DNA Helicases: Mechanisms and Functions

DNA 解旋酶：机制和功能

• 批准号: 8323299
• 负责人: Kevin Douglas Raney
• 金额: $ 27.61万
• 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/8323299
• 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protein interaction; protein structure; quadruplex DNA; recombinational repair; research study; single molecule; tool; unpublished works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）：解旋酶是普遍存在的酶，几乎涉及 DNA 和 RNA 代谢的各个方面。该项目重点关注该酶家族中最大的一类，即超家族 1B (SF1B)。有限的结构信息减缓了我们对此类酶的理解进展。 SF1B 解旋酶将 ATP 水解与 DNA 解旋结合起来，但此过程中的限速步骤尚不清楚。已知特定的氨基酸基序会与 DNA 接触，但这些基序的动态作用仅是推测的。 SF1B 解旋酶与其他蛋白质（例如单链结合蛋白）相互作用，但这些相互作用的生化和生物学作用在很大程度上尚未得到解决。填补我们知识空白的重要性与解旋酶在 DNA 代谢中发挥的许多作用有关，包括复制、修复和重组。解旋酶活性的分子缺陷与许多以基因组不稳定、过早衰老和癌症为特征的人类遗传疾病直接相关。因此，我们必须了解这些酶的机制，以便了解分子水平的缺陷如何导致此类毁灭性疾病。来自噬菌体 T4 的 Dda 解旋酶已作为 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，该机制涉及包含标准解旋酶基序的结构域以及未表征的新结构域。解旋酶测定和 DNA 足迹将用于测试这一机制。我们将确定 DNA 解旋过程中 ATP 水解的动力学机制，以确定该过程中的总体限速步骤，目前尚不清楚。通过构象变化驱动解旋酶的蛋白质结构域将通过快速化学足迹方法进行检查，揭示 DNA 在活性位点内是紧密结合还是松散结合。高迁移率蛋白质基序将通过氢-氘交换来鉴定，以确定蛋白质结构和动力学之间的关系。解旋酶学中尚未解答的主要问题之一涉及酶与每条 DNA 链之间的相互作用。 X 射线晶体学、质谱和动力学方法的结合将用于识别酶表面上的所有 DNA 结合位点。这些新的DNA结合位点的结构-功能关系将通过DNA解旋实验来确定。将使用单分子方法研究解旋酶从 DNA 中去除蛋白质的机制。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并检查该酶取代 DNA 结合蛋白质的能力来确定。对本提案中提出的问题的回答将推动该领域的深度（解旋酶酶学）和广度（解旋酶与蛋白质伙伴的相互作用），每一个都将有助于理解这些酶在 DNA 代谢的正常和致病途径中所发挥的作用。这项工作将为研究其他类别的解旋酶提供实验和概念工具。