Repair of Environmentally Induced Mitochondrial DNA Damage

项目摘要/摘要 这一建议将代谢功能障碍与线粒体DNA修复的调节联系起来。 (线粒体DNA)由环境毒物造成的损伤。环境物质，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 过程中，在细胞中产生活性氧物种(ROS)。ROS会对细胞DNA造成损害，如果不是 如果修复得当，可能会引发基因组不稳定和代谢性疾病的进展，包括 神经退行性疾病、衰老和癌症。线粒体DNA比其核对应物更容易感染 氧化应激。碱基切除修复(BER)途径修复细胞核和 线粒体隔间。称为dna糖基酶的特殊酶在初始化过程中起着关键作用。 通过切除受损的碱基并通过Essential调解修复过程的其他方面 蛋白质：蛋白质相互作用。 我们将确定两种DNA糖基酶NEIL1和NEIL2在骨肉瘤修复中的作用和调节。 线粒体DNA。我们推测Neil酶与线粒体形成独特而独特的复合体。 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体形成驱动 在复制/转录分叉之前进行修复，并通过(去)乙酰化来调节。要解决这个问题 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分析。用蛋白质涂布、小角X射线进行实验 散射和X射线结晶学将被用来确定形成的络合物的结构 NEL1、MTSSB、POLγ和闪烁以及NEIL2、TFAM和POLγ之间。接下来，我们将测试一下 (去)乙酰化对Neil功能的影响。线粒体中高水平的乙酰辅酶A驱动化学物质 蛋白质的乙酰化，我们的初步数据表明NEIL2是以这种方式修饰的。脱乙酰化 NAD+依赖的sirtuin酶对Neil蛋白的调节作用 这里。细胞代谢、线粒体功能障碍和环境毒物导致的 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酶在以下条件下的定位 环境诱导的氧化应激及其对线粒体呼吸、膜电位、 和形态。这将阐明氧化导致的基本的核-线粒体串扰 压力。 线粒体DNA修复是一个萌芽领域，Neil酶被置于修复过程的前沿 我们最近的发现。通过解决Neil复合体的形成、活动的调节和 定位，拟议的研究将提供必要的分子洞察力，以了解修复如何 环境毒物引起的线粒体DNA损伤可以防止突变，并提供了一种新的联系 线粒体代谢功能与线粒体DNA修复之间的关系。

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.