G-quadruplex DNA as a chemical signaling agent

 描述(申请人提供)：四链DNA，或G4DNA，由四个鸟嘌呤组成，它们在一个平面环上形成Hoogsteen氢键，称为G四重体。这些G四联体(或四分体)的多个堆叠结合形成高度稳定的结构。最近，几种方法提供了令人信服的证据，证实细胞中的G4DNA影响DNA代谢，包括转录和复制。最近的工作提供了G4DNA和G4RNA序列的形成与一些神经退行性疾病(如ALS)之间的直接联系。目前正在加紧开发与G4DNA序列结合的试剂，用于治疗各种癌症。G4DNA序列在整个基因组中都存在，但优先定位于某些区域，如原癌基因启动子、端粒和线粒体DNA。尽管人们对G4DNA进行了密集的研究，但这些结构赋予生物功能的机制(S)在很大程度上还不清楚。我们已经应用蛋白质组筛选来发现与G4DNA结合的新蛋白质。已知包括DHX36在内的主要蛋白质在细胞应激条件下组装成称为应激颗粒的细胞质结构。这些蛋白质的位置及其在翻译调控中的已知作用使我们对G4DNA的功能提出了一个新的假说。我们认为，从受损的线粒体和核基因组中取出的内源性G4DNA可以完整地进入细胞质。这种被摘除的G4DNA结合到参与翻译的蛋白质上，通过形成应激颗粒来启动“应激反应”。我们的假设提供了一种新的机制，通过这种机制，细胞可以在氧化应激过程中对DNA损伤的影响做出反应。因此，切除的G4DNA可以作为一种化学信号媒介，提醒细胞注意DNA损伤。为了验证这一假设，我们将确定G4DNA在细胞质中的数量和特性作为氧化细胞应激的函数(目标1)。在目标2中，我们将确定G4DNA是否调节DHX36的翻译调节活性，以及G4DNA和DHX36是否与应激颗粒蛋白共定位。目的3将重点研究不同类型的G4DNA对DHX36酶活性的影响，以测试G4DNA影响蛋白质的可能机制。不同的G4DNA结构将在报告翻译的生物筛查中进行评估。这项工作将直接影响我们对以G4DNA为靶点的小分子机制的理解，这些小分子已被提议用于癌症治疗。这项研究将影响与癌症病因学相关的多个研究领域，包括DNA损伤反应、线粒体和端粒生物学、先天免疫、翻译调控和细胞信号转导。

 DESCRIPTION (provided by applicant): Quadruplex DNA, or G4DNA, is made of four guanines that form Hoogsteen hydrogen bonds in a planar ring which is referred to as a G quartet. Multiple stacks of these G quartets (or tetrads) associate to form highly stable structures. Recently, several methodologies have provided convincing evidence confirming that G4DNA in cells affects DNA metabolism including transcription and replication. Recent work has provided a direct link between formation of G4DNA and G4RNA sequences and some neurodegenerative diseases such as ALS. Intense efforts are underway to develop agents that bind to G4DNA sequences for treatment of various cancers. G4DNA sequences are found throughout the genome, but are localized preferentially to certain regions such as promoters of proto-oncogenes, telomeres, and mitochondrial DNA. Despite the intense research focused on G4DNA, the mechanism(s) through which these structures impart biological function are largely unknown. We have applied a proteomic screen to discover new proteins that bind to G4DNA. The major proteins identified, including DHX36, are known to assemble into cytoplasmic structures termed stress granules under conditions of cellular stress. The location of these proteins and their known roles in regulation of translation leads us to a new hypothesis for the function of G4DNA. We propose that endogenous G4DNA excised from damaged mitochondrial and nuclear genomes can enter the cytosol intact. This excised G4DNA binds to proteins involved in translation to initiate a "stress response" through formation of stress granules. Our hypothesis provides a new mechanism by which cells can respond to the effects of DNA damage during oxidative stress. Hence, excised G4DNA can serve as a chemical signaling agent, alerting the cell to DNA damage. To test this hypothesis, we will determine the quantity and identity of G4DNA in the cytoplasm as a function of oxidative cell stress (Aim 1). In Aim 2, we will determine if G4DNA modulates the translation regulatory activity of DHX36 and if G4DNA and DHX36 co-localize with stress granule proteins. Aim 3 will focus on the consequences of different types of G4DNA on the enzymatic activity of DHX36 to test possible mechanisms by which G4DNA affects the protein. Different G4DNA structures will be evaluated in a biological screen that reports on translation. This work will have a direct impact on our understanding of the mechanisms of small molecules that are targeted to G4DNA and have been proposed as therapies for cancer treatment. Multiple research areas of relevance to cancer etiology will be impacted by this research including DNA damage response, mitochondrial and telomere biology, innate immunity, translation regulation, and cell signaling.