Functions and Mechanisms of Helicases and G-Quadruplex Nucleic Acids

解旋酶和 G-四链体核酸的功能和机制

• 批准号: 9277158
• 负责人: Kevin Douglas Raney
• 金额: $ 29.37万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9277158
• 关键词: ATP Hydrolysis; Affect; Binding Proteins; Biochemical; Biological Process; CRISPR/Cas technology; Cells; Cellular Stress; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Structures; DNA; DNA Damage; DNA Repair; DNA Sequence; DNA Structure; Data; Enzymes; Eukaryota; Family; G-Quartets; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Goals; Guanine; Heart Diseases; Hydrogen Bonding; Innate Immune Response; Location; Malignant Neoplasms; Metabolism; Methods; Mitochondria; Mitochondrial DNA; Molecular Motors; Multiprotein Complexes; Mutation; Nuclear; Nucleic Acids; Phase; Play; Post-Translational Protein Processing; Proteins; Proteomics; Proto-Oncogenes; RNA; RNA Helicase; Reaction; Regulation; Research; Risk; Role; Signal Pathway; Signal Transduction; Site; Stress; Structure; Testing; Translations; helicase; histone modification; human disease; improved outcome; malignant breast neoplasm; nervous system disorder; nucleic acid metabolism; promoter; quadruplex DNA; recombinase; repaired; response; telomere

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.

项目摘要 解旋酶是分子运动蛋白，使用ATP水解中的能量来操纵DNA和RNA 核酸代谢的所有阶段。在许多不同的解旋酶中已经发现了许多突变 与包括癌症，心脏病和神经系统疾病在内的人类疾病有关的。这 解旋酶的主要功能是放松双链DNA，但已经发现了其他关键功能 哪些生化机制尚不清楚。解旋酶从DNA和次级展开蛋白质 DNA中的结构，例如G-四链体DNA（G4DNA），这对于维持基因组至关重要 稳定。 G4DNA由四个鸟嘌呤制成，在平面环中形成hoogsteen氢键 称为G四重奏。这些G四重奏的多个堆栈合并以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 在所有真核生物中都已经确定了解旋酶，并被确定为在识别和 G4DNA结构的展开。 PIF1中的突变会增加某些形式的乳腺癌的风险。这 该项目的总体目标是确定PIF1和其他解旋酶推蛋白的机制 从DNA和展开的临界DNA结构（例如G4DNA）。我们将确定解旋酶如何受到影响 通过它们相互作用的蛋白质，例如单链结合蛋白和重组酶。 G4DNA序列在整个基因组中都发现，但优先局部到某些区域 例如原始基因，端粒和线粒体DNA的启动子。通过的机制 这些结构赋予生物学功能，在很大程度上未知。我们设计了一种检查 通过使用CRISPR-CAS9靶向策略，实际上在基因组中的任何地点的表现组。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，重组和其他活动。我们已经将蛋白质组学屏幕应用于 发现与G4DNA结合的新蛋白质。确定的主要蛋白质，包括RNA解旋酶DHX36， 已知会在细胞应激条件下组装成被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节中的已知作用导致我们检验了一个假设 G4DNA的一个功能。我们的数据支持以下结论：G4DNA是从损坏的线粒体中切除的 和核基因组，可以进入细胞质完整，以促进应激颗粒的形成。我们的 现在的目标是确定从基因组中去除的G4DNA的特定序列，该机制是 切除G4DNA，以及切除的G4DNA影响翻译的特定功能。这 长期目标是了解G4DNA信号与其他信号通路的信号如何与其他信号通路相交 例如DNA损伤反应和先天免疫反应。