DNA Helicases: Mechanisms and Functions

DNA 解旋酶：机制和功能

• 批准号: 8539805
• 负责人: Kevin Douglas Raney
• 金额: $ 26.63万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539805
• 关键词: ATP Hydrolysis; Accounting; Active Sites; Address; Amino Acid Motifs; Bacteriophage T4; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Chemicals; Coupling; DNA; DNA Binding; DNA Footprint; DNA annealing; DNA-Binding Proteins; DNA-Protein Interaction; Data; Defect; Deuterium; Disease; Enzymatic Biochemistry; Enzymes; Exhibits; Family; Funding; Genomic Instability; Hereditary Disease; Homologous Gene; Human Genetics; Hydrogen; Individual; Investigation; Kinetics; Knowledge; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Metabolism; Methods; Molecular; Mutagenesis; Mutation; Pathway interactions; Play; Premature aging syndrome; Process; Protein Dynamics; Proteins; RNA; Reaction; Reporting; Research; Research Project Grants; Role; SH3 Domains; Structure; Structure-Activity Relationship; Surface; Tertiary Protein Structure; Testing; Time; Work; dimer; enzyme coupling mechanism; enzyme mechanism; helicase; melting; member; novel; protein protein interaction; protein structure; quadruplex DNA; recombinational repair; research study; single molecule; tool; unpublished works

DESCRIPTION (provided by applicant): Helicases are ubiquitous enzymes involved in virtually every aspect of DNA and RNA metabolism. This project focuses on one of the largest classes of this family of enzymes, superfamily 1B (SF1B). Limited structural information has slowed progress of our understanding of this class of enzymes. SF1B helicases couple ATP hydrolysis to DNA unwinding, but the rate limiting steps in this process are unknown. Specific amino acid motifs are known to make contact with DNA, but the dynamic role of these motifs has only been inferred. SF1B helicases interact with other proteins such as single-stranded binding proteins, but the biochemical and biological roles of these interactions are largely unaddressed. The importance of filling in these gaps in our knowledge relates to the many roles that helicases play in DNA metabolism including replication, repair, and recombination. Molecular defects in helicase activity have been directly linked to numerous human genetic diseases characterized by genome instability, premature aging, and cancer. Therefore, it is critical that we understand the mechanisms of these enzymes in order to understand how defects at the molecular level can lead to such devastating diseases. Dda helicase from bacteriophage T4 has served as the prototypical model system for the SF1B helicases. New structural data for Dda has led us to propose a mechano-chemical coupling mechanism that involves domains that include the standard helicase motifs along with novel domains that are uncharacterized. Helicase assays and DNA footprinting will be used to test this mechanism. We will determine the kinetic mechanism for ATP hydrolysis during DNA unwinding to determine the overall rate-limiting step in the process, which is currently unknown. Protein domains that are proposed to drive the helicase through conformational changes will be examined by rapid chemical footprinting methods that reveal whether DNA is bound tightly or loosely within the active site. High mobility protein motifs will be identified by hydrogen-deuterium exchange in order to determine the relationship between protein structure and dynamics. One of the major unanswered questions in helicase enzymology relates to the interaction between the enzyme and each individual strand of DNA. A combination of x-ray crystallographic, mass spectrometric and kinetic approaches will be used to identify all of the DNA binding sites on the surface of the enzyme. The structure-function relationship of these novel DNA binding sites will be determined through DNA unwinding experiments. The mechanism by which helicases remove proteins from DNA will be investigated using single molecule approaches. The role of protein-protein interactions will be determined by creating a tethered, dimeric form of the helicase and examining the ability of this enzyme to displace DNA-bound proteins. Answers to the questions posed in this proposal will advance the field in depth (helicase enzymology) and breadth (helicase interactions with protein partners), each of which will facilitate understanding of the role that these enzymes play in normal and pathogenic pathways of DNA metabolism. This work will provide experimental and conceptual tools to investigate other classes of helicases.

描述（由申请人提供）：解旋酶是无处不在的酶，几乎参与了DNA和RNA代谢的各个方面。该项目着重于该酶家族的最大类别之一，即超家族1B（SF1B）。有限的结构信息减慢了我们对这类酶的理解。 SF1B解旋酶将ATP水解与DNA融化，但在此过程中的限制步骤尚不清楚。已知特定的氨基酸基序与DNA接触，但是仅推断出这些基序的动态作用。 SF1B解旋酶与其他蛋白质（例如单链结合蛋白）相互作用，但是这些相互作用的生化和生物学作用在很大程度上是未经压力的。在我们的知识中填补这些空白的重要性与解旋酶在DNA代谢中扮演的许多角色有关，包括复制，修复和重组。解旋酶活性中的分子缺陷已与许多人类遗传疾病直接相关，其特征是基因组不稳定性，过早衰老和癌症。因此，至关重要的是，我们了解这些酶的机制，以了解分子水平的缺陷如何导致这种毁灭性疾病。噬菌体T4的DDA解旋酶已成为SF1B解旋酶的原型模型系统。 DDA的新结构数据使我们提出了一种机械化学耦合机制，该机制涉及包括标准解旋酶基序的域以及未经表征的新域。解旋酶测定和DNA足迹将用于测试该机制。我们将确定在DNA休息期间ATP水解的动力学机制，以确定该过程中的总速率限制步骤，目前尚不清楚。提议通过构象变化驱动解旋酶的蛋白质结构域将通过快速的化学足迹方法来检查，这些方法揭示了DNA是在活性位点内紧密还是松散结合的。高迁移率蛋白基序将通过氢 - 居民交换来识别，以确定蛋白质结构与动力学之间的关系。解旋酶酶学中的主要未解决问题之一与酶与DNA的每个单独的链之间的相互作用有关。 X射线晶体学，质谱和动力学方法的组合将用于识别酶表面上的所有DNA结合位点。这些新型DNA结合位点的结构功能关系将通过DNA放松实验确定。解旋酶从DNA中去除蛋白质的机制将使用单分子方法研究。蛋白质蛋白质相互作用的作用将通过创建解旋酶的束缚，二聚体形式来确定，并检查该酶取代DNA结合蛋白的能力。回答本提案中提出的问题的答案将使该领域深度（解旋酶酶学）和广度（与蛋白质伴侣的解旋酶相互作用），每种酶都将有助于理解这些酶在DNA代谢的正常和致病途径中起的作用。这项工作将提供实验和概念工具来研究其他类别的解旋酶。