this manner. Deacetylation of the NEIL proteins by the NAD+-dependent sirtuin enzymes regulates protein function and will be explored here. Cellular metabolism, mitochondrial dysfunction, and environmental toxicants that cause an increase in ROS levels adversely impact the bioavailability of key metabolites (NAD+) required for deacetylation; a pivotal aspect of our research. Lastly, we will study the localization of the NEIL enzymes under conditions of environmentally induced oxidative stress and their impact on mitochondrial respiration, membrane potential, and morphology. This will shed light on essential nuclear-mitochondrial crosstalk that results from oxidative stress. MtDNA repair is a budding field, with the NEIL enzymes placed at the forefront of the repair process by our recent discoveries. By addressing critical questions of NEIL complex formation, regulation of activity, and localization, the proposed studies will provide the molecular insight necessary for understanding how the repair of mtDNA damage caused by environmental toxicants prevents mutagenesis and offers a novel connection between mitochondrial metabolic function and mtDNA repair.