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，但线粒体响应或适应损伤的机制尚不清楚。最近已经证明，脓毒症患者的存活率可以通过其线粒体生物反应的强度来预测。缺氧是引起线粒体生物合成的多种刺激之一，它使许多心肺、感染和肿瘤疾病复杂化。这种适应性反应的机制尚不清楚，但重要的是，许多已知增加线粒体生物合成的刺激物使用活性氧（ROS）作为第二信使。拟议的研究将在分子和功能水平上探索一种调节线粒体生物合成的新途径。传统观念认为，维持DNA的完整性是正常细胞功能所必需的。然而，有新的证据表明，至少对于核基因，控制DNA损伤和修复可能是必要的正常转录调控。例如，在肺血管细胞中，缺氧引起缺氧诱导基因的缺氧反应元件（HRE）内的ROS依赖性碱基修饰。由于病变局限于与转录活性核小体相关的HRE，并且由于通过在VEGF启动子的HRE中引入修饰的碱基来模拟缺氧的作用导致增强的DNA柔性、改变的转录复合物组装和更稳健的报告基因表达，有人提出，ROS-介导的DNA损伤和修复可用于改变关键DNA序列的拓扑结构，以使调节蛋白结合并促进转录。我们建议测试的假设，控制氧化DNA损伤和修复的D-环区域促进mtDNA的转录和复制。利用已建立的改变线粒体DNA修复效率的策略，我们将：（1）测试缺氧诱导的线粒体DNA D环区氧化损伤的操作协同调节缺氧条件下线粒体DNA复制和转录的假设，（2）确定是否需要转录因子结合缺氧引起的D环区氧化碱基修饰的形成和修复。如果控制DNA损伤和修复的概念是有效的线粒体DNA的转录和复制在缺氧，这将是一个重大的进步，了解线粒体基因表达是如何调节健康和疾病，包括一些疾病，其中缺氧和线粒体功能障碍已被定罪。它还将有助于更详细地了解氧化剂信号传导和线粒体适应途径之间的联系，从而指出在许多具有此类异常的疾病中纠正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许多病理非常重要。这些研究的完成将揭示活性氧调节mtDNA复制和转录的全新机制。这些研究对于理解正常ROS依赖性过程与多种疾病（包括癌症、心血管疾病、糖尿病和神经退行性疾病）的mtDNA不稳定性特征之间的分子联系具有重要意义。

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.