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

描述（由申请人提供）：线粒体功能障碍继发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机制尚不清楚。最近有研究表明，脓毒症患者的生存可以通过其线粒体生物源反应的强度来预测。缺氧是引起线粒体生物发生的多种刺激之一，它使许多心肺、感染性和肿瘤疾病复杂化。这种适应性反应的机制尚不清楚，但重要的是，许多已知的刺激增加线粒体生物发生使用活性氧（ROS）作为第二信使。提出的研究将在分子和功能水平上探索调节线粒体生物发生的新途径。传统观念认为，维持DNA的完整性是细胞正常功能所必需的。然而，越来越多的证据表明，至少对核基因来说，受控制的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，缺氧会导致缺氧诱导基因的缺氧反应元件（HREs）中ROS依赖的碱基修饰。由于损伤仅限于与转录活性核小体相关的HREs，并且由于通过在VEGF启动子的HRE中引入修饰碱基来模拟缺氧的影响，导致DNA柔韧性增强，转录复合物组装改变和更强健的报告基因表达，因此有人提出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，从而使调节蛋白结合并促进转录。我们提出验证在d环区控制DNA氧化损伤和修复促进mtDNA转录和复制的假设。利用现有的改变mtDNA修复效率的策略，我们将：(1)验证对缺氧诱导的mtDNA d环区氧化损伤的操纵协调调节缺氧条件下mtDNA复制和转录的假设；(2)确定缺氧引起的d环区氧化碱基修饰的形成和修复是否需要转录因子结合。如果在缺氧条件下控制DNA损伤和修复控制mtDNA转录和复制的概念是有效的，它将代表着理解线粒体基因表达如何在健康和疾病中被调节的重大进步，包括一些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信号和控制线粒体适应的途径之间的联系，从而指出纠正线粒体生物能量缺陷的新策略。

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.