HELICASE CATALYZED DNA UNWINDING

解旋酶催化 DNA 解旋

• 批准号: 8014458
• 负责人: Timothy M Lohman
• 金额: $ 5.9万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-24 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8014458
• 关键词: ATP Hydrolysis; Bacteriophage T4; Base Pairing; Binding; Bloom Syndrome; Chemicals; Complement; Complex; Coupled; DNA; DNA Binding; DNA biosynthesis; DNA helicase E; Drug Delivery Systems; Enzymes; Escherichia coli; Family member; Filament; Fluorescence; Goals; Grant; Human; Hydrolysis; In Vitro; Kinetics; Link; Metabolism; Methods; Molecular; Molecular Conformation; Molecular Motors; Motor; Movement; Mutagenesis; Mutation; Nature; Organism; Play; Process; Progress Reports; Proteins; Reaction; Research; Role; Single-Stranded DNA; Testing; Thermodynamics; chemical kinetics; dimer; enzyme mechanism; helicase; human DNA; human disease; in vitro activity; melting; monomer; nucleic acid metabolism; protein function; public health relevance; recombinational repair; single molecule; stopped-flow fluorescence; translocase

DESCRIPTION (provided by applicant): DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA metabolism in all organisms. We are studying the kinetic mechanisms of DNA unwinding and DNA translocation of three non-hexameric SF1 DNA helicases from E. coli, Rep, UvrD, and RecBCD, which function in replication, repair, and recombination, respectively. RecBCD is a hetero-trimeric complex containing two SF1 helicases (B and D). Our pre-steady state kinetic studies indicate that Rep and UvrD helicases function as oligomers in vitro, even though monomers of these enzymes can translocate efficiently along ss-DNA. We have discovered an important regulatory domain within these monomers (2B domain) that, when removed, activates helicase activity of a Rep monomer in vitro. Although the 2B domain is not needed for translocation, large movements of the 2B domain are coupled to DNA binding and ATP hydrolysis, hence we will study the role of the 2B domain in monomer ssDNA translocation and DNA unwinding by oligomers. We will use mutagenesis and transient kinetic approaches (stopped-flow and chemical quenched-flow) and methods of analysis that we have developed to examine the details of how the monomeric molecular motor functions in translocation. We will also test current hypothesis for how the oligomeric helicase complexes unwind DNA, with the goal of developing a full kinetic mechanism for unwinding. SF1 helicases also function to disrupt protein-DNA complexes, and we will study the mechanism by which UvrD disrupts RecA-ssDNA filaments. DNA binding, ATP hydrolysis and DNA unwinding by RecBCD and RecBC helicases will also be examined mechanistically. Thermodynamic and kinetic studies will be used to understand how these proteins destabilize (melt) DNA base pairs in a Mg2+dependent, but ATP-independent reaction. We will also examine the mechanism by which accessory proteins, MutL for UvrD and PriC for Rep, stimulate DNA unwinding. Our ensemble studies will be complemented by single molecule studies of DNA binding, translocation and unwinding by these helicases. The overall goal is to obtain a molecular understanding of the kinetic mechanism(s) by which these molecular motors translocate along and unwind DNA and how these processes are coupled to ATP binding and hydrolysis. PUBLIC HEALTH RELEVANCE: DNA helicases and translocases play fundamental roles in all aspects of DNA metabolism, including DNA replication, recombination and repair in all organisms including humans. Mutations in a number of human DNA helicases are linked to several human diseases, including Werner's and Bloom's syndromes. Because of their pivotal roles in nucleic acid metabolism, these enzymes are prime targets for drugs that may inhibit them specifically, and it is critical to understand their mechanisms of action.

描述（由申请人提供）：DNA解旋酶是ATP依赖性运动蛋白，它们放弃双链DNA以形成所有生物体中DNA代谢所需的单个链（SS）DNA中间体。我们正在研究来自大肠杆菌，REP，UVRD和RECBCD的三种非尚面SF1 DNA解旋酶的DNA放松和DNA易位的动力学机制，它们分别在复制，修复和重组中起作用。 RECBCD是一种包含两个SF1解旋酶（B和D）的异晶络合物。我们的稳态状态动力学研究表明，尽管这些酶的单体可以沿SS-DNA有效地转移，但REP和UVRD解旋酶在体外充当寡聚物。我们已经发现了这些单体（2B结构域）内的一个重要的调节结构域，该结构域（2b结构域）在体外激活Rep单体的解旋酶活性。尽管不需要2B结构域才能进行易位，但是2B结构域的大运动与DNA结合和ATP水解耦合，因此我们将研究2B结构域在单体SSDNA易位中的作用，并由低聚物降低DNA。我们将使用诱变和瞬态动力学方法（停止流量和化学淬灭流）以及我们开发的分析方法，以研究单体分子运动在易位中的功能的细节。我们还将检验当前的假设，以了解寡聚旋转酶络合物如何解开DNA，目的是开发完整的动力学机制以放松身心。 SF1解旋酶还起作用可破坏蛋白质-DNA复合物，我们将研究UVRD破坏Reca-Ssdna丝的机制。 RECBCD和RECBC解旋酶也将进行机械检查，将DNA结合，ATP水解和DNA解开。热力学和动力学研究将用于了解这些蛋白如何在Mg2+依赖性但非ATP无依赖性反应中不稳定（熔体）DNA碱基对。我们还将检查辅助蛋白（用于UVRD的MUTL和REP的PriC）的机制，可以刺激DNA的解开。我们的整体研究将通过对这些解旋酶的DNA结合，易位和放松的单分子研究来补充。总体目标是获得对动力学机制的分子理解，这些分子电动机沿该机制沿着并放开DNA以及如何将这些过程与ATP结合和水解耦合。 公共卫生相关性：DNA解旋酶和易位酶在DNA代谢的各个方面都起着基本作用，包括包括人类在内的所有生物中的DNA复制，重组和修复。许多人DNA解旋酶的突变与包括Werner's和Bloom综合征在内的几种人类疾病有关。由于它们在核酸代谢中具有关键作用，因此这些酶是可能抑制它们的药物的主要靶标，因此了解其作用机理至关重要。