Functions and Mechanisms of Helicases and G-Quadruplex Nucleic Acids

项目摘要 解旋酶是分子马达蛋白，其使用来自ATP水解的能量来操纵DNA和RNA， 核酸代谢的所有阶段。在许多不同的解旋酶中已经鉴定出许多突变 与癌症、心脏病和神经系统疾病等人类疾病有关。的 解旋酶的主要功能是解旋双链体DNA，但已经发现解旋酶的其他关键功能。 其生化机制尚不清楚。解旋酶从DNA中置换蛋白质， DNA中的结构，如G-四链体DNA（G4DNA），在反应中对维持基因组 稳定G4DNA由四个鸟嘌呤组成，它们在平面环中形成Hoogsteen氢键， 称为G四重奏。这些G四重态的多个堆叠结合形成高度稳定的结构。 G4DNA影响DNA代谢，包括转录、重组和复制。Pif1家族 解旋酶已在所有真核生物中被鉴定，并已被鉴定为在识别和 G4DNA结构的解折叠。Pif1突变会增加某些形式乳腺癌的风险。的 这个项目的总体目标是确定Pif1和其他解旋酶推动蛋白质的机制 并展开关键的DNA结构，如G4DNA。我们将确定解旋酶如何受到影响 通过与它们相互作用的蛋白质如单链结合蛋白和重组酶。 G4DNA序列存在于整个基因组中，但优先定位于某些区域 如原癌基因、端粒和线粒体DNA的启动子。通过何种机制 这些赋予生物学功能的结构在很大程度上是未知的。我们设计了一种方法来检查 通过使用CRISPR-Cas9靶向策略，在基因组中的几乎任何位点上检测表观蛋白质组。我们将确定 围绕G4DNA位点的蛋白质和组蛋白修饰，以了解这些序列如何 影响基因表达、重组和其它活动。我们应用蛋白质组学筛选， 发现与G4DNA结合的新蛋白质。鉴定的主要蛋白质，包括RNA解旋酶DHX36， 已知在细胞应激条件下组装成称为应激颗粒的细胞质结构。 这些蛋白质的位置和它们在翻译调节中的已知作用使我们测试了一个假设， G4DNA的功能之一。我们的数据支持G4DNA从受损线粒体中切除的结论。 和核基因组，并可以完整地进入细胞质，在那里它促进应力颗粒的形成。我们 现在的目标是确定从基因组中去除的G4DNA的特定序列，其机制是 G4DNA被切除的位置，以及切除的G4DNA影响翻译的特定功能。的 长期目标是了解G4DNA信号如何与其他信号通路重叠和交叉 例如DNA损伤反应和先天免疫反应。

Project Summary Helicases are molecular motor proteins that use energy from hydrolysis of ATP to manipulate DNA and RNA in all phases of nucleic acid metabolism. Numerous mutations have been identified in many different helicases that are associated with human diseases including cancer, heart disease, and neurological disorders. The primary function of helicases is to unwind duplex DNA, but other critical functions have been discovered for which biochemical mechanisms are unknown. Helicases displace proteins from DNA and unfold secondary structures in DNA such as G-quadruplex DNA (G4DNA) in reactions that are critical for maintaining genomic stability. 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 associate to form highly stable structures. G4DNA affects DNA metabolism including transcription, recombination, and replication. The Pif1 family of helicases has been identified in all eukaryotes and has been identified as playing a key role in recognition and unfolding of G4DNA structures. Mutation in Pif1 can increase the risk for some forms of breast cancer. The overall goals of this project are to determine the mechanism(s) by which Pif1 and other helicases push proteins from DNA and unfold critical DNA structures such as G4DNA. We will determine how helicases are affected by proteins with which they interact such as single-stranded binding proteins and recombinases. G4DNA sequences are found throughout the genome, but are localized preferentially to certain regions such as promoters of proto-oncogenes, telomeres, and mitochondrial DNA. The mechanism(s) through which these structures impart biological function are largely unknown. We have devised a method to examine the epiproteome at practically any site in the genome by using a CRISPR-Cas9 targeting strategy. We will identify the proteins and histone modifications that surround G4DNA sites in order to understand how these sequences influence gene expression, recombination, and other activities. We have applied a proteomic screen to discover new proteins that bind to G4DNA. The major proteins identified, including the RNA helicase 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 led us to test a hypothesis for one function of G4DNA. Our data supports the conclusion that G4DNA is excised from damaged mitochondrial and nuclear genomes and can enter the cytoplasm intact where it facilitates formation of stress granules. Our goals now are to determine the specific sequences of G4DNA removed from the genome, the mechanism by which the G4DNA is excised, and the specific functions by which excised G4DNA affects translation. The long-term goal is to understand how signaling by G4DNA overlaps and intersects with other signaling pathways such as the DNA damage response and innate immune response.