Role of oxidative modifications to D-loop region in mTDNA replication in hypoxia

描述(由申请人提供)：继发于线粒体功能障碍的生物能量缺陷发生在许多严重疾病中，但线粒体对损伤的反应或适应损伤的机制尚不清楚。最近已经证明，败血症患者的存活可以通过他们线粒体生物反应的强度来预测。缺氧是导致线粒体生物发生的多种刺激因素之一，它使许多心肺、感染性和肿瘤性疾病复杂化。这种适应性反应的机制尚不清楚，但重要的是，许多已知的增加线粒体生物发生的刺激使用活性氧物种(ROS)作为第二信使。这项拟议的研究将在分子和功能水平上探索一种调控线粒体生物发生的新途径。传统概念认为，维持DNA完整性是细胞正常功能所必需的。然而，有新的证据表明，至少对于核基因来说，受控的DNA损伤和修复可能是正常转录调控所必需的。例如，在肺血管细胞中，低氧导致低氧诱导基因的低氧反应元件(HRE)内的ROS依赖的碱基修饰。由于这些损伤仅限于与转录活性核小体相关的HRE，而且由于通过在VEGF启动子的HRE中引入修饰碱基来模拟缺氧的影响，导致DNA灵活性增强，转录复合体组装改变，报告基因表达更强劲，因此有人认为，ROS介导的DNA损伤和修复可能有助于改变关键DNA序列的拓扑结构，以实现调节蛋白结合和促进转录。我们建议检验这一假说，即控制D-loop区域的氧化DNA损伤和修复有助于mtDNA的转录和复制。使用已建立的改变mtDNA修复效率的策略，我们将：(1)检验操纵缺氧对mtDNA D-loop区域的氧化损伤协调调节mtDNA复制和转录的假设，以及(2)确定转录因子结合是否需要形成和修复缺氧引起的D-loop区域的氧化碱基修饰。如果控制DNA损伤和修复的概念在低氧条件下控制线粒体DNA转录和复制的概念是有效的，这将代表着在理解线粒体基因表达在健康和疾病中如何调节方面的重大进展，包括一些已被认为与缺氧和线粒体功能障碍有关的疾病。它还将有助于更详细地了解氧化剂信号和调控线粒体适应的途径之间的联系，从而指出纠正许多具有此类异常的疾病中的线粒体生物能量缺陷的新策略。 公共卫生相关性：了解线粒体基因组转录和复制的机制对于解释和治疗与线粒体功能障碍相关的一些病理非常重要。拟议研究的完成将揭示一种全新的机制，通过这种机制，活性氧物种可以调节线粒体DNA的复制和转录。这些研究对于理解正常的ROS依赖过程与多种疾病的线粒体DNA不稳定之间的分子联系具有重要意义，这些疾病包括癌症、心血管疾病、糖尿病和神经退行性疾病。

DESCRIPTION (provided by applicant): Bioenergetic defects secondary to mitochondrial dysfunction occur in many severe illnesses, but the mechanisms by which mitochondria respond to, or accommodate the damage are not well understood. Very recently it has been demonstrated that survival in septic patients can be predicted by the strength of their mitochondrial biogenic response. Hypoxia, which complicates many cardiopulmonary, infectious, and neoplastic disorders, is one of multiple stimuli causing mitochondrial biogenesis. The mechanism underlying this adaptive response is unknown, but it is important that many stimuli known to increase mitochondrial biogenesis use reactive oxygen species (ROS) as second messengers. The proposed research will explore, at molecular and functional levels, a novel pathway regulating mitochondrial biogenesis. Traditional concepts hold that maintenance of DNA integrity is required for proper cell function. However, there is emerging evidence that, at least for nuclear genes, controlled DNA damage and repair may be necessary for normal transcriptional regulation. In lung vascular cells, for example, hypoxia causes ROS- dependent base modifications within hypoxic response elements (HREs) of hypoxia inducible genes. Because the lesions are restricted to HREs associated with transcriptionally-active nucleosomes and since mimicking the effect of hypoxia by introducing modified bases in the HRE of the VEGF promoter leads to enhanced DNA flexibility, altered transcription complex assembly and more robust reporter gene expression, it has been proposed that ROS-mediated DNA damage and repair may serve to alter the topology of key DNA sequences to enable regulatory protein binding and facilitate transcription. We propose to test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region facilitates mtDNA transcription and replication. Using established strategies to alter the mtDNA repair efficiency, we will: (1) test the hypothesis that manipulation of hypoxia-induced oxidative damage to the mtDNA D-loop region coordinately regulates mtDNA replication and transcription in hypoxia, and, (2) determine whether formation and repair of hypoxia-caused oxidative base modifications in the D-loop region are required for transcription factor binding. If the concept that controlled DNA damage and repair govern mtDNA transcription and replication in hypoxia is valid, it will represent a significant advance in understanding how mitochondrial gene expression is regulated in health and diseases, including a number of disorders in which hypoxia and mitochondrial dysfunction have been incriminated. It will also contribute to a more detailed appreciation of the link between oxidant signaling and pathways governing mitochondrial adaptation, and thus point to new strategies for correcting mitochondrial bioenergetic defects in many disorders with such abnormalities. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms of mitochondrial genome transcription and replication is very important for the explanation and treatment of a number of pathologies associated with mitochondrial dysfunction. Completion of the proposed studies will reveal a fundamentally new mechanism by which reactive oxygen species regulate replication and transcription of mtDNA. These studies are significant with respect to understanding a molecular link between a normal ROS-dependent process and the mtDNA instability characteristic of a variety of diseases including cancer, cardiovascular disease, diabetes and neurodegenerative diseases.