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

基本信息

批准号：
9892786
负责人：
Kevin Douglas Raney
金额：
$ 12.96万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
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/9892786
关键词：
ATP Hydrolysis Affect Binding Proteins Biochemical Biological Process CRISPR/Cas technology Cells Cellular Stress Cytoplasm 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 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 stress granule telomere

项目摘要

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.

项目概要解旋酶是分子马达蛋白，利用 ATP 水解产生的能量来操纵 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。这解旋酶的主要功能是解旋双链 DNA，但还发现了其他关键功能其中生化机制尚不清楚。解旋酶从 DNA 中取代蛋白质并展开次级 DNA 结构，例如反应中的 G 四链体 DNA (G4DNA)，对于维持基因组至关重要稳定。 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 四重奏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得到鉴定，并被确定在识别和解旋酶中发挥着关键作用。 G4DNA 结构的展开。 Pif1 突变会增加患某些乳腺癌的风险。这该项目的总体目标是确定 Pif1 和其他解旋酶推动蛋白质的机制从 DNA 中提取并展开关键的 DNA 结构，例如 G4DNA。我们将确定解旋酶如何受到影响通过与其相互作用的蛋白质，例如单链结合蛋白和重组酶。 G4DNA 序列遍布整个基因组，但优先定位于某些区域例如原癌基因、端粒和线粒体 DNA 的启动子。所通过的机制这些赋予生物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，可以在基因组中的几乎任何位点进行表观蛋白质组分析。我们将确定 G4DNA 位点周围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。我们已经应用蛋白质组筛选发现与 G4DNA 结合的新蛋白质。鉴定出的主要蛋白质，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。这些蛋白质的位置及其在翻译调节中的已知作用使我们检验了以下假设： G4DNA的一项功能。我们的数据支持以下结论：G4DNA 是从受损的线粒体中切除的和核基因组，并且可以完整地进入细胞质，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 的具体序列，其机制是 G4DNA 被切除的情况，以及切除的 G4DNA 影响翻译的具体功能。这长期目标是了解 G4DNA 信号传导如何与其他信号传导途径重叠和交叉例如DNA损伤反应和先天免疫反应。